JPH11246301A - Organ preserving solution - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an organ preserving solution substitutable to hydroxyethyl starch by including hyaluronic acid (salt) and useful for transplantation in which a survival ratio of cells during preserving an organ is kept by improving a defect accompanying to its use. SOLUTION: This organ preserving solution preferably having 6.5-7.8 pH, 240-360 mOsm/kg is obtained by including (A) hyaluronic acid (salt) and (B) other components adjusting osmosis, pH, etc., as necessary. The hyaluronic acid is usable the one obtained from an extract from crista galli, glass body, umbilical cord, etc., or one kind of bacterium, e.g. the one obtained from a cultured material of a hyaluronic acid produced bacterium of genus Streptococcus, and preferably the one obtained from the cultured material of hyaluronic acid produced bacterium is used. As the hyaluronic acid salt, preferably a sodium salt or a potassium salt is used. Preferably, an average molecular weight of the component A is >=10×10<4> , more preferably (10-250)×10<4> . Preferably, a concentration of the component A is 0.05-1.0 wt.%.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an organ preservation solution, and more particularly to an organ preservation solution for transplantation containing hyaluronic acid and / or a physiologically acceptable salt thereof (hereinafter collectively referred to as hyaluronic acids). Related to the solution.

[0002]

BACKGROUND OF THE INVENTION Today,
Organ transplantation has been applied clinically as the ultimate treatment for refractory end-stage organ disorders. In the case of liver transplantation, the five-year survival rate reaches about 70%, and this high result has been supported by improvements in surgical methods, development of organ preservation solutions and immunosuppressants, and development of organ supply systems. Many organ preservation solutions have been studied, including Euro-collins Solution (ECS). At present, for example, EP-11 solution is used as an eyeball preservation solution, and Universi is said to be the most excellent organ preservation solution in liver, kidney, pancreas, lung and the like.
ty of Wisconsin Solution (UWS) is widely used. Clinically, UWS's organ preservation time averaged 24 hours for the kidney, 17 hours for the liver and pancreas, and 4 hours for the heart (JH Southard, Hiroshima, 1993). . If the preservation time can be further extended, organs can be procured from remote locations, and elective transplant operations can be performed. It has great potential to contribute to improvement.

[0003] An organ preservation solution is a polymer for preventing cell / tissue swelling during flush-out by the solution and cell swelling / tissue edema due to low temperature during organ preservation. An electrolyte is blended. Many studies have so far been made on, for example, serum albumin, dextran, polyvinylpyrrolidone, Ficoll, and polyethylene glycol. In UWS, which is said to be the best organ preservation solution, hydroxyethyl starch (HES) is used as a polymer electrolyte of the organ preservation solution. HES is a high molecular weight polysaccharide having α-1,4 and α-1,6 bonds of glucose in which a hydroxyl group is substituted with a hydroxyethyl group, and plays a role as a shape maintaining substance in UWS. However, since HES is originally an ex vivo component, it has been difficult to guarantee safety including biocompatibility and toxicity.

On the other hand, hyaluronic acid is a linear high-molecular-weight mucopolysaccharide having extremely high viscosity and moisturizing properties, which is present in a large amount in connective tissues of mammals, and is essentially non-antigenic and biocompatible. Therefore, it is used as a therapeutic drug for osteoarthritis of the knee and an auxiliary agent for ophthalmic surgery.

The present inventors have conducted intensive studies on various polymer substances which can be substituted for HES, which is currently mainly used as the above-mentioned polymer electrolyte, and which can improve disadvantages associated with their use. The use of hyaluronic acids surprisingly maintained the survival rate of cells during organ preservation, and found that the above-mentioned problems could be solved. Thus, the present invention was completed.

[0006]

That is, the present invention provides:
(1) an organ preservation solution for transplantation containing hyaluronic acids, (2) the solution according to (1), wherein the average molecular weight of the hyaluronic acids is 100,000 or more, and (3) a concentration of the hyaluronic acids of 0.05 to The solution according to (2), which is 1.0% by weight.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
The hyaluronic acid in the present invention is obtained from an extract from a cockscomb, vitreous body, umbilical cord or the like or a certain bacterium, such as a culture of a hyaluronic acid-producing bacterium of the genus Streptococcus. Can be used. However, hyaluronic acid obtained from a culture of a hyaluronic acid-producing bacterium is preferable because a high molecular weight hyaluronic acid can be obtained with high purity. Hyaluronic acid may be a free acid or a physiologically acceptable salt thereof. Particularly preferred physiologically acceptable salts are those in the form of sodium or potassium salts which are compatible with the other components added to the organ preservation solution of the present invention.

