DK154253B - Perfusate for keeping of organs for transplantation, method for preparation of the perfusate and method for keeping an organ with use of the perfusate - Google Patents
Perfusate for keeping of organs for transplantation, method for preparation of the perfusate and method for keeping an organ with use of the perfusate Download PDFInfo
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Den foreliggende opfindelse angår et perfusat til brug ved opbevaring af organer til transplantation, hvilket perfusat indeholder en Ringer's opløsning, hvori der er opløst albumin, og mindst en emulgeret, flydende, 5 eventuelt substitueret perfluorcarbonforbindelse og mindst et emulgeringsmiddel. Opfindelsen angår også en fremgangsmåde til fremstilling af perfusatet og en opbevaringsmetode, ved hvilken man anvender det omhandlede perfusat, og nærmere bestemt en fremgangsmåde til opbe-10 varing af et organ, der skal transplanteres,.hvorved man som perfusat anvender en blanding bestående af en modificeret Ringer's opløsning, der tjener som primær saltopløsning, albumin og en emulsion i form af en per-fluorcarbonforbindelse.The present invention relates to a perfusate for use in the storage of organs for transplantation, which contains a Ringer's solution containing dissolved albumin and at least one emulsified, liquid, optionally substituted perfluorocarbon compound and at least one emulsifier. The invention also relates to a method for preparing the perfusate and a storage method using the present perfusate, and more particularly to a method for storing an organ to be transplanted, using as a perfusate a mixture of a perfusate. modified Ringer's solution serving as primary saline, albumin and an emulsion in the form of a perfluorocarbon compound.
15 I de senere år er der sket mærkbare tekniske fremskridt med hensyn til at kunne opbevare et organ, der skal benyttes til transplantation. Specielt er der sket tekniske fremskridt med hensyn til opbevaring af nyrer, og dette har medført en udbredelse af 20 nyretransplantationer, ved hvilke der anvendes nyrer fra afdøde personer. Dette fremskridt skyldes i høj grad forbedringer af de anvendte perfusater. F. eks. kan de kliniske metoder til opbevaring af nyrer til transplantationsformål i store træk klassificeres i 25 to kategorier, hvoraf den ene er hypotermisk opbevaring (ved nedsænkning) (Lancet, 2, 1219 - 1222 (1969)), mens den anden er hypotermisk kontinuerlig per fusion (Lancet, 2, 536 (1967)). I praksis anvender man den førstnævnte metode til kortvarige opbevaringer, mens 30 den sidstnævnte metode anvendes til opbevaringer af længere varighed. Når der sker en stadig udbredelse af nyretransplantationer med nyrer fra afdøde personer, vil langvarige opbevaringer, dvs. opbevaringer ved perfusion, få stadig større betydning.15 In recent years, there have been noticeable technical advances in being able to store a body to be used for transplantation. Specifically, technical progress has been made with regard to renal preservation, and this has led to the proliferation of 20 kidney transplants using deceased person's kidneys. This progress is largely due to improvements in the perfusates used. For example, the clinical methods for renal transplant storage can be broadly classified into 25 categories, one of which is hypothermic storage (by immersion) (Lancet, 2, 1219 - 1222 (1969)) while the other is hypothermic continuous per fusion (Lancet, 2, 536 (1967)). In practice, the former method is used for short-term storage, while the latter method is used for longer-term storage. When renal transplants are still being propagated with the kidneys of deceased persons, prolonged storage, ie. storage by perfusion is becoming increasingly important.
35 Ued nyretransplantationer, hvor der anvendes nyrer fra afdøde personer, stammer disse nyrer fortrinsvis 235 Without kidney transplants using the kidneys of deceased persons, these kidneys preferably originate from 2
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fra personer, som har været udsat for ulykkestilfælde.from people who have suffered an accident.
De nødvendige foranstaltninger, som skal træffes i det tidsrum, der forløber fra en sådan nyre stilles til rådighed og indtil transplantationen kan finde 5 sted, omfatter undersøgelser af histokompatibiliteten imellem nyren og patienten, udvælgelse af en egnet recipient, transport af nyren samt forberedelser til transplantationen af denne. Af disse årsager er det nødvendigt at opbevare den afgivne nyre så længe ID som muligt. Der har foreløbig været udført mange forsøg på at opbevare nyrer fra afdøde personer i længere tidsrum. Disse forsøg har primært drejet sig om udvikling og forbedring af perfusater.The necessary measures to be taken during the period from which such a kidney is made available and until the transplant can take place include studies of histocompatibility between the kidney and the patient, selection of a suitable recipient, transport of the kidney and preparation for the transplant. of this. For these reasons, it is necessary to keep the kidney delivered for as long as possible. So far, many attempts have been made to store the kidneys of deceased persons for extended periods of time. These trials have primarily focused on the development and improvement of perfusates.
Den stadig mere udbredte kliniske anvendelse af den 15 hypotermiske kontinuerte perfusion kan føres til bage til det forskningsarbejde, som udførtes af Belzer et al. (Lancet 536, 2, (1967)). Det lykkedes for disse forskere at opbevare en hundenyre i 72 timer ved som perfusat at anvende et plasma, der var befriet 20 for fibrinogen i så stor udstrækning som muligt, og som var fremstillet ved frysning og efterfølgende smeltning af et kryoudfældet plasma. Siden offentliggørelsen af deres forskningsrapport har man først og fremmest anvendt kryoudfældet plasma som perfusat til opbevaring 25 af nyrer.The increasingly widespread clinical application of the 15 hypothermic continuous perfusion can be traced back to the research work carried out by Belzer et al. (Lancet 536, 2, (1967)). These researchers succeeded in storing a canine kidney for 72 hours using as perfusate a plasma that was liberated to the greatest extent possible by fibrinogen and which was prepared by freezing and subsequently melting a cryoprecipitated plasma. Since the publication of their research report, cryoprecipitated plasma has primarily been used as perfusate for the storage of 25 kidneys.
I 1974 udviklede Toledo-Pereyra et al. (Surg, Gynecol.In 1974, Toledo-Pereyra et al. (Surg, Gynecol.
