FI65009C - PERFUSIONSVAETSKA FOER KONSERVERING AV ETT ORGAN SOM SKALL TRANSPLANTERAS OCH KONSERVERINGSFOERFARAS - Google Patents

Description

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Pmtantti- ja r.kl.t.H Killitti. >. | hT ^ .. ___ och nglrtwrtyflwn '* Amatoi ud ^ d od> 30.11.83 (32) (33) (31) * ΤΤ * «* Τ weMw .- * efcd prtoriMc (71) The Green Cross Corporation, 1-1 + 7, Chuo-l-chcme, Joto-ku, Osaka,

Japan-Japan (JP) (72) Kazumasa Yokoyama, Toyonaka-shi, Masakazu Ivai, Fujiidera-shi,

Hiroyuki Okamoto, Akashi-shi, Noboru Yamada, Ashiya-shi, J apani-Japan (JP) (7I +) Oy Kolster Ab (5I +) Perfusion solution for preservation of a transplanted organ and method of preservation - Perfusions for preservation of organ intended for transplantation and preservation

The invention relates to a liquid for perfusion preservation of a transplantable organ containing a modified Ringer's solution, to a process for preparing this liquid and to a process for preserving the transplantable organ using as perfusate a mixture comprising Ringer's solution, albumin and perfluorocarbon emulsion used as a saline solution.

In recent years, technological advances in organ preservation for transplantation have been significant: in particular, developments in methods for preserving the kidneys have strongly promoted kidney transplantation from the deceased. This development has been greatly facilitated by improvements in perfusates. For example, clinical methods for preserving the kidney for transplantation are basically divided into two categories, one being hypothermic storage (by immersion) (Lancet 2 (1969) 1219-122 and the other by hypothermic continuous perfusion (Lancet 2 (1967) 536).) In practice 2 65009 the former is used The importance of long-term preservation, i.e., perfusate preservation, increases with the further development of kidney transplantation from corpses.

In the transplantation of kidneys from the deceased, since most kidneys are donated from victims, the necessary steps between deceased kidney removal and transplantation include histological compatibility tests between the deceased kidney and patient, selection of a suitable recipient, donated kidney transplantation, and transplant preparation. For these reasons, it is necessary to preserve the donated kidney for as long as possible. In fact, as part of studies of kidney transplantation from the deceased, several attempts have been made to date to preserve the deceased kidney for a long time. One of the main targets of these efforts is the development and improvement of perfusates.

The increasing clinical use of hypothermic continuous perfusion is based on the research work of Belzer et al (Lancet 536 (1967)). They managed to preserve the dog’s kidney for 72 hours using as fibrogen-purified plasma as possible as a perfusate prepared by freezing and thawing cryoprecipitated plasma. Since the publication of their research results, cryoprecipitated plasma has been mainly used as a perfusate for the kidneys.

In 1974, Toledo-Pereyra et al. (Surg, Gynecol. Obstet 138 (1974) 901-905) developed a new perfusate from silica gel-treated cryoprecipitated plasma that lacks fibrinogen β-lipoprotein but still retains conventional cryopreservation. as precipitated plasma and in which the insoluble fat fraction, i.e. triglycerides, the proportion is reduced by a third. Conventional cryoprecipitated plasma poses a high risk of blood clot formation during perfusion due to residues of fibrinogen, lipoprotein, or neutral fat globules, whereas this risk is greatly reduced by the action of silica gel-treated cryoprecipitated plasma developed by Joledo-Pereyra et al. However, these cryoprecipitated plasmas still have serious disadvantages. First, the preparative procedure is not yet fully established and the composition of the treated plasma tends to vary, making it difficult to obtain preparations with constant protein content and electrolyte composition. Secondly, there is still a slight risk of the formation of fat plugs during perfusion due to the incomplete removal of the insoluble fat fraction. To reduce this risk, it is necessary to use an expensive membrane oxidizer. Third, an important and substantial drawback is the lack of appropriate deactivation treatment against the hepatitis virus, which poses a potential risk of infection caused by the hepatitis virus. Although cryoprecipitated plasma is an excellent perfusate for kidney preservation, it has several of the disadvantages outlined above.

It has been shown that in order to overcome the above-mentioned difficulties associated with cryoprecipitated plasma, efforts have been made to use albumin solution as a perfusate and that results have been obtained in animal experiments. Grundman et al (Transpl., 17, 299-305) perfused the dog's kidney with 6% albumin solution and successfully preserved the kidney for 96 hours. Their perfusate was Krebs Ringer's solution containing 6% (w / v) human albumin. The dog's kidney perfused for 96 hours at low temperature was autotransplanted and the success rate was greater than 80%, indicating that the albumin solution is an excellent perfusate and can be advantageously compared to cryoprecipitated plasma.