The hyaluronic acids used in the present invention preferably have an average molecular weight of 100,000 or more.
In the case of high molecular substances such as hyaluronic acids, it is difficult to uniformly define the molecular weight because the molecular weight distribution is not monodisperse but polydisperse, but the average molecular weight is preferably in the range of 100,000 to 2.5 million. Is within. If the average molecular weight is less than 100,000, since it is a low molecule such as glucose, it is taken into the cells of the organ, and there is a concern that cells and tissues may swell. On the other hand, if the average molecular weight is 2.5 million or more, the viscosity of the organ preservation solution increases and the stickiness becomes sticky, so that the operability at the time of organ handling decreases.

[0009] The concentration of hyaluronic acids is 0.05-1.
It is preferably 0% by weight. When the concentration of the hyaluronic acids is 0.05% by weight or less, the protective effect of the cells is reduced. On the other hand, if it exceeds 1.0% by weight, the viscosity increases and the stickiness and operability decrease.

[0010] In the organ preservation solution of the present invention, other components for adjusting osmotic pressure, pH and the like are added as necessary. As other components used, usually, sodium ion, potassium ion, calcium ion, magnesium ion and the like can be mentioned as cations, and chloride ion, phosphate ion, sulfate ion, and citrate ion can be mentioned as anions. Rara. And it may further include raffinose.

Each of the above components is formulated at an appropriate concentration, the pH is adjusted to 6.5 to 7.8, and the osmotic pressure is adjusted to 240 to
When adjusted to the range of 360 mOsm / kg, the organ preservation solution of the present invention can be provided. Preferably, each component is added as potassium phosphate or magnesium sulfate.

As a method for producing the organ preservation solution of the present invention, for example, lactobionic acid, potassium phosphate,
Add magnesium sulfate, adenosine, glutathione, sodium hyaluronate, raffinose, and allopurinol, dissolve in water for injection at room temperature, adjust the pH with sodium hydroxide, and finally sterilize by filtration. The organ preservation solution thus obtained usually has a pH of 6.5 to 7.8 and an osmotic pressure of 240 to 360 mOsm / kg.

[0013]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to specific examples of organ preservation solutions for transplantation containing hyaluronic acids, but the present invention is not limited to these examples. Absent. The following organ preservation solution was used. Here, the organ preservation solution was manufactured according to the following formulation. In addition, a commercially available organ preservation solution was used.

Organ preservation solution In a 2 liter flask, 100 mmol of lactobionic acid, 25 mmol of potassium phosphate, 5 mmol of magnesium sulfate, 5 mmol of adenosine, 3 mmol of glutathione, sodium hyaluronate (average molecular weight of 6
3) and 69.45 mmol of raffinose were weighed and dissolved in an appropriate amount of water for injection at room temperature. Next, 1 mmol of allopurinol was dissolved in a small amount of 1N sodium hydroxide and added to it. This solution was adjusted to pH 7.4 with 1N sodium hydroxide to make the total volume 1 liter. The concentration of sodium hyaluronate at this time was
0.3% by weight. This is sterilized by filtration and 200 ml
The vial was aseptically dispensed and filled into a vial. Its osmotic pressure was 318 mOsm / kg and its viscosity was 4.5 mPa · s.

Organ Preservation Solution In a 2 liter flask, 100 mmol of lactobionic acid, 25 mmol of potassium phosphate, 5 mmol of magnesium sulfate, 5 mmol of adenosine, 3 mmol of glutathione, sodium hyaluronate (average molecular weight 2
2) and 56.59 mmol of raffinose were weighed and dissolved in an appropriate amount of water for injection at room temperature. Next, 1 mmol of allopurinol was dissolved in a small amount of 1N sodium hydroxide and added to it. This solution was adjusted to pH 7.4 with 1N sodium hydroxide to make the total volume 1 liter. The concentration of sodium hyaluronate at this time was
0.2% by weight. This is sterilized by filtration and 200 ml
The vial was aseptically dispensed and filled into a vial. Its osmotic pressure was 318 mOsm / kg and viscosity was 3.8 mPa · s.

Organ preservation solution In a 2 liter flask, 100 mmol of lactobionic acid, 25 mmol of potassium phosphate, 5 mmol of magnesium sulfate, 5 mmol of adenosine, 3 mmol of glutathione, and 80 HES 70000 (manufactured by Kyorin Pharmaceutical)
g and raffinose (17.63 mmol) were weighed and dissolved in an appropriate amount of water for injection at room temperature. Next, 1 mmol of allopurinol was dissolved in a small amount of 1N sodium hydroxide and added to it. This solution was adjusted to pH 7.4 with 1N sodium hydroxide to make the total volume 1 liter. This was sterilized by filtration, and aseptically dispensed and filled into a 200 ml vial. The osmotic pressure of this is 316 mOsm / kg,
The viscosity was 3.2 mPa · s.

Organ preservation solution Organ preservation solution: Viaspan (manufactured by Alcon Japan, UWS) The osmotic pressure of this is 321 mOsm / kg, and the viscosity is 3.8 mPa · s.

Example 1 Preparation of Rat Free Hepatocytes According to a conventional method, Wistar rats were anesthetized by intraperitoneal injection of pentobarbital, and the liver was perfused with collagenase for about 15 minutes. Thus, a rat free hepatocyte solution was obtained.