Obstet, 138, 901 - 905 (1974)) et nyt perfusat bestående af silicagel-behandlet kryoudfældet plasma. Dette plasma er befriet for (3-lipoprotein hidrørende fra 30 det fibrinogen, som stadig findes i det konventionelle kryoudfældede plasma. I det nye perfusat er niveauet af uopløselige fedtstoffer, dvs. triglycerider, endvi- 3Obstet, 138, 901-905 (1974)) a novel perfusate consisting of silica gel-treated cryoprecipitated plasma. This plasma is liberated from (3-lipoprotein derived from the fibrinogen still found in the conventional cryoprecipitated plasma. In the new perfusate, the level of insoluble fats, i.e. triglycerides, is also 3
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dere reduceret til en tredjedel. Med det konventionelle kryoudfældede plasma er der stor risiko for forekomst af emboli under perfusionen, hvilket skyldes det residuelle fibrinogen, lipoprotein eller neutra- 5 le fedtdråber. Denne risiko reduceres væsentligt, når man anvender det ovennævnte kryoudfældede plasma, der er behandlet med silicagel. Imidlertid er der andre problemer knyttet til anvendelsen af dette kryoudfældede plasma. For det første er den præparative procedure 10 ikke fuldstændig fastlagt, og sammensætningen af det fremstillede plasma udviser tendens til variation, hvilket gør det vanskeligt at opnå præparater med konstant proteinindhold og konstant elektrolytsammensætning. For det andet er der stadig en vis risiko for 15 dannelse af emboli under perfusionen på grund af en ufuldstændig fjernelse af den uopløselige fedtfraktion. For at reducere denne risiko er det nødvendigt at benytte en kostbar membran-oxygenator. For det tredje mangler man en egnet inaktiveringsbehandling over 20 for hepatitis-virus, hvilket indebærer risiko for en infektion med dette virus, og manglen på en sådan behandling er et væsentligt problem. Selv om det kryoudfældede plasma er et fremragende perfusat til opbevaring af nyrer, er der således visse ulemper forbun-25 det med anvendelsen af dette plasma.you reduced to a third. With the conventional cryoprecipitated plasma, there is a high risk of the occurrence of embolism during perfusion due to the residual fibrinogen, lipoprotein or neutral fat droplets. This risk is significantly reduced when the above-mentioned cryoprecipitated plasma treated with silica gel is used. However, there are other problems associated with the use of this cryoprecipitated plasma. First, preparative procedure 10 has not been fully established and the composition of the plasma produced tends to vary, making it difficult to obtain constant protein content and constant electrolyte composition. Second, there is still some risk of emboli formation during perfusion due to incomplete removal of the insoluble fat fraction. To reduce this risk, it is necessary to use a costly membrane oxygenator. Third, a suitable inactivation treatment over 20 for hepatitis virus is lacking, which involves the risk of infection with this virus, and the lack of such treatment is a major problem. Thus, although the cryoprecipitated plasma is an excellent perfusate for renal storage, there are certain disadvantages associated with the use of this plasma.
Det anførtes, at man for at overvinde de ovennævnte vanskeligheder, som er forbundet med det kryoudfældede plasma, gjorde et forsøg på at anvende en albuminopløsning som perfusat, og herved opnåede man heldige re-30 sultater ved dyreforsøg. Grundman et al. (Transpi., 17, 299 - 305 (1974)) perfuserede en hundenyre med en 6?ό albuminopløsning, og det lykkedes at opbevare nyren i 96 timer. Det benyttede perfusat var en Krebs Ringer's opløsning indeholdende 6% (ω/ν) humant albumin.It was stated that in order to overcome the aforementioned difficulties associated with the cryoprecipitated plasma, an attempt was made to use an albumin solution as perfusate, thereby obtaining successful results in animal experiments. Grundman et al. (Transpi., 17, 299-305 (1974)) perfused a canine kidney with a 6? Albumin solution and managed to store the kidney for 96 hours. The perfusate used was a Krebs Ringer's solution containing 6% (ω / ν) human albumin.
35 Hundenyren undergik perfusion i 96 timer ved lav tem peratur, hvorefter den blev autotransplanteret,og over- 4The canine kidney was perfused for 96 hours at low temperature, then autotransplanted, and over 4 hours.
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levelsesprocenten ved transplantationen var over 80, hvilket viste, at albuminopløsningen er et fremragende perfusat, som i sammenligning med det kryoudfældede plasma er meget fordelagtigt.the level of transplantation was over 80, which showed that the albumin solution is an excellent perfusate, which is very advantageous compared to the cryoprecipitated plasma.
5 Opbevaring af en nyre i længere tid kræver et perfusat, der har et vist kolloidt osmotisk tryk ved lave temperaturer såvel som en passende, elektrolytsammensætning, og det skal desuden indeholde en vis mængde oxygen. Når nyren opbevares ved lave temperaturer, 10 forbruger den en væsentligt mindre mængde oxygen,Storage of a kidney for a prolonged period requires a perfusate having a certain colloidal osmotic pressure at low temperatures as well as an appropriate electrolyte composition, and it must also contain a certain amount of oxygen. When stored at low temperatures, the kidney consumes a substantially smaller amount of oxygen,
end når den opbevares ved stuetemperatur, men den kræver dog en vis mængde oxygen for at kunne opretholde sin'funktion. Ifølge en klassisk undersøgelse foretaget af Levy (American Journal of Physiology 197, 1111 -15 1114, (1959)) er en nyres oxygenforbrug ved 8 - 10 °Cthan when stored at room temperature, but it does require a certain amount of oxygen to maintain its function. According to a classic study by Levy (American Journal of Physiology 197, 1111 -151114, (1959)), a kidney's oxygen consumption is at 8 - 10 ° C.
omkring 3 ^ul/g/minut, hvilket svarer til omkring 20¾ af forbruget ved stuetemperatur. Ved opbevaring af en nyre er det således generel praksis, at man perfuserer den med et perfusat, mens den oxygeneres.about 3 µl / g / minute, which corresponds to about 20¾ of the consumption at room temperature. Thus, in the storage of a kidney, it is common practice to perfuse it with a perfusate while oxygenating.