Long-term preservation of the kidney requires a perfusate which has a certain colloidal osmotic pressure at low temperatures and which has a suitable electrolyte composition and in addition contains a certain amount of oxygen. When stored at low temperatures, the kidney consumes significantly less oxygen than at room temperature, but still requires a certain amount of oxygen to maintain its function. According to classical studies of the plate (American Journal Physiology 197 (1959) 1111-1114, the oxygen consumption of the kidney at 8-10 ° C is about 3 μg / g / min, which corresponds to about 20% of the consumption at room temperature. it is common practice to perfuse it, in which case the perfusate is oxidized.

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A saturated organic perfluoro compound (hereinafter abbreviated as PFC) is a liquid that is capable of dissolving a substantial volume of oxygen and acts as an oxygen carrier when used as an emulsion. Developmental studies are underway for artificial blood that takes advantage of the aforementioned characteristic of the PFC emulsion. The PFC emulsion acts well as an oxygen carrier either in vivo or in vitro (in an extracorporeal system such as a perfusion system). This property suggests that the PFC emulsion can be advantageously used as an oxygen carrier for kidney storage. Not only is it a good oxygen solvent, the chosen PFC is biologically inert and does not cause direct toxicity to tissues. Thus, when used as a perfusate to preserve the kidney, there are no toxicity problems.

Studies to preserve the kidney using PFC emulsion have only recently begun and are based on the work of Nakaya et al. (Proceedings of the Symposium on Perfluorochemical Blood, Kyoto 1976, 187-201). They studied renal viability by biologically perfusing a dog’s kidney with FC-43 emulsion (perfluorotri-butylamine emulsion) for 9 hours at room temperature. Compared to the control group perfused with Ringer's solution, the kidney perfused with FC-43 emulsion retained well the function of mitochondrial function and high activity of key glycolytic and gluconeogenetic enzymes, indicating that oxygen transport by FC-43 material effectively affects the function of perfused kidney. However, the aforementioned researchers focused their research primarily on the biological aspect, but not on the transplantation of a preserved kidney. Bercowitz was the person who published the results of rabbit kidney transplantation preserved by perfusion using PFC emulsion (J. Surg. Res. 20 (1976) 595-600). He perfused the dog’s kidney for 24 hours at low temperature with a PFC emulsion containing albumin and cryoprecipitated plasma. The preserved kidney was autotransplanted and then examined for successful organ transplantation. This was found to be 100% in all five cases, while the success rate was 62 * in five of the eight control cases where the perfusate did not contain FC-43 emulsion, clearly demonstrating the benefit of the FC-43 system.

5 65009 These of the above publications showed that PFC emulsion is an effective perfusate for maintaining the kidney at both low and room temperatures. However, the storage times disclosed in both publications are not long enough to demonstrate the usefulness of the storage method. In particular, the albumin-containing FC-43 emulsion is not stable enough to allow normalized preparation.

Under the above conditions, the present inventors performed studies with PFC-43-containing perfusate for the long-term preservation of organs, especially the kidneys, for transplantation. Based on these studies, the present invention was obtained.

For the perfusion preservation of a transplanted organ according to the present invention, which perfusate contains Ringer's solution, it is characterized in that it contains potassium ions at a concentration of 8-20 meg / liter, albumin dissolved therein at a concentration of 1-8% (w / v), emulsified liquid A perfluorocarbon compound containing 9 to 12 carbon atoms in a concentration of 7.5 to 12.5% (w / v) and an emulsifier.

The basic Ringer1 solution in the modified Ringer's solution used in the present invention is not subject to any special requirements, but may be conventional or modified by Locke, Tyrode, Earle, Hanks or Krebs. The standard Ringer's solution is an aqueous solution for injection (pH 5.0-7.5) containing 0.53-0.58% (w / v) chlorine and 0.030-0.036% (w / v) calcium chloride (Pharmacopoeia of Japan). , 10th ed., 1981, p. 1262). The modification according to the invention consists in raising the potassium ion content from the usual value of 4-6 meq / liter to 8-20 meq / liter, preferably to 8-15 meq / liter.

