JPH04149101A - Transplanted organ-preserving solution in perfusion and transplanted organ preservation in perfusion - Google Patents

Abstract

PURPOSE:To obtain the subject preserving solution capable of sufficiently supplementing oxygen or nutrition necessary by perfusion without possible risk of reduction in ATPase activity or angiopathy, etc., by containing a liposome in which hemoglobin adjusted at a specific concentration is enclosed. CONSTITUTION:The objective perfusion-preserving solution contains a liposome in which hemoglobin is enclosed and the concentration of hemoglobin is adjusted to 7-15wt.% and the colloid osmotic pressure is preferably adjusted to 25-35mmHg without possible risk such as reduction in ATPase activity apt to occur in a low temperature perfusing preservation method or angiopathy, etc., in a case of applying perfluorocarbon emulsion, and is used to preserve a transplanted organ enabling sufficiently supplementing oxygen or nutrition necessary by perfusion. Besides, said preserving solution is preferably perfused at a temperature of 35-38 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a perfusion preservation solution used for preservation of transplanted organs.

(Conventional techniques and problems) It has been over 30 years since kidney transplantation was first performed clinically, and during this time there have been improvements in surgical techniques, consideration of eligibility criteria for transplant patients, advances in immunological tests, and development of immunosuppressants. As a result, transplant results have improved significantly, and transplantation has already become established as a common medical treatment in Europe and the United States. Nowadays, transplants of not only kidneys but also hearts, livers, pancreases, etc. are becoming more common. Advances in organ preservation methods have greatly contributed to this improvement in organ transplant results.Especially in recent years, the rapid increase in the number of transplant cases has led to a shortage of organ supplies, and the ability of organs to function properly after transplantation. In the event of failure, retransplantation is extremely difficult.Therefore, there is a strong need to establish a method that can preserve the transplanted organ for a long time without reducing its function.

The organ preservation method currently used clinically is the low-temperature immersion method (Lancet 212+9-1222 (1969)).
:l and cold perfusion preservation method [Lancet 2536 (1
967))).

In recent years, as cadaveric kidney transplantation has become widespread, low-temperature perfusion preservation methods that allow organs to be preserved for long periods of time have become increasingly important, and the development and improvement of fluids used in this preservation method are actively underway. It is being said.

The purpose of cold perfusion preservation is to preserve organs in conditions closer to physiological conditions. In other words, low-temperature perfusion preservation is a method of suppressing metabolism by keeping tissues at low temperatures (generally below 10°C), providing necessary oxygen and nutrients through perfusion, and removing metabolites. .

Here, it is generally known that when blood itself is used as a perfusate, increases in viscosity, tissue deposition of free hemoglobin due to red blood cell destruction, and thrombus formation due to platelets, lipids, and fibrinogen occur.

Berza et al. freeze-thawed plasma and removed as much fibrinogen as possible to produce cryoprecipitated plasma (Cryoprecipitated plasma).
We successfully preserved canine kidneys for 72 hours using a perfusate containing plasma. [Lanc
et 2.536. (1,967)] Furthermore, Toledo-Verreira et al. completely removed fibrinoken and β-lipoprotein remaining in cryoprecipitated plasma treated with silica gel, and further reduced the amount of triglyceride, which is an insoluble lipid fraction, to 173%. A reduced perfusate was developed.

However, since the insoluble lipid fraction is not completely removed from these low-temperature precipitated plasmas, there is a risk of lipid embolism occurring during perfusion. ), it has the disadvantage of being associated with the risk of hepatitis virus infection.

To compensate for these shortcomings, Gertmann et al. used a perfusate containing 6% human albumin in Krebs-Ringer's solution to perfuse Daiken at low temperature for 96 hours, performed autologous transplantation, and achieved over 80% success. We succeeded in obtaining a high rate of arrival. (Grundman et al; Transpl,
, 17299-305 (1974)) However,
This low-temperature perfusion preservation method using a perfusate has the following problems.