20 En mættet organisk perfluorforbindelse (i det følgende forkortet PFC) er en væske, som er i stand til at opløse et betragteligt volumen oxygen, og som virker som en bærer for oxygen, når den anvendes i form af en emulsion. Der udføres en del forskning vedrørende 25 kunstigt blod, som gør brug af den ovennævnte karak-teriske egenskab ved PFC-emulsionen. Denne PFC-emul-sion fungerer udmærket som bærer for oxygen såvel in vivo som in vitro (i et system, der befinder sig udenfor legemet, såsom et perfusionssystem). En sådan egen-50 skab muliggør, at PFC-emulsionen med fordel kan anvendes som en bærer for oxygen ved opbevaring af nyrer.A saturated organic perfluoro compound (hereinafter abbreviated PFC) is a liquid which is capable of dissolving a considerable volume of oxygen and which acts as a carrier for oxygen when used in the form of an emulsion. Some research is being conducted on artificial blood using the above-mentioned characteristic of the PFC emulsion. This PFC emulsion works very well as a carrier for oxygen both in vivo and in vitro (in a system located outside the body, such as a perfusion system). Such a feature allows the PFC emulsion to be advantageously used as a carrier for oxygen in the storage of kidneys.
Foruden at være et godt opløsningsmiddel for oxygen er den udvalgte PFC biologisk inert og frembyder ikke nogen problemer med hensyn til direkte toxicitet over-55 for vævet. Når man derfor anvender PFC som perfusat 5In addition to being a good solvent for oxygen, the selected PFC is biologically inert and does not present any direct toxicity problems over the tissue. Therefore, when using PFC as perfusate 5
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ved opbevaring af nyrer, optræder der ikke nogen problemer med hensyn til toxicitet.in renal storage, no toxicity problems occur.
Undersøgelser over opbevaring af en nyre ved anvendelse af en PFC-emulsion er påbegyndt inden for de senere 5 år og kan føres tilbage til et forskningsarbejde afStudies on renal storage using a PFC emulsion have begun within the last 5 years and can be traced back to research work by
Nakaya et al. (Proceedings of Symposium on Perfluorochemical Artificial Blood, Kyoto 1976, 187 - 201).Nakaya et al. (Proceedings of Symposium on Perfluorochemical Artificial Blood, Kyoto 1976, 187 - 201).
Disse forskere undersøgte levedygtigheden af en nyre ad biologisk vej ved at perfusere en kaninnyre med 10 en FC-43-emulsion (en perfluortributylaminemulsion) i 9 timer ved stuetemperatur. Sammenlignet med en kontrolgruppe perfuseret med Ringer’s opløsning opretholdt nyren, der var perfuseret med FC-43-emulsionen, en udmærket mitochondriefunktion og højere aktivitetsni-15 veauer med hensyn til glycolytiske og gluconeogene- tiske nøgleenzymer, hvilket viste, at oxygentransporten ved hjælp af FC-43 har en effektiv indflydelse på opretholdelsen af den perfuserede nyres funktioner. Imidlertid koncentrerede de ovennævnte forskere 20 sig fortrinsvis om det biologiske aspekt og ikke om transplantation af en opbevaret nyre. Resultaterne af en transplantation af en hundenyre, der var opbevaret ved perfusion ved hjælp af en PFC-emulsion, blev rapporteret af Bercowitz (J. Surg. Res., 2£, 595 - 600 25 (1976)). Han perfuserede en hundenyre i 24 timer ved lav temperatur med en PFC-emulsion indeholdende albumin og et kryoudfældet plasma. Den opbevarede nyre blev derefter autotransplanteret, og overlevelses-graden i forbindelse med transplantationen blev un-30 dersøgt. Overlevelsesgraden viste sig at være 100¾ i alle fem tilfælde, hvorimod den var 62¾ i fem kontroltilfælde ud af otte, hvor perfusatet ikke indeholdt nogen FC-43-emulsion. Dette illustrerer klart fordelene ved FC-43-systemet.These researchers examined the viability of a kidney by biological pathway by perfusing a rabbit kidney with an FC-43 emulsion (a perfluorotributylamine emulsion) for 9 hours at room temperature. Compared to a control group perfused with Ringer's solution, the kidney perfused with the FC-43 emulsion maintained an excellent mitochondrial function and higher activity levels in glycolytic and gluconeogenetic key enzymes, demonstrating that oxygen transport by FC 43 has an effective influence on the maintenance of the functions of the perfused kidney. However, the aforementioned researchers 20 concentrated primarily on the biological aspect and not on transplantation of a stored kidney. The results of a transplantation of a canine kidney stored by perfusion by means of a PFC emulsion were reported by Bercowitz (J. Surg. Res., £ 2, 595-600 (1976)). He perfused a dog kidney for 24 hours at low temperature with a PFC emulsion containing albumin and a cryoprecipitated plasma. The stored kidney was then autotransplanted and the rate of survival associated with the transplant was examined. The survival rate was found to be 100¾ in all five cases, whereas it was 62¾ in five control cases out of eight, where the perfusate contained no FC-43 emulsion. This clearly illustrates the benefits of the FC-43 system.
35 De to ovennævnte rapporter viser, at PFC-emulsionen er et effektivt perfusat til opbevaring af en nyreThe two above reports show that the PFC emulsion is an effective perfusate for the storage of a kidney.
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6 ved såvel lave temperaturer som ved stuetemperatur.6 at low temperatures as well as at room temperature.
De opbevaringsperioder, som er beskrevet i de ovennævnte to rapporter, er imidlertid ikke tilstrækkeligt lange til at sikre, at de omtalte opbevaringsmetoder kan an-5 vendes i praksis. Specielt er FC-43-emulsionen, der indeholder albumin, ikke tilstrækkeligt stabil til at tilvejebringe et normaliseret præparat.However, the storage periods described in the above two reports are not long enough to ensure that the storage methods mentioned can be used in practice. In particular, the FC-43 emulsion containing albumin is not sufficiently stable to provide a normalized composition.
På grundlag af de ovennævnte forhold har man derfor foretaget studier af et PFC-43-holdigt perfusat til 10 langvarig opbevaring af et organ, specielt en nyre, til brug ved transplantation. Resultaterne af disse studier danner baggrund for den foreliggende opfindelse.Therefore, on the basis of the above-mentioned conditions, studies have been carried out on a PFC-43-containing perfusate for long-term storage of an organ, especially a kidney, for use in transplantation. The results of these studies form the basis of the present invention.