This increased potassium ion concentration was used to bring the potassium ion level more closely into line with the potassium ion concentration in the extracellular fluid. By selecting this potassium ion content, it is possible to obtain a sharp increase in the success rate of organ transplantation, especially in the kidney, which has been maintained for a long time by perfusion. The modified Ringer's solution of this invention has an osmolarity of 290-300 m Osm / liter and a pH of 7.1-7.7 at 20 ° C. Table I shows, for comparison, the salt compositions of some typical Ringer's solutions modified in accordance with this invention together with normal plasma, which is a prototype of a conventional Ringer's solution.

table 1

Examples of the electrolyte composition of a modified Ringer's solution. Unit meq / liter

According to the invention, the modified Ringer's Normal solution___ plasma no.

Ions 1 2 3

Na + 112 142 116 142 K + 11 8 10 5

Mg ++ 7 2 6 2

Ca ++ 5

Cl "118 103 15 103 HC03" 3 27 10 27 HP04 "2 2 101 2 SO4" 7 6

Glucose 33 139

Lactose 5 The perfusion solution of the present invention for organ preservation is prepared by mixing a PFC emulsion with a modified Ringer's solution and albumin so that the concentrations of PFC material and albumin reach predetermined values, respectively.

The PFC emulsion used in the invention is prepared from a liquid perfluorocarbon compound capable of absorbing oxygen by a known method, such as disclosed in U.S. Patent 3,958,014, 3,911,138, 3,962,439 or DE 6,630,586. These patents disclose that a stable emulsion suitable for use as an artificial blood can be prepared by emulsifying a perfluorocarbon compound with a phospholipid used as an emulsifying agent (U.S. Patent 3,958,014), a phospholipid and a Cg22 fat as additives (U.S. Patent 3,962,439); or using a polyoxyethylene-polyoxy-7,6009 propylene copolymer (molecular weight 2000-20,000) as a nonionic emulsifier (U.S. Patent 3,911,138); or using polyoxyethylene alkyl ether or polyoxyalkyl allyl ether or using together the aforementioned fatty acids, a nonionic emulsifier and the aforementioned additional emulsifier (DE application 2,630,589).

Emulsifiable perfluorocarbons are those which do not cause adverse effects on organs or tissues and are saturated perfluorocarbons, preferably having a total of 9 to 12 carbon atoms, some or all of which form at least one saturated alicyclic ring, heterocyclic ring heterotype atom and / or oxygen atom, an aliphatic tertiary amine together with a nitrogen atom or an aliphatic ether together with an oxygen atom or atoms.

One group of perfluoro compounds used in the invention are perfluoroalkanes, perfluorocycloalkanes and perfluoro (alkylcycloalkanes), including, for example, perfluoro (C8-21 alkanes) | such as perfluorodecane and perfluorododecane? perfluoro (C 1-6 alkylcyclohexanes) / such as perfluoro (methylpropylcyclohexanes), perfluoro (butylcyclohexanes), perfluoro (trimethylcyclohexanes), perfluoro (ethylpropylcyclohexanec); ).

Another group includes perfluoro (alkyl-saturated-heterocyclic) compounds, including, for example, perfluoro (alkyl tetrahydropyrans), such as perfluoro (butyltetrahydropyrans), perfluoro (pentyltetrahydropyrans), and perfluoro (hexyltetrahydropyrans); perfluoroalkyltetrahydrofurans such as perfluoro (pentyltetrahydrofurans), perfluoro (hexyltetrahydrofurans) and perfluoro (heptyltetrahydrofurans); perfluoro (N-alkylpiperidines), such as perfluoro (N-pentylpiperidines), perfluoro (N-hexylpiperidines) and perfluoro (N-butylpiperidines); and perfluoro (N-alkylmorpholines) such as perfluoro (N-pentylmorpholines), perfluoro (N-hexylmorpholines) and perfluoro (N-heptylmorpholines).

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The third group comprises perfluoro (tert-amines), which include, for example, perfluoro (tributylamine), perfluoro (diethylhexylamine), perfluoro (dipropylbutylamine), and perfluoro (diethylcyclohexylamine); and perfluoro (dioxa-starting nits), i.e. perfluoro (alkylene glycolide alkyl ethers such as perfluoro (3,8-dioxa-2,9-dimethyldecane) or perfluoro (tetramethylene glycol diisopropyl ether), perfluoro (3,7-dioxa-2,8-dimethylnonane) or perfluoro (trimethylene glycol) isopropyl ether) and perfluoro (4,6-dioxa-5,5-dimethylnonane) or perfluoro (isopropylene glycol di-n-propyl ether).

The particle size of the PFC emulsion thus prepared in the modified Ringer's solution is 0.05-0.3 micrometers, it contains 10-50% (w / v) PFC material, 2.0-5% (w / v) emulsifies and, if necessary, 0.1-1.0% (w / v) of additional emulsifier and can be stored in a stable state for long periods of time.