That is, cells maintain their shape by operating Na pumps without consuming ATP. However, when the tissue becomes cold, the activity of ATPase in the vascular endothelium decreases, so energy is not supplied and the Na pump stops working, causing cells to undergo cell division. As the preserved organ lengthens, the vascular endothelium and its surroundings become more prominent, which is thought to cause microcirculatory disturbances after transplantation. [Jacbsen
I^, Pegg DE: Kiclncy, In+
Organ preservation for tr
ansplantation Karow AM, Pe
gg DE (eds), p556-558. Marc
el Dekker Inc., New York
, 198]) On the other hand, fully fluorinated organic compounds (perfluorocarbon compounds) are liquids that dissolve oxygen very well and act as oxygen carriers in the form of emulsions. Using this perfluorocarbon emulsion, Nakaya et al. perfused rabbit kidneys at room temperature for 9 hours and examined the viability of the kidneys using a biochemical method. (Proceeding of Sym
posium on perfluoro-chemi
cal artificial blood, Kyo
Berkowitz et al. perfused the kidney with a perfluorocarbon emulsion stabilized with albumin and examined the results after transplantation. As a result, they reported that the kidney engraftment rate was nearly 100%, which was a good result. [J, Surg, Res, 20595
600 (1976)) However, when this perfluorocarbon emulsion is applied as a perfusate, in order to sufficiently dissolve oxygen in the perfusate, the partial pressure of oxygen in the perfusate is lowered by using an oxygenator such as a membrane type or bubbling type. must be circulated while maintaining a sufficiently high temperature, which inevitably complicates the apparatus.In addition, Southard et al.
It has been reported that increasing the oxygen partial pressure of the perfusate reduces mitochondrial function in vascular endothelial cells. (S
outside JHetaloToxicity o
f oxygen to m1tochondrial
Respiratory activity in
hypothermical perfused ca
nine kids, Transplantat
ion 29:459.1980) Therefore, when a perfluorocarbon emulsion is applied as a perfusate, there is a risk of damage due to excessive oxygenation and damage to blood vessels due to perfusion pressure.

(Problems to be Solved by the Invention) Therefore, the present invention solves the problem of ATPa observed in low-temperature perfusion preservation method.
To provide a new perfusion preservation solution that has risks such as decreased se activity and vascular disorders when perfluorocarbon emulsions are applied, and which can sufficiently support necessary oxygen and nutrients through perfusion. The purpose is to

(Means for Solving the Problems) Therefore, the present invention for solving the above problems is characterized in that it contains a liposome in which hemoglobin is encapsulated, and the hemoglobin concentration is adjusted to 7 to 15% by weight. Transplant organ perfusion preservation solution.

It is preferable that the colloid osmotic pressure of the preservation solution is adjusted to 25 to 35 mmHg.

In addition, when perfusing the preservation solution, 35 to 3
Preferably, it is carried out at a temperature of 8°C.

The present invention will be explained in detail below.

The transplant organ perfusion preservation solution (hereinafter simply referred to as preservation solution) according to the present invention is characterized by containing liposomes in which hemoglobin is encapsulated.

Liposomes encapsulating hemoglobin (hereinafter referred to as hemoglobin-encapsulating liposomes) and methods for producing the same have already been disclosed in JP-A-52-1.51758 and JP-A-58-1.
No. 83625, No. 61-37735, No. 62-1.7
No. 8521, No. 63-209746, No. 63-211
．． No. 230, No. 63275522, No. 64-61.4
No. 26, No. 6475418, JP-A-1-1.8024
It is disclosed in Publication No. 5.

The hemoglobin-containing liposome will be explained below.