Det er således et formål med den foreliggende opfindelse at tilvejebringe et PFC-perfusat samt en metode til effek-15 tiv langvarig opbevaring af organer til transplantation.Thus, it is an object of the present invention to provide a PFC perfusate as well as a method for effective long-term storage of organs for transplantation.
Opfindelsen består nærmere bestemt af et perfusat til opbevaring af et organ til transplantation, hvilket perfusat indeholder en Ringer's opløsning, hvori der er opløst albumin og mindst en emulgeret, flydende, eventuelt substitue-20 ret perfluorcarbonforbindelse og mindst et emulgeringsmiddel, og perfusatet ifølge opfin-delsen er ejendommeligt ved, at den anvendte Ringer's opløsning er modificeret på en sådan måde, at den indeholder kaliumioner i en koncentration på mellem 8 og 20 m.ækv./liter.More particularly, the invention consists of a perfusate for storing a transplant organ, which contains a Ringer's solution containing dissolved albumin and at least one emulsified, liquid, optionally substituted perfluorocarbon compound and at least one emulsifier, and the perfusate of the invention. The property is peculiar in that the Ringer's solution used is modified in such a way that it contains potassium ions at a concentration of between 8 and 20 mEq / liter.
25 Den Ringer's opløsning, som er udgangspunktet for den modificerede Ringer's opløsning, er ikke underkastet nogen specielle begrænsninger, men kan være en sædvanlig opløsning eller en opløsning modificeret af Locke,25 The Ringer's solution, which is the starting point for the modified Ringer's solution, is not subject to any particular limitations, but may be a conventional solution or a solution modified by Locke,
Tyrode, Earle, Hanks eller Krebs. Modifikationen består 30 i, at koncentrationen af kaliumioner forøges fra det normale niveau på 4 -6 m.ækv./liter til mellem 8 og 20, fortrinsvis mellem 8 og 15 m.ækv./liter. Denne forøgede koncentration er valgt med det formål at opnå 7Tyrode, Earle, Hanks or Cancer. The modification consists in increasing the concentration of potassium ions from the normal level of 4 -6 m.f./liter to between 8 and 20, preferably between 8 and 15 m.f./liter. This increased concentration is selected for the purpose of obtaining 7
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en nærmere approximering af kaliumion-niveauet i den ekstracellulære væske. Ued at vælge en sådan koncentration af kaliumioner bliver det muligt at opnå en kraftig forøgelse af overlevelsesgraden for et organ, 5 specielt en nyre, som er opbevaret i længere tid ved perfusion. Den modificerede Ringer's opløsning har en osmolaritet på 290 - 300 mOsm/liter og en pH-værdi på mellem 7,1 og 7,7 ved 20 °C. I den efterfølgende tabel 1 er vist saltsammensætningen af nogle typiske modifi-10 cerede opløsninger, og til sammenligning er vist salt sammensætningen af et normalt plasma, som er en prototype på en normal Ringer's opløsning.a closer approximation of the potassium ion level in the extracellular fluid. By choosing such a concentration of potassium ions, it is possible to achieve a substantial increase in the survival rate of an organ, especially a kidney, which is stored for a long time by perfusion. The modified Ringer's solution has an osmolarity of 290 - 300 mOsm / liter and a pH of between 7.1 and 7.7 at 20 ° C. The following Table 1 shows the salt composition of some typical modified solutions, and in comparison the salt composition of a normal plasma, which is a prototype of a normal Ringer's solution, is shown.
88
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TABEL 1TABLE 1
Eksempler på elektrolytsammensætningen af en modificeret Ringer's opløsning. Enhed: m.ækv./literExamples of the electrolyte composition of a modified Ringer's solution. Unit: m.q. / liter
Modificeret Ringer's opløsning til anvendelse i perfusatet ifølge opfindelsen Normalt N. plasmaModified Ringer's solution for use in the perfusate of the invention Normal N. plasma
Ion _1__2__3___Ion _1__2__3___
Na+ 112 142 llé 142 K+ 11 8 10 5Na + 112 142 llé 142 K + 11 8 10 5
Mg++ 7 2 6 2Mg ++ 7 2 6 2
Ca++ 3Ca ++ 3
Cl" 118 103 13 103 HC03" 3 27 10 27 HP04" 2 2 101 2 S04“ 7 6Cl "118 103 13 103 HC03" 3 27 10 27 HP04 "2 2 101 2 SO4" 7 6
Glucose 33 139Glucose 33 139
Lactose 5 5 9Lactose 5 5 9
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Perfusionsvæsken til opbevaring af et organ fremstilles ifølge opfindelsen ved, at man blander en Ringer's opløsning, som er modificeret med hensyn til mængden af kaliumioner ved forøgelse af denne mængde til mellem 5 8 og 20 m.ækv./liter, en opløsning af albumin i den modificerede Ringer's opløsning og en stabil emulsion af en perfluorcarbonforbindelse i den modificerede Ringer's opløsning, således at koncentrationen af albumin er mellem 1 og 8 So (w/v), og således at koncentrationen 10 af perfliuorcarbonforbindelsen er mellem 7,5 og 12,5 % (w/v).The perfusion fluid for storing an organ is prepared according to the invention by mixing a Ringer's solution, which is modified in terms of the amount of potassium ions by increasing this amount to between 5 and 20 m / e, a solution of albumin in the modified Ringer's solution and a stable emulsion of a perfluorocarbon compound in the modified Ringer's solution such that the concentration of albumin is between 1 and 8 So (w / v) and so that the concentration 10 of the perfluorocarbon compound is between 7.5 and 12.5 % (w / v).