The present organ transfusion perfusate is prepared by mixing the above-mentioned PFC emulsion, commercial bovine serum albumin or human albumin and the above-mentioned modified Ringer's solution before use so that the resulting liquid contains 7.5-12.5% (w / v) PFC. material, 1-8% (w / v) albumin. If necessary, pharmaceutical agents such as procaine hydrochloride, heparin, phenoxybenzamine, insulin, dexamethasone ("Decadron"), methylprednisolone, antibiotics and urokinases may be added. The above PFC concentration limits have been set to ensure the success of organ transplantation when the organ has been preserved by fluid perfusion prior to organ transplantation. The concentration of albumin within the above-mentioned limits is suitable for adjusting the osmotic pressure of the colloid so that the perfusion fluid can be kept physiologically isotonic.

The storage of the movable member is carried out in a conventional manner by oxidation with oxygen by means of a conventional device. For example, 400 ml of a 25% (w / v) PFC emulsion, 360 ml of a modified Ringer's solution and 240 ml of a 25% (w / v) human albumin solution in a modified Ringer's solution are mixed together to give a total of 1 liter. The resulting mixture 9 65009 is introduced into a perfusate tank and perfused through the movable member with oxidation with the same gas mixture (95-98% oxygen and 5-2% carbon dioxide) or with oxygen alone by means of a membrane-type or bubbling-type oxidizer. The perfusate has a circulation rate of 15-50 ml / g kidney / hour and the perfusate was oxidized with a gas mixture containing 95% O 2 and 5% CO 2 at a flow rate of 1500 ml / min. If necessary, the above-mentioned pharmaceutical agents can be added to the perfusate without adversely affecting the perfusate. In perfusion storage, it is also possible to circulate the perfusate fed to the arterial side of the organ storage device by means of a pulsating pump. For long-term storage, it is advantageous to freshly perfuse the perfusate. In addition to perfusion storage, the perfusate of this invention can be used in surgical table surgery.

The method of the present invention allows the organ to be stored very safely for a long time up to the time of transplantation. Due to the very high success rate of organ transplantation, the presented method guarantees the commercial success of organ transplantation in practical medical treatment.

The following are illustrative examples of methods for preparing each component of the perfusate presented and a procedure for preparing the perfusate. The term "% (w / v)" in this brochure and in the claims means the proportion by weight of material in grams per 100 ml of liquid obtained.

Preparation Example 1

Preparation of fluorocarbon emulsion:

300 g of a polyoxyethylene-polyoxypropylene copolymer (molecular weight 8350) was dissolved in eight liters of modified Ringer's solution (shown later). 3 kg of perfluorodecalin was added and the resulting mixture was stirred in a mixer to form a coarse emulsion. The coarse emulsion was fed to the tank of a spray-type emulsifier (Monton-Gaul type homogenizer) to recycle the emulsion. Emulsification was performed at 35-5 ° C

O

at a temperature of 200-500 kp / cm. The concentration of perfluorodecalin in the fine emulsion obtained was 31.3% (w / v) and the average diameter of the particles was 0.09 to 0.1 micrometers as measured by a centrifugal sedimentation method. After 6 months of storage of the emulsion at 4 ° C, no co-growth of particles was observed and the mean diameter of the particles remained essentially unchanged.

Preparation of albumin solution;

A commercial preparation of human serum albumin was dissolved in 25% (w / v) of the above modified Ringer's solution.

Preparation of modified Ringer's solution:

The modified Ringer's solution was prepared by dissolving in distilled water in grams per liter 6.51 NaCl, 0.4 MgSO 4, 0.8 KCl, Q, 24 NaHCO 3, 0.14 Na 2 HPO 4 and 6.0 glucose to give a solution having the following electrolyte composition as indicated. meq / liter in units: 112 Na +, 11 K +, 7 Mg ++, 118 Cl, 3 HCO-j, 2 HPO 2, 33 glucose.

Mixing the ingredient solutions: 57 ml of the modified Ringer * solution and 330 ml of the fluorocarbon emulsion were mixed thoroughly. The mixture was sterilized by heating in a sterilizer at 115 ° C for 12 minutes. The sterilized mixture was mixed with 100 ml of albumin solution passed through a bacterial filter. The resulting mixture was stored in a cold room at 1-10 ° C and used as a perfusion fluid, if necessary, to preserve the organ for transplantation.