There are no particular restrictions on the lipid that forms the liposome membrane, and any natural or synthetic lipid can be used as long as it forms a liposome. In particular, phospholipids are preferably used, examples of which include phosphatinylethanolamine, phosphatidylserine, sphingomyelin, and phosphatinorcholine, which are derived from egg yolk, soybeans, and other natural materials. , or those obtained by organic chemical synthesis means can be used alone or in combination as the main component. In addition, cholesterol as a membrane stabilizer,
Sterols such as cholestanol, and pear-shaped substances such as phosphatic acid, ncetyl phosphate, and higher fatty acids may be added.

In the preservation solution of the present invention, the hemoglobin concentration is 7.
It is necessary to adjust it to ~15%. By adjusting to such a value, even if the tissue is kept at room temperature and its metabolism becomes active, sufficient oxygen can be supplied to compensate for this.

As an attempt to use hemoglobin as an oxygen transport substance and apply it to a transplant tissue perfusion preservation solution, stabilized hemoglobin in which polyoxine ethylene is bonded to hemoglobin is known. [Toshikatsu Iwasaki, Takashi Yamakawa, Yuji Shishita]
Artificial Organs 17(2), 704-707 (1988)
] As described above, in the preservation solution of the present invention, hemoglobin is encapsulated within liposomes, so the colloid osmotic pressure is not affected by the hemoglobin concentration. Therefore,
By increasing hemoglobin concentration to 7-15%,
It becomes possible to supply sufficient oxygen even at room temperature. Furthermore, the production of methemoglobin during perfusion can be effectively suppressed.

If a preservation solution containing polyoxene ethylene-bound hemoglobin as a main component is applied to the dough, the hemoglobin concentration is lower than 6 to maintain the colloid osmotic pressure allowed by the tissue, since the hemoglobin is not encapsulated. There was a problem in that the oxygen transport capacity was limited to about 100% by weight, and the oxygen transport ability was insufficient for perfusion at room temperature. Additionally, it has been pointed out that methemoglobin production is significant (20% in 24 hours) when this preservation solution is perfused at room temperature. It was necessary to suppress the metabolism by keeping the tissue at a certain low temperature (20°C).

The concentration and viscosity of the hemoglobin solution incorporated into the liposome are not particularly limited, but the viscosity is preferably 10 to 3.000 cp (4°C) and the hemoglobin concentration is 30 to 60% (W/W). If the colloid osmotic pressure is extremely low compared to the normal colloid osmotic pressure of biological blood, there is a risk that cells outside the blood vessels will float.
If it is too high, cells outside the blood vessels may become dehydrated. The preservation solution of the present invention has had its osmotic pressure adjusted by, for example, adding a colloid osmotic pressure adjusting agent;
Specifically, it is desired that the weight be adjusted to 25 to 35 mm and 11 g.

Such colloid osmotic pressure regulators include proteins derived from natural plasma such as albumin and globulin, or substances such as gum acacia, modified ceratin, polyvinylpyrrolidone, dextran, polyethylene glycol, calphomethyl cellulose, and human quinoethyldenbuno. ＼Technical water-soluble polymer compounds are included.

The viscosity of the preservation solution is preferably adjusted to a viscosity equal to or lower than that of natural blood, ie, 4.5 cp or less, at a shear rate of 333 sec and a temperature of 37°C.

Furthermore, in the engineered blood of the present invention, the electrolyte composition is
It is preferable that the composition be adjusted to approximately the same composition as natural blood using an electrolyte solution such as Ringer's solution, Lactated Linker's solution, or Collins' solution.

Next, the method for producing the preservation solution of the present invention will be explained.

Although known methods can be used to produce hemoglobin-containing liposomes, it is preferable to add distilled water to a homogeneous mixed powder obtained by freeze-drying liposome membrane-forming lipids,
Hydrate the lipids by heating to ~80°C, add an aqueous hemoglobin solution to the hydrate, and stir at 5-10°C using a high-speed stirrer until the average particle size of the suspended particles in the mixture is 1.80. ~300n
This is achieved by stirring until m.