PFC-emulsionen, som anvendes i perfusionsvæsken ifølge den foreliggende opfindelse, fremstilles ud fra en flydende perfluorcarbonforbindelse, som er i stand til 15 at absorbere oxygen. Fremstillingsmetoden er kendt og er f.eks. beskrevet i US patentskrifterne nr. 3 958 014, 3 911 138 og nr. 3 962 439 i tysk offentliggørelsesskrift nr. 2 630 586. Det anføres i disse skrifter, at man kan fremstille en stabil emulsion, som er egnet 20 til anvendelse som kunstigt blod, ved at emulgere en perfluorcarbonforbindelse ved hjælp af et phosphorlipid som emulgeringsmiddel (US patentskrift nr. 3 958 014), med et phosphorlipid og en fedtsyre med 8-22 carbona-atomer som hjælpemiddel (US patentskrift nr. 3 962 439) 25 eller ved at anvende en polyoxyethylen-polyoxypropylen- copolymer (molekylvægt 2000-20000) som ikke-ionisk emulgeringsmiddel (US patentskrift nr. 3 911 138). Man kan også anvende en polyoxyethylen-alkylether eller poly-oxyalkyl-allylether, eller man kan samtidigt anvende 30 de ovenfor anførte fedtsyrer, de ikke-ioniske emulge ringsmidler og den ovennævnte fedtsyre, der tjener som hjælpemiddel ved emulgeringen (tysk offentliggørelses-skrift nr. 2 630 586).The PFC emulsion used in the perfusion liquid of the present invention is prepared from a liquid perfluorocarbon compound capable of absorbing oxygen. The method of preparation is known and is e.g. U.S. Patent Nos. 3,958,014, 3,911,138, and 3,962,439 to German Patent Specification No. 2,630,586. It is stated in these publications that a stable emulsion suitable for use as an artificial agent can be prepared. blood, by emulsifying a perfluorocarbon compound using a phosphorus lipid as an emulsifier (U.S. Patent No. 3,958,014), with a phosphorus lipid and a fatty acid having 8-22 carbon atoms as an adjuvant (U.S. Patent No. 3,962,439) or by using a polyoxyethylene-polyoxypropylene copolymer (molecular weight 2000-20000) as a nonionic emulsifier (U.S. Patent No. 3,911,138). A polyoxyethylene alkyl ether or polyoxyalkyl allyl ether may also be used, or the fatty acids listed above, the nonionic emulsifying agents and the above fatty acid which serve as the aid of the emulsification may be used simultaneously (German publication no. 2 630 586).
Perfluorcarbonforbindelserne, der skal emulgeres, er 10The perfluorocarbon compounds to be emulsified are 10
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sådanne, som ikke giver uheldige bivirkninger på organer eller væv, og der er tale om perfluoralkaner, mættede perfluorcarbonforbindelser, der fortrinsvis indeholder 9-12 carbonatomer, hvoraf i det mindste en del danner 5 mindst en mættet alicyclisk ring, en heterocyclisk ring sammen med et nitrogen og/eller et oxygenatom, en ali-fatisk tertiær amin sammen med et nitrogenatom eller en alifatisk ether sammen med et eller flere oxygen-atomer.those which do not cause adverse effects on organs or tissues, and are perfluoroalkanes, saturated perfluorocarbon compounds, preferably containing 9-12 carbon atoms, at least a portion of which forms at least one saturated alicyclic ring, a heterocyclic ring together with a nitrogen and / or an oxygen atom, an aliphatic tertiary amine together with a nitrogen atom or an aliphatic ether together with one or more oxygen atoms.
10 Den første gruppe af perfluorcarbonforbindelser anvendt i den foreliggende opfindelse omfatter perfluoralkaner, perfluorcycloalkaner og perfluoralkylcycloalkaner, hvilken gruppe eksempelvis omfatter perfluor (Cg_^2-al|<a-ner) såsom perfluordecan og perfluordodecan, perfluor 15 alkylcycloalkaner) såsom perfluor(methylpropylcyclo- hexaner), perfluor(butylcyclohexaner), perfluor-(trimethylcyclohexaner), perfluor(ethylpropylcyclo-hexaner) og perfluor(pentylcyclohexaner), perfluorde-calin, perfluor(methyldecaliner) og perfluor(dimethyl-20 decaliner).The first group of perfluorocarbon compounds used in the present invention comprise perfluoroalkanes, perfluorocycloalkanes and perfluoroalkylcycloalkanes, which group comprises, for example, perfluoro (C ), perfluoro (butylcyclohexanes), perfluoro (trimethylcyclohexanes), perfluoro (ethylpropylcyclohexanes) and perfluoro (pentylcyclohexanes), perfluorodecalin, perfluoro (methyldecalines) and perfluoro (dimethyl-decalines).
Den anden gruppe omfatter perfluor(alkyl-mættet heterocyclisk ) -forbindelser , der f. eks. kan være perfluor-(alkyltetrahydropyraner) såsom perfluor(butyltetrahy-dropyraner), perfluor(pentyltetrahydropyraner) og per-25 fluor(hexyltetrahydropyraner), perfluor(alkyltetra- hydrofuraner) såsom perfluor(pentyltetrahydrofuraner), perfluor(hexyltetrahydrofuraner) og per fluor(heptyl-tetrahydrofuraner), perfluor(N-alkylpiperidiner) såsom perfluor(N-pentylpiperidiner), perfluor(N-hexyl-30 piperidiner) og perfluor(N-butylpiperidiner) samt per- fluor(N-alkylmorpholiner) såsom perfluor(N-pentyl-morpholiner), perfluor(N-hexylmorpholiner) og perfluor-(N-heptylmorpholiner).The second group comprises perfluoro (alkyl-saturated heterocyclic) compounds which may be, for example, perfluoro (alkyl tetrahydropyrans) such as perfluoro (butyl tetrahydropyranes), perfluoro (pentyl tetrahydropyranes) and perfluoro (hexyl tetrahydropyranes), perfluoro (hexyl tetrahydropyranes), perfluoro - hydrofurans) such as perfluoro (pentyl tetrahydrofurans), perfluoro (hexyl tetrahydrofurans) and per fluorine (heptyl tetrahydrofurans), perfluoro (N-alkylpiperidines) such as perfluoro (N-pentylpiperidines), perfluoro (N-hexyl-piperidines) butylpiperidines) and perfluoro (N-alkylmorpholines) such as perfluoro (N-pentyl-morpholines), perfluoro (N-hexylmorpholines) and perfluoro (N-heptylmorpholines).