Preparation Example 2

Preparation of perfluorocarbon emulsion:

To eight liters of the modified Ringer's solution was dissolved 330 g of polyoxyethylene octyl ether having an average molecular weight of 3,500. To the solution were added 40 g of soybean phospholipid and 2 g of potassium oleate. The mixture was stirred in a mixer to form a suspension. The suspension was mixed with 3 kg of perfluorine (tributylamine) and mixed in a mixer to form a coarse emulsion. The coarse emulsion was further emulsified as described in Example 1. The fine emulsion thus obtained was divided into flasks and sterilized by heating in a rotary sterilizer at 115 ° C for 12 minutes. The concentration of perfluorocarbon in the sterilized emulsion was 29.7% (w / v).

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No particle aggregation was observed when stored at 4 ° C for 6 months.

Mixture of ingredient liquids;

550 ml of the modified Ringer's solution used in Preparation Example 1, 100 ml of albumin solution and the above fluorocarbon emulsion were mixed together. The mixed liquid was stored in a cold room at 1-10 ° C and used as a perfusion fluid to preserve the organ for transplantation, if necessary.

Preparation Example 3

The procedures of Preparative Example 1 were repeated except that perfluoromethylpropylcyclohexane was used instead of perfluorodecalin. The properties of the obtained perfusion liquid were similar to the perfusion liquid obtained according to Preparation Example 1.

Preparation Example 4

The procedures of Example 1 were repeated except that perfluorobutylcyclohexane, perfluorotrimethylcyclohexane, perfluoromethylpropylcyclohexane, perfluoromethyldecalin, perfluorohexyltetrahydropyran, perfluoropentyltetrahydrofuran, perfluoropentyltetrahydrofuran, perfluoroheptylethanethydrogen The properties of the obtained perfusion liquids were similar to those obtained in Example 1.

Preparation Example 5

The procedures of Preparative Example 1 were repeated except that perfluoro-N, N-dibutylmonomethylamine, perfluoro-N, N-diethylpentylamine, perfluoro-N, N-dipropylbutylamine, perfluorotripropylamine, perfluoro-N, N-diethylamidine cyclohexyl , perfluoro-N-hexylpiperidine, perfluoro-N-butylpiperidine, perfluoro-N-pentylmorpholine, perfluoro-N-hexylmorpholine and perfluoro-N-heptylmorpholine were each used in place of perfluorodecalin. The perfusion fluids obtained were similar to those obtained in Preparative Example 1.

Preparation Example 6

The procedures of Preparative Example 1 were repeated except that perfluorotributylamine was used instead of perfluorodecalin. The properties of the obtained perfusion liquid were similar to those of Preparation Example 1.

Preparation Example 7

The procedure of Bleaching Example 1 was repeated except that a polyoxyethylene-polyoxypropylene copolymer having a molecular weight of 15,800 was used instead of a copolymer having a molecular weight of 8,350. The perfusion liquid obtained had similar properties to the liquid obtained in Example 1.

The following are examples of comparative experiments to demonstrate the efficacy of the perfusion solutions of this invention in animal experiments in the transplantation of perfusion-preserved kidneys. The success or failure of an organ transplant was determined by monitoring the function of the transplanted kidney after the contralateral normal kidney had been removed. In the tables, the term "early action" means that urinary excretion was observed within two days after contralateral renal excretion; the term "late activity" means that no urinary excretion was observed for two days after contralateral renal excretion, but thereafter renal function gradually improved until urinary excretion was observed and the test animal was rescued; the term "necrosis" refers to the destruction of tissues in local areas of the transplanted kidney prior to removal of the contralateral kidney (experiment discontinued); and "A.T.N." (acute tubular necrosis) means that after removal of the contralateral kidney, the animal suffered from anuria, loss of appetite, and recurrent vomiting until the animal died of acute renal failure.

Experimental Example 1

Optimal PFC concentration in the storage liquid.

It is necessary and essential to determine the most suitable PFC concentration for perfusion, because as the PFC content of the PFC emulsion increases, the oxygen transfer capacity of the emulsion increases, while on the other hand, the viscosity increases considerably at low temperatures. To determine the optimal PFC concentration during perfusion, a series of perfusion fluids were prepared with varying PFC concentrations using Collin's modified saline (MCS) (The Lancet 2 (1966) 1219-1222) f, which is said to be the hypothermic storage of 6,500 most preferred organs. and whose electrolyte composition corresponds to the intracellular fluid. Various amounts of the fluorocarbon emulsion obtained according to Preparation Example 2 were added to the M.C.S. solution so that the final PFC concentrations obtained in the perfusion fluids were 5, 7.5, 10, 12 and 15% (w / v). Experiments were performed to preserve the kidneys of dogs for transplantation by perfusing the above fluids at 5-80 ° C at perfusion solutions flow rates of 15-18 ml / g / hour for 24 hours under oxidation with oxygen-containing gas (95% CH 2, 5% CO 2) at a flow rate of 300-500 ml. / min. 5 dogs per group were used and the kidneys were autotransplanted. On the seventh day after autotransplantation, the contralateral normal kidney of each dog was removed and the subsequent days of life of each dog were counted. If the dog lived for 4 weeks or more, the transplant was considered successful because in such a case, BUN (blood urea nitrogen) and creatine levels remained normal and urinary excretion was observed. From the values obtained, the optimal PFC concentration in the perfusate was determined.