Mixing and stirring of the liposome membrane-forming lipid and hemoglobin aqueous solution is carried out by stirring using a stirring type cell crusher (Waring blender) until the outer diameter of the suspended particles becomes 180 to 300 nm. Stirring is performed at a rotation speed of 10.
It is desirable to conduct the heating at 000 to 20.00 rpm for 6 to 10 minutes.

The liposome suspension thus obtained is washed and concentrated according to a conventional method.

The washed and concentrated liposome suspension has a colloid osmotic pressure of 25-35 ++++nF1g and a hemoglobin concentration of 7.
A colloid osmotic pressure regulator and an electrolyte solution are added and mixed so that the concentration is 15% to obtain the preservation solution of the present invention. In addition,
The colloid osmotic pressure regulator may be added and dissolved in the aqueous suspension of liposomes in the form of raw material powder.

Next, a method for preserving a transplanted organ using the preservation solution of the present invention will be explained.

That is, a transplanted organ donated by an organ donor is washed with modified Collins' solution or lactated Ringer's solution, then connected to the preservation solution of the present invention using a commercially available perfusion preservation device, and kept at 35 to 38° C. for a predetermined period of time. Maintained with perfusion (approximately 24-72 hours). The circulation speed at this time is
Usually 10-200+r+f/g (kidney) for 7 hours. After perfusion preservation, the tissue is washed with modified Ringer's solution or lactated Ringer's solution, and then used for transplantation.

Next, the present invention will be explained in more detail with reference to Examples and Comparative Examples.

〔Example〕

■Preparation of posome suspension containing hemoglobin Add an equal amount of distilled water to 108 g of purified soybean phosphatidylconone, cholesterol, myristic acid, and vitamin E (presome, Nippon Keikahige 'J) with a hydrogenation rate of 90%. In addition, hydration treatment at 600C for 60 minutes was performed. 60 mj of red blood cell membrane-removed hemoglobin solution with a hemoglobin concentration of 50% (W/W) was added to the obtained hydrated lipid, and the mixture was cooled to 5°C using a high-speed stirrer [Ashihomo Mixer, manufactured by Tokushu Kika Kogyo Co., Ltd.]. , 1. Stirring treatment was performed at 0.000 rpm for 60 minutes.

The obtained liposome suspension was diluted 10 times with physiological saline, and sterilization and particle size control were performed using a 045μ filter.The filtrate was treated with a plasma separator, and no particles were incorporated into the liposomes. Hemoglobin and microparticles were removed. Physiological saline was added to the filtrate until hemoglobin was no longer detected, circulation washing was repeated, and the filtrate was finally concentrated until the hemoglobin concentration was 5%. The volume of the obtained Liposome V suspension is 1200m+? Met.

■Adjustment of modified Ringer's solution Na C16,51gefSMgSOn 0.4 g/
f.

KCl0.8 g#5NaHCO30,24g#,Na
2HP 040.14 FZ/l, glucose 6.0
Dissolved in distilled water in the proportion of g#, the electrolyte composition is Na=1
12, K-11, Mg=7, Cr=118, Hco=3
, HPO4=2.5O4=7, and glucose=33 (each unit is m E q /l'). ■Commercially available Hydroquine Dildenbun (Sarinhesu Kyorin Seiyaku Tosei) was dissolved in the solution obtained in ■, and then
The modified Linkel's solution obtained in
/V) to prepare a perfusate (Example).

The colloid osmotic pressure of the obtained perfusate was 28 mmHg, the quality osmotic pressure was 290 m05 m, and the viscosity was 3.2 cp (sliding speed 3
835 ec-', temperature 37°C).

[Comparative example: Perfusion preservation solution containing polyoquinoethylene-bound hemoglohine]

Monomethquine polyoxyethylene succinic acid monoester (average molecular weight 5000) 5q, N-hydroxysuccinimide 0.23q, zinclohexynolcarbonimide 0
42g was dissolved in 3QQm/(7)N,N dimethylformamide and stirred at room temperature overnight.