Den tredje gruppe omfatter perfluor(tert-aminer), 35 som eksempelvis kan være per fluor(tributylamin), 11The third group comprises perfluoro (tert-amines), which may be, for example, per fluoro (tributylamine), 11
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perfluor(diethylhexylamin), perfluor(dipropylbutyl-amin) og perfluor(diethylcyclohexylamin), og per-fluor (dioxaalkaner), dvs. perfluor(alkylenglycol-dialkylethere), såsom perfluor(3,8-dioxa-2,9-dimethyl-5 decan) eller perfluor(tetramethylenglycol-diiso- propylether), perfluor(3,7-dioxa-2,8-dimethylnonan) eller perfluor(trimethylenglycol-diisopropylether) og perfluor(4,6-dioxa-5,5-dimethylnonan) eller per-fluor ( isopropylenglycol-di-n-propylether).perfluoro (diethylhexylamine), perfluoro (dipropylbutylamine) and perfluoro (diethylcyclohexylamine), and perfluoro (dioxal alkanes), i.e. perfluoro (alkylene glycol dialkyl ethers) such as perfluoro (3,8-dioxa-2,9-dimethyl-decane) or perfluoro (tetramethylene glycol-diisopropyl ether), perfluoro (3,7-dioxa-2,8-dimethylnonane) or perfluoro (trimethylene glycol diisopropyl ether) and perfluoro (4,6-dioxa-5,5-dimethylnonane) or perfluoro (isopropylene glycol di-n-propyl ether).
10 Den således fremstillede emulsion af PFC i den modificerede Ringer's opløsning har en partikelstørrelse på mellem 0,05 og 0,3 ^um, den indeholder fra 10 - 50¾ (w/v) PFC, 2-5¾ (vi//v) af et emulgeringsmiddel og om nødvendigt 0,1 - 1,0¾ (w/v) af et emulgerende hjælpe-15 middel, og den kan opbevares i stabil tilstand i en længere periode.The emulsion of PFC thus prepared in the modified Ringer's solution has a particle size of between 0.05 and 0.3 µm, containing from 10 - 50¾ (w / v) PFC, 2-5¾ (vi // v) of an emulsifier and, if necessary, 0.1 - 1.0¾ (w / v) of an emulsifying auxiliary, and it can be stored in a stable state for a prolonged period.
Det omhandlede perfusat til opbevaring af et organ til transplantation fremstilles inden anvendelsen ved blanding af den ovennævnte PFC-emulsion med et kommercielt oksealbu-20 min eller humant albumin opløst i den modificerede Ringer's opløsning og den ovennævnte modificerede Ringer's opløsning, saledes at den resulterende væske kan indeholde fra 7,5 - 12,5¾ (w/v) PFC og fra 1 - 8¾ (w/v) albumin. Om nødvendigt kan man tilsætte farmaceutiske midler såsom procain, 25 hydrochlorid, heparin, phenoxybenzamin, insulin, dexamethason ("Decadron"), methylprednisolon, antibiotika og urokinase . De ovenfor givne grænser for PFC-kon-centrationen er opstillet med henblik på at sikre overlevelse efter transplantation af organet, der har været 30 opbevaret ved perfusion af væsken. Koncentrationen af albumin inden for de ovenfor angivne grænser er passende til justering af det kolloide osmotiske tryk, således at perfusionsvæsken kan holdes fysiologisk iso-tonisk.The present perfusate for storing a transplant organ is prepared prior to use by mixing the above-mentioned PFC emulsion with a commercial oxalbum or human albumin dissolved in the modified Ringer's solution and the above-modified modified Ringer's solution so that the resulting liquid can contain from 7.5 - 12.5¾ (w / v) PFC and from 1 - 8¾ (w / v) albumin. If necessary, pharmaceutical agents such as procaine, hydrochloride, heparin, phenoxybenzamine, insulin, dexamethasone ("Decadron"), methylprednisolone, antibiotics and urokinase may be added. The above limits for PFC concentration have been set up to ensure post-transplant survival of the organ which has been stored by perfusion of the fluid. The concentration of albumin within the above limits is appropriate for adjusting the colloidal osmotic pressure so that the perfusion fluid can be kept physiologically isotonic.
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Opbevaring af et organ, der skal transplanteres, gennemføres på sædvanlig måde under iltning med oxygen ved hjælp af et velkendt udstyr. Eksempelvis blander man 400 ml af en 25 % (w/v) PFC-emulsion i den modificerede 5 Ringer's opløsning, 360 ml af den modificerede Ringer's opløsning og 240 ml af en 25 % (w/v) opløsning af humant albumin i den modificerede Ringer's opløsning, således at der opnås et totalt volumen på 1 liter. Den resulterende blanding ledes til en perfusatbeholder og perfu-10 seres igennem organet, der skal transplanteres, mens der sker en oxygenering med en blandet gas (95-98 % oxygen og 5-2 % carbondioxid) eller oxygen alene ved hjælp af en oxygena-tor af membrantypen eller gennem-boblingstypen. Cirkulationshastigheden af perfusatet 15 er 15-50 ml/g nyre,time, og perfusatet oxygeneres med 95 % 0^ og 5 % CO2 med en gennemstrømningshastighed på 30-1500 ml/minut. Om nødvendigt kan man sætte de '>> / ovennævnte farmaceutiske midler til perfusatet, uden at der sker nogen forringelse af dette. Ved opbevaring 20 ved perfusion er det også muligt ved hjælp af en pulserende pumpe at recirkulere perfusatet, som ledes til beholderen på arteriesiden af. et udstyr til organopbevaring. Ved opbevaring over længere tidsperioder er det ønskeligt, at man nve.d.. pa-ssende mellemrum kan ud-25 skifte perfusatet.Storage of an organ to be transplanted is carried out in the usual way during oxygenation with oxygen by a well-known equipment. For example, 400 ml of a 25% (w / v) PFC emulsion is mixed in the modified 5 Ringer's solution, 360 ml of the modified Ringer's solution and 240 ml of a 25% (w / v) solution of human albumin in the modified Ringer's solution so that a total volume of 1 liter is obtained. The resulting mixture is passed to a perfusate vessel and perfused through the organ to be transplanted while oxygenation is performed with a mixed gas (95-98% oxygen and 5-2% carbon dioxide) or oxygen by means of an oxygen alone. membrane type or bubbling type. The circulation rate of the perfusate 15 is 15-50 ml / g kidney, hour, and the perfusate is oxygenated with 95% O 2 and 5% CO 2 at a flow rate of 30-1500 ml / minute. If necessary, the above-mentioned pharmaceutical agents can be added to the perfusate without any deterioration thereof. In storage 20 by perfusion, it is also possible to recycle with the aid of a pulsating pump the perfusate which is fed to the vessel on the arterial side of. an organ storage equipment. When stored over extended periods of time, it is desirable that at appropriate intervals, the perfusate can be replaced.