The experiments were normally performed according to the following procedure:

Anesthesia: Sodium pentobarbital (30 mg / kg, i.v. injection), and endotracheal administration of halothane and oxygen.

Incision of the midline of the abdomen.

Surgical isolation of the left kidney.

Cannulation (ureter, renal artery and vein).

All-wash with cold M. C. S. solution or cold Ringer's lactate solution.

Connection to perfusion circuit. Oxidation storage with oxidation perfusate at 5-8 ° C for 24 hours.

Wash (M.C.S. or lactated Ringer's solution).

Autotransplantation into the femoral cavity.

Delayed contralateral renal removal.

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Table 2 Effect of PFC concentration in perfusate on organ transplant success_

Dog Perfusate Lifespan Success Remarks No. (days) 1 Alive Early Action 2 - Necrosis 3 M.C.S. 5 2/5 A.T.N. ++ (Anuria) 4 Alive Early Action 5 4__A.T.N. (Anuria) _ 6 - Necrosis 7 Alive Delayed action 8 5% PFC 5 3/5 A.T.N. (Anuria) in M.C.S 9 Alive Early Action 10 Alive Late Action_ 11 Alive Early Action 12 Alive Early Action 13 7.5% PPC Alive 4/5 Early Action in M.C.S 14 7 A.T.N. (Anuria) 15 Alive__Early Action_ 16 Alive Early Action 17 5 A.T.N. (Anuria) 18 10% PPC Alive 4/5 Early Activity in M.C.S. 19 Alive Early Activity 20 Alive Early Activity 21 Alive Early Activity 22 Alive Early Activity 23 12.5% PFC Alive 4/5 Early Activity in M.C.S. 24 5 A.T.N. (Anuria) 25 Alive Early Action ~ 26 8 A.T.N. (Hematuria) 27 Alive Early Action (Hema) 28 15% PPC 4 2/5 A.T.N. (Anuria) 29 M.C.S.. Alive Early Action (Hema) 30 7 A.T.N. (Anuria) 15 65009 + Alive. That is, longer than 4 weeks.

| | A.T.N. (acute tubular necrosis)

The results obtained are shown in Table 2. As can be seen from Table 2, a high success rate of organ transplantation was obtained at a PFC concentration of 7.5-12.5% (w / v). If the PFC concentration is greater than 12.5% (w / v) and reaches 15% (w / v), haematuria was observed in all cases after restoration of blood flow after organ transplantation. Two dogs that survived for more than 2 weeks developed hematuria for several days after contralateral renal removal, after which they recovered and survived. From the above results, it was concluded that a perfusion fluid with a PFC concentration adjusted to 10% (w / v) is most effective for perfusion storage of the removed kidney.

Experimental Example 2

Basic Salt Solution:

Long-term, low-temperature perfusion-storage experiments were performed to select an appropriate stock solution. The saline solutions studied were M.C.S. (shown above), Ringer's solution and a modified Ringer's solution (M.R.S) prepared according to Preparation Example 1. Perfusion fluids for storage were prepared by adding the perfluorocarbon emulsion of Preparation Example 2 and bovine serum albumin to saline to give a final concentration of 10% (w / v) PFC and 6% (w / v) albumin. Perfusion was performed on dog kidneys (5 individuals per group) at 5-8 ° C for 72 hours following the procedure of Experimental Example 1. Following transplantation and circulatory restoration, studies were performed on renal blood flow, renal color and appearance, urinary excretion, and symptoms of contralateral renal removal performed on day 7 post-transplant. Perfuate (2000 ml in total) was not freshened during 72 hours of perfusion, but was circulated at a rate of 15-18 ml / g / h. Except in cases where M.R.S. was used as basal saline, the results in transplantation were very unfavorable and almost all experimental animals died of acute renal failure during the 10 years of 65,5009 cycles after contralateral renal removal. On the other hand, in cases where M.R.S. used as stock saline, all animals invariably recovered without complications.

Experimental Example 3

Effect of albumin concentration in perfusate on canine kidney.