Evaluation method of stock solution by trypan blue exclusion test (TB test) Cells are stained with trypan blue, the total number of cells and the number of dead cells are counted under a microscope, and the survival rate and cell density are calculated. The higher the cell viability (%), the higher the performance of the organ preservation solution. In addition, cell viability (%) and cell density (ce
lls / ml) is calculated by the following equation. Cell viability (%) = (total cell number−dead cell number) / total cell number × 100 cell density (cells / ml) = (total cell number−dead cell number) /
0.1 x 1000 x dilution ratio

Evaluation method of stock solution by thiobarbituric acid test (TBAtest) Rat free hepatocyte solution was appropriately diluted, and 1
1 ml of 0% trichloroacetic acid and 2 ml of 0.7% thiobarbituric acid are added and mixed, heated at 100 ° C. for 20 minutes, centrifuged at 1000 × G for 25 minutes at room temperature, and the absorbance of the supernatant is measured at 530 nm. . A high TBA value (abs / cells) indicates that lipid peroxidation is progressing, which is not good for organ preservation. In addition, the TBA value (abs / ce
lls) is calculated by the following equation. TBA (abs / cells) = absorbance / cell density

Evaluation of Storage Solution Using Rat Free Hepatocytes 1-1. As in 1-4, 2 ml of the above organ preservation solution was separately added to 1 ml of the rat free hepatocyte solution, and stored at 4 ° C. for 48 hours under light shielding. After storage, use the above TB test and TBA test
Was done. The survival rate (%) of rat free hepatocytes is TB test
And the TBA value (abs / cells) was determined from the TBA test. Table 1 shows the results.

[0022]

[Table 1]

From Table 1, it can be seen that both the survival rate of rat free hepatocytes and the TBA value were the same as those of Experiment No. 1. 1-1 and No. 1 1-2
Is the experiment No. Addition of HES of 1-3 and Experiment No. 1-
Since a value equivalent to or higher than UWS (manufactured by Nippon Alcon) of No. 4 was obtained, it was shown that the addition of hyaluronic acid was highly effective as an organ preservation solution. In addition, with the average molecular weight of hyaluronic acid of the organ preservation solution of 600,000 and the average molecular weight of hyaluronic acid of the organ preservation solution of 200,000, a higher survival rate and a lower TBA value were obtained as compared with the addition of HES to the organ preservation solution.

Reference Example 1 Four 2 liter flasks were prepared, and 100 mmol of lactobionic acid, 25 mmol of potassium phosphate, 5 mmol of magnesium sulfate, 5 mmol of adenosine, and 3 mmol of glutathione were weighed. Was dissolved in an appropriate amount of water for injection at room temperature. Next, 1 mmol of allopurinol was dissolved in a small amount of 1N sodium hydroxide. Then, raffinose was not added (Experiment No. 2-1), 37.56 mmol (Experiment No. 2-2), and 83.40 mmol (Experiment No. 2-3) were added to four 2-liter flasks, respectively. 162.12 mmol (Experiment No. 2-4). These solutions were adjusted to pH 7.4 with 1N sodium hydroxide to make the total volume 1 liter. Each of them was sterilized by filtration, and aseptically dispensed and filled into 200 ml vials. And the osmotic pressure of these and the organ preservation solution was measured. The osmotic pressure was determined by experiment N
o. 2-1 to No. 239, 280, 32 for 2-4
5, and 407 mOsm / kg. 2-
The osmotic pressure of the organ preservation solution of No. 5 was 318 mOsm / kg.

Evaluation of preservation solution using rat free hepatocytes 1 ml of rat free hepatocyte solution was compared with 2 ml of organ preservation solution having different osmotic pressure depending on the above four raffinose concentrations.
And stored at 4 ° C. for 48 hours under light shielding. After storage, the above TB test and TBA test were performed on these four types of organ preservation solutions. The survival rate (%) of rat free hepatocytes was determined from the TB test, and the TBA value (abs / cells) was determined from the TBA test. Table 2 shows the results.

[0026]

[Table 2]

From Table 2, it can be seen that the survival rate and TBA value of rat free hepatocytes were effective at an osmotic pressure of 239 to 407 mOsm / kg, and particularly high at 239 to 325 mOsm / kg.

[0028]

As described above, the organ preservation solution of the present invention is attained by including hyaluronic acids in the organ preservation solution, and is equivalent to or conventionally equivalent to an organ preservation solution to which HES has been added. It has the effect of preserving organs more.

Claims (3)

[Claims] 1. An organ preservation solution for transplantation comprising hyaluronic acid and / or a physiologically acceptable salt thereof. 2. The hyaluronic acid and / or a physiologically acceptable salt thereof has an average molecular weight of 100,000 or more.
The solution as described. 3. The solution according to claim 2, wherein the concentration of hyaluronic acid and / or a physiologically acceptable salt thereof is 0.05 to 1.0% by weight.