Med den foreliggende opfindelse er det blevet muligt at opbevare et organ meget sikkert i en lang tidsperiode, indtil organet skal bruges til transplantation.With the present invention, it has become possible to store a organ very securely for a long period of time until the organ is to be used for transplantation.
Eftersom overlevelsesgraden er meget høj, vil den omhand-30 lede metode sikre en kommercielt succesfuld organtrans plantation inden for praktisk medicinsk behandling.Since the survival rate is very high, the method in question will ensure a commercially successful organ transplantation in the field of practical medical treatment.
I det følgende gives nogle illustrative eksempler på fremgangsmåden til fremstilling af hver af de indgående komponenter i det omhandlede perfusat, ligesom fremstil- 13In the following, some illustrative examples of the process for preparing each of the constituent components of the present perfusate are given, as well as the preparation thereof.
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lingsproceduren til fremstilling af selve perfusatet beskrives. Symbolet "% (w/v) angiver den vægtmængde af et materiale i gram, der indgår i 100 ml af den resulterende væske.The procedure for preparing the perfusate itself is described. The symbol "% (w / v) indicates the amount of weight of a material in grams contained in 100 ml of the resulting liquid.
5 Præparation 15 Preparation 1
Fremstilling af en fluorcarbon-emulsion I 8 liter af en modificeret Ringer's opløsning (beskrives senere) opløstes 300 g af en polyoxyethylen-polyoxy-propylen-copolymer (molekylvægt 8350). Efter tilsætning 10 af 3 kg perfluordecalin blev den resulterende blanding omrørt i en mixer til dannelse af en grov emulsion.Preparation of a Fluorocarbon Emulsion In 8 liters of a modified Ringer's solution (described later), 300 g of a polyoxyethylene-polyoxy-propylene copolymer (molecular weight 8350) was dissolved. After adding 10 kg of perfluorodecaline 3 kg, the resulting mixture was stirred in a blender to form a coarse emulsion.
Denne grove emulsion overførtes til beholderen i en emulgator af stråletypen (homogenisator af Monton-Gaulin-type) for cirkulation af emulsionen. Emulgeringen gen-15 nemførtes ved en temperatur på 35 - 5 °C under et tryk på 200 - 500 kg/cm . Koncentrationen af perfluordecalin i den resulterende fine emulsion var 31,3% (\i//v), og den gennemsnitlige partikeldiameter var mellem 0,09 og 0,1 ium målt ved centrifugal sedimentation. Efter 6 må-20 neders opbevaring af emulsionen ved 4 °C kunne der ikke iagttages nogen sammenvoksning af partiklerne, og den gennemsnitlige partikeldiameter forblev i det væsentlige uforandret.This coarse emulsion was transferred to the container in a jet-type emulsifier (Monton-Gaulin-type homogenizer) to circulate the emulsion. The emulsification was carried out at a temperature of 35-5 ° C under a pressure of 200-500 kg / cm. The concentration of perfluorodecaline in the resulting fine emulsion was 31.3% (µ in v) and the mean particle diameter was between 0.09 and 0.1 µm as measured by centrifugal sedimentation. After 6 months of storage of the emulsion at 4 ° C, no aggregation of the particles could be observed and the average particle diameter remained essentially unchanged.
Fremstilling af albuminopløsningen 25 Et kommercielt præparat af humant serumalbumin blev opløst i en koncentration på 25% (w/v) i den modificerede Ringer's opløsning, som beskrives i det følgende .Preparation of the Albumin Solution 25 A commercial preparation of human serum albumin was dissolved at a concentration of 25% (w / v) in the modified Ringer's solution, which is described below.
Fremstilling af en modificeret Ringer's opløsning 30 Man fremstillede en modificeret Ringer's opløsning ved i destilleret vand at opløse følgende forbindelser 14Preparation of a modified Ringer's solution 30 A modified Ringer's solution was prepared by dissolving in distilled water the following compounds 14
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(angivet i g/liter): 6,51 NaCl, 0,4 MgSO^, 0,8 KC1, 0,24 NaHCO^, 0,14 Na2HP0^ og 6,0 glucose. Herved opnåedes en opløsning med følgende elektrolytsammensætning (angivet i m-ækv./liter): 112 Na+, 11 K+, 7 Mg++, 118 5 Cl-, 3 HCO^-, 2 HPO^--, 7 SO^-- og 33 glucose. De ovenfor beskrevne procedurer repræsenterer kendt teknik.(indicated in g / liter): 6.51 NaCl, 0.4 MgSO 4, 0.8 KCl, 0.24 NaHCO 3, 0.14 Na 2 HPO 4, and 6.0 glucose. There was thus obtained a solution of the following electrolyte composition (given in m-eq / liter): 112 Na +, 11 K +, 7 Mg ++, 118 5 Cl-, 3 HCO 2 -, 2 HPO 2 -, 7 SO 2 - and 33 glucose. The procedures described above represent prior art.
Blanding af de indgående væsker til dannelse af perfu-satet ifølge opfindelsen_ 570 ml af den modificerede Ringer's opløsning og 330 10 ml af fluorcarbon-emulsionen blev blandet og omrørt grundigt. Blandingen blev steriliseret ved opvarmning i et sterilisationsapparatur til 115 °C i 12 minutter.Mixing of the incoming liquids to form the perfusate of the invention - 570 ml of the modified Ringer's solution and 330 ml of the fluorocarbon emulsion were mixed and stirred thoroughly. The mixture was sterilized by heating in a sterilizer to 115 ° C for 12 minutes.