The perfusates used in this experiment were 10% PFC emulsions prepared in a modified Ringer's solution using the PFC emulsion of Preparation Example 6 and containing varying amounts of human albumin. The kidneys were perfused for 72 hours at 5-8 ° C by oxidizing the oxygen-containing gas used in Experimental Example 1 at a flow rate of 300-500 ml / min and then autotransplanted as described in Experimental Example 1. The flow rate of perfusion fluid was maintained between 15-18 ml / g / h during perfusion. Contralateral renal removal was performed one week after autograft.

The results are shown in Table 3.

Table 3

Dog Albumin- Kidney weight- After contralateral renal removal ητο concentration increase per% perfusion Success (%) during (%) 1 73.5 No activity Death 2 0 49.4 No activity Death 3 53.1 Delayed survival __sample 2 days__ 4 38.4 Delayed survival Survival 2 days 5 2 52.9 No activity Death 6 49.8 No activity Death_ 7 37.6 No activity Death 8 4 20.3 Early activity Survived 9 41.6 Early Action Survived 10 13.7 Early Action Survived 11 6 30.6 Early Action Survived 12 ___ 34.4__Early Action Survived 13 11.9 Early Action Survived “14 8 22.8 Early Action Survived 15 30.5 ELI activities Died 17 65009

Experimental Example 4

Prolonged, low-temperature perfusion of the kidney for transplantation.

Dog kidney transplantation experiments were performed as described in Experimental Example 1. Each kidney was stored for one week by perfusion at 5-8 ° C and the perfusion fluid flow rate was 15-18 mg / g / h with oxidation at a gas flow rate of 300-500 ml / min. The perfusion liquid was prepared using the M.R.S. solution of Preparation Example 1 as the basic saline solution and adding bovine serum albumin and the PFC emulsion of Preparation Example 2 or the PFC emulsion prepared according to Preparation Example 2 except that perfluorodecalin was used for perfluoro-tributylamine. The concentrations of albumin and PFC material in the perfusion fluid were 6% (w / v) and 10% (w / v), respectively. One week after perfusion storage, the kidneys were autotransplanted according to Experimental Example 1. Contralateral renal removal was performed one week after autograft and symptoms were observed. The results are shown in Table 4. Plasma creatine and BUN levels in surviving animals returned to normal within 3 weeks.

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Table 4 • -—-----—

Example Perfusate Kidney Contralateral no> weight gain after renal removal perfusion__,% Function Ctanning 1 34.3 Early Survival activity 2 M.R.S. 46.9 No activity Died albumin 6% (w / v) 3 20.1 Delayed Survived activity 2 days 4 Ferf luor itr ibutyl 18.7 Delayed Died amine 10% (w / v) activity 3 days 5 33.5 Early Survived ______Activity__ 6 19.3 Early Survived Activity 7 MRS 40.1 Delayed Death albumin 6% (w / v) activity 3 days 8 Ferfluorodecalin 40.9 Early Survived 10% (w / v) activity 9 23.5 Early Survived activity 10 21.6 No activity Died

Experimental Example 5 Using canine kidneys, transplantation experiments were performed according to Experimental Example 1. Each kidney was stored at 18-22 ° C for 12 or 24 hours by perfusing the perfusion fluid at a flow rate of 40-50 ml / g / h with oxidation with a mixed gas (95% C 1-5% CO 2) at a flow rate of 300-500 ml / min.

The perfusion liquid was prepared using the M.R.S. solution of Preparation Example 1 as a base salt solution and adding the PFC emulsion of Preparation Example 2 and bovine serum albumin. The PFC and albumin concentrations in the perfusion fluid were 10% (w / v) and 6% (w / v), respectively. After 12 or 24 hours of storage, the kidneys were autotransplanted. Contralateral renal removal was performed one week after is 65009 autograft and symptoms were observed. Table 5 shows the results of the above experiments together with a control experiment in which no PFC material was used and a control experiment in which M.C.S solution was used as the base salt solution.

Table 5

Example Perfusate Perfusion Contralateral Renal Removal no. duration, after h

Action Success 1 M.R.S. No activity Death 2 albumin 12 "" 6% (w / v) 3 ""

4 "H

5 M.C.S. No activity Died 6 albumin 12 6% (w / v) 7 PFC 10% "(w / v) 8 ___" "9 MRS Early activity Survived 10 albumin 12 Early 6% (w / v) survived 11 PFC 10% (w / w) Early activity Survived 12 Early ____ activity__Survived_ 13 MRS Early activity Survived 14 albumin 24 No activity Died 15 PFC 10% (w / w) Early activity Survived 16 Late activity Survived

Claims (14)