Filter the generated synchrohequinurea and add 6QQ to the filtrate.
ml of ethyl ether was added, and the formed monomethoxypolyethylene glycol mono (zakunimine rusacunonate) crystals were filtered, washed with ether, and dried.
．． White crystals of f3g were obtained.

Mocrohin 0.59 to pyridoxal phosphate binding 10
To a solution dissolved in 0 mn boric acid buffer (pH 8,5), 0.59% of the above active ester was added under water cooling.

After stirring for 4 hours under water cooling, ultrafiltration was repeated using an ultrafiltration membrane with a molecular weight blocking of 30,000 to remove unreacted active esters or their decomposition products. Obtained.

The colloid osmotic pressure of the obtained modified hemoglobin solution was 25 m
m1g, quality osmotic pressure is 29 Q m05m, viscosity is 32
Cp (slip rate 3835 ec-', temperature 37°C).

[Methemoglohine production experiment] Using two porofiber type artificial persimmon caviox ■ (manufactured by Terumo Corporation), pure oxygen was added to one side, and 5.2% oxygen (oxygen partial pressure 40 mmHg) was added to the other side B for 200 ml. !
It flowed at a speed of / minute. In this system, the clear stream preservation solutions of Examples and Comparative Examples were perfused at a rate of 1.00 ml/min at 37° C., and the methemoglobin ratio was measured over time by visible absorption. As a result, in the example in which the perfusion preservation solution of the comparative example was perfused, the value increased by 20% compared to the initial value in 24 hours, whereas in the case of perfusion with the perfusion preservation solution of the example, it increased by 12% compared to the initial value. only rose.

[Transplant Kidney Perfusion Preservation Experiment] A transplant kidney perfusion preservation experiment was conducted using the perfusion preservation solutions of Examples and Comparative Examples. The experiment was conducted in the following steps.

Ten adult mongrels weighing 1.0 kg were anesthetized by intravenously injecting bentoparbital sodium at a rate of 30 mg/kg, and the left kidney was removed through abdominal midline incision. After inserting a cannula into the ureter, artery, and vein of the excised kidney, Rei's modified Collins solution was introduced into the cannula for washing, and then the perfusion preservation solution of the example and comparative example was introduced into the cannula. Perfusion was performed for 48 hours at 37°C.

After storage, wash again using modified Collins solution,
This was autotransplanted into the thigh. Furthermore, the contralateral kidney was removed 1 week after transplantation.

After that, those that survived for 60 days or more were judged to be alive, and 6
Those who died in less than 0 days and were accompanied by renal insufficiency were judged to have died due to poor renal preservation. As a result, in the case where the perfusion preservation solution of the example was used, 9 out of 10 animals survived, whereas in the case where the perfusion preservation solution of the comparative example was used, only 4 out of 10 animals survived. .

(Effects of the Invention) As detailed above, the transplant organ perfusion preservation solution of the present invention contains liposomes with hemoglobin encapsulated therein, and the hemoglobin concentration is adjusted to 7 to 15% by weight. AT, which is seen in cold perfusion preservation method, is characterized by
Although there are risks such as a decrease in Pase activity and vascular disorders when a perfluorocarbon emulsion is applied, it has the effect of being able to sufficiently support necessary oxygen and nutrients through perfusion.

Claims (3)

[Claims] (1) A transplanted organ perfusion preservation solution, characterized in that it contains a liposome in which hemoglobin is encapsulated, and the hemoglobin concentration is adjusted to 7 to 15% by weight. (2) The transplanted organ perfusion preservation solution according to claim 1, wherein the colloid osmotic pressure is adjusted to 25 to 35 mmHg. (3) A method for perfusion preservation of a transplanted organ, which comprises perfusing the transplantation organ perfusion preservation solution according to claim 1 or 2 at a temperature of 35 to 38°C.