20 65009 A liquid for perfusion preservation of a transplanted organ, comprising a modified Ringer's solution, characterized in that it contains potassium ions at a concentration of 8-20 meq / liter, albumin dissolved therein at a concentration of 1-8% (w / v), an emulsified liquid 9-12 a carbon-containing perfluorocarbon compound in a concentration of 7.5 to 12.5% (w / v) and an emulsifier. Liquid according to Claim 1, characterized in that the perfluorocarbon compound is at least one compound selected from the group consisting of perfluoroalkylcyclohexanes having 3 to 5 carbon atoms in the alkyl group, perfluorodecalin, perfluoromethyldecalin, perfluoroalkyltetrahydropyrans containing 4 to 6 carbon atoms in the alkyl group, , Perfluoroalkylpiperidines having 4 to 6 carbon atoms in the alkyl group, perfluoroalkylmorpholines containing 5 to 7 carbon atoms in the alkyl group, perfluorotriakylamines, perfluoro (diethylcyclohexylamine) and perfluoro (dioxaalkane). Liquid according to Claim 1, characterized in that the perfluorocarbon compound is perfluoroalkylcyclohexane, perfluorodecalin or perfluoromethyldecalin containing 3 to 5 carbon atoms in the alkyl group. Liquid according to Claim 1, characterized in that the perfluorocarbon compound is perfluoroalkyltetrahydropyran having 4 to 6 carbon atoms in the alkyl group, perfluoroalkyltetrahydrofuran having 5 to 7 carbon atoms in the alkyl group, perfluoroalkylpiperidine containing 4 to 6 carbon atoms in the alkyl group or 5 to 7 carbon atoms in the alkyl group. Liquid according to Claim 1, characterized in that the perfluorocycloalkane is perfluorodecalin, perfluoro (methyldecalin) or perfluoro (dimethyldecalin). 21 65009 Liquid according to Claim 3, characterized in that the perluorocycloalkane is perfluoro (butylcyclohexane), perfluoro (methylpropylcyclohexane), perfluoro (trimethylcyclohexane), perfluoro (ethylpropylcyclohexane) or perfluoro (pentylcyclo) cyclohexane. Liquid according to Claim 4, characterized in that the perfluoro-saturated heterocyclic compound is perfluoro (butyltetrahydropyran), perfluoro (pentyltetrahydrofuran), perfluoro (hexyltetrahydrofuran), perfluoro (heptyl-tetrahydrofuran), perfluoro (N-hexyl) piper , perfluoro (N-pentylmorpholine), perfluoro (N-hexylmorpholine) or perfluoro- (N-heptylmorpholine). Liquid according to Claim 1, characterized in that the perfluorocarbon compound is perfluorotriakylamine, perfluoro (diethylcyclohexylamine) or perfluoro (dioxaalkane). Liquid according to Claim 8, characterized in that the perfluorocarbon compound is a perfluoro-tert-amine which is perfluoro (diethylhexylamine), perfluoro (dipropylbutylamine), perfluoro (diethylcyclohexylamine) or perfluoro (tri-butylamine). Liquid according to Claim 8, characterized in that the perfluorocarbon compound is perfluoro (dioxaalkane), which is perfluoro (tetramethylene glycol diisobutyl ether), perfluoro (trimethylene glycol diisobutyl ether) or perfluoro (isopropylidene) glycol diol. Liquid according to Claim 1, characterized in that the emulsifier is a polyoxyethylene-polyoxypropylene copolymer, a polyoxyethylene alkyl ether, a polyoxyalkyl allyl ether or a phospholipid or a mixture thereof. A liquid according to claim 1, characterized in that the C8-22 fatty acid is present together with the emulsifier, said fatty acid being an additional emulsifier. 65009 13. A method of preparing a liquid for perfusion preservation of a transplanted organ, comprising albumin, a Ringer's solution modified to increase the potassium ion content to 8-20 meq / liter, and a stable perfluorocarbon compound having 9-12 carbon atoms. mixing the emulsion together so that the albumin content is 1-8% (w / v) and the perfluorocarbon compound content is 7.5 to 12.5% (w / v). A method of preserving an organ to be transplanted, wherein the organ is perfused with a perfusion fluid containing a perfluorocarbon compound while oxidizing, characterized in that the perfusion fluid is a mixture containing an effective amount of albumin, Ringer's solution modified to increase its potassium ion content to 8-20 meq / liter and a stable emulsion of a perfluorocarbon compound having 9 to 12 carbon atoms, having an albumin content of 1 to 8% (w / v) and a perfluorocarbon compound content of 7.5 to 12.5% (w / v) based on the perfusion solution. 65009