Den steriliserede alanding blev blandet med 100 ml af albuminopløsning, som forinden var ledt igennem et bak-15 teriefilter. Den resulterende blanding blev opbevaret koldt (ved 1-10 °C) og anvendt efter behov som perfusionsvæske til opbevaring af et organ til transplantation.The sterilized alander was mixed with 100 ml of albumin solution which had previously been passed through a bacterial filter. The resulting mixture was stored cold (at 1-10 ° C) and used as needed as perfusion fluid to store a transplant organ.
Præparation 2Preparation 2
Fremstilling af en perfluorcarbon-emulsion 20 330 g polyoxyethylen-octylether med en gennemsnitlig molekylvægt på 3500 blev opløst i 8 liter af den modificerede Ringer's opløsning·. Til opløsningen sattes 40 g sojabønne-phosphorlipid og 2 g kaliumoleat. Blandingen blev omrørt i en mixer til dannelse af en sus-25 pension. Denne suspension blev blandet med 3 kg per-fluortributylamin og omrørt i en mixer til dannelse af en grov emulsion. Denne grove emulsion blev yderligere emulgeret på samme måde som i præparation 1. Den herved opnåede fine emulsion blev fyldt på hættefla-3Ό sker og steriliseret ved opvarmning i et roterende sterilisationsapparatur ved 115 °C i 12 minutter.Preparation of a Perfluorocarbon Emulsion 20 330 g of polyoxyethylene octyl ether with an average molecular weight of 3500 was dissolved in 8 liters of the modified Ringer's solution. To the solution was added 40 g of soybean phosphorus lipid and 2 g of potassium oleate. The mixture was stirred in a mixer to form a suspension. This suspension was mixed with 3 kg of perfluorotributylamine and stirred in a mixer to form a coarse emulsion. This coarse emulsion was further emulsified in the same manner as in Preparation 1. The resultant fine emulsion was loaded onto cap flakes and sterilized by heating in a rotary sterilization apparatus at 115 ° C for 12 minutes.
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Koncentrationen af perfluorcarbonforbindelsen i den steriliserede emulsion var 29,7 % (w/v). Efter opbevaring ved 4 °C i 6 måneder viste emulsionen ingen tegn på sammenhobning af partiklerne. Denne procedure repræsente-5 rer kendt teknik.The concentration of the perfluorocarbon compound in the sterilized emulsion was 29.7% (w / v). After storage at 4 ° C for 6 months, the emulsion showed no evidence of aggregation of the particles. This procedure represents prior art.
Blanding af de indgående væsker til dannelse af perfu-atet ifølge opfindelsen____Mixture of the incoming liquids to form the perfume of the invention
Man blandede 550 ml af den modificerede Ringer's opløsning, som anvendtes i præparation 1, 100 ml albu-10 minopløsning og 350 ml af den ovenfor beskrevne fluor-carbon-emulsion. Den blandede væske blev opbevaret pa et køligt sted ved en temperatur på 1 - 10 °C og anvendt efter behov som perfusionsvæske til opbevaring af et organ, der skal transplanteres.Mixed 550 ml of the modified Ringer's solution used in Preparation 1, 100 ml of elu 10 min solution and 350 ml of the fluorocarbon emulsion described above. The mixed fluid was stored in a cool place at a temperature of 1 - 10 ° C and used as a perfusion fluid as needed to store an organ to be transplanted.
15 Præparation 3Preparation 3
De i præparation 1 beskrevne procedurer blev gentaget med den undtagelse, at man anvendte perfluormethylpropyl-cyclohexan i stedet for perfluordecalin. Den opnåede per fusionsvæske havde egenskaber, der svarede til egen-20 skaberne af den i præparation 1 opnåede væske.The procedures described in Preparation 1 were repeated with the exception that perfluoromethylpropyl-cyclohexane was used instead of perfluorodecaline. The obtained per fusion fluid had properties similar to the properties of the liquid obtained in Preparation 1.
Præparation 4Preparation 4
De i præparation 1 beskrevne procedurer blev gentaget med den undtagelse, at man i stedet for perfluordecalin anvendte henholdsvis perfluorbutylcyclohexan, perfluor-25 trimethylcyclohexan, perfluorethylpropylcyclohexan, perfluormethyldecalin, perfluorhexyltetrahydropyran, perfluorpentyltetrahydrofuran, perfluorheptyltetra-hydrofuran og perfluordecan. De opnåede perfusionsvæsker havde egenskaber, der svarede til egenskaberne 30 af væsken fra præparation 1.The procedures described in Preparation 1 were repeated with the exception that instead of perfluorodecalin, perfluorobutylcyclohexane, perfluoro-trimethylcyclohexane, perfluoroethylpropylcyclohexane, perfluoromethane fluoro-tetrahydro-fluoro-ethaneduhydro-tetrahydro-fluoro-ethanol, The perfusion fluids obtained had properties similar to the properties of the liquid from Preparation 1.
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Præparation 5Preparation 5
De i præparation 1 beskrevne procedurer blev gentaget med den undtagelse, at man i stedet for perfluordeca- lin anvendte henholdsvis perfluor-N,N-dibutylmonome-5 thylamin, perfluor-N,N-diethylpentylamin, perfluor- N,N-dipropylbutylamin, perfluortripropylamin, perfluor-N,N-diethylcyclohexylamin, perfluor-N-pentylpiperidin, perfluor-N-hexylpiperidin, perfluor-N-butylpiperidin, perfluor-N-pentylmorpholin, perfluor-N-hexylmorpholin 10 og perfluor-N-heptylmorpholin. De opnåede perfusions væsker havde egenskaber, der svarede til egenskaberne af perfusionsvæsken fra præparation 1.The procedures described in Preparation 1 were repeated with the exception that perfluoro-N, N-dibutyl monomethylamine, perfluoro-N, N-diethylpentylamine, perfluoro-N, N-dipropylbutylamine, perfluorotripropylamine were used instead of perfluorecetin. , perfluoro-N, N-diethylcyclohexylamine, perfluoro-N-pentylpiperidine, perfluoro-N-hexylpiperidine, perfluoro-N-butylpiperidine, perfluoro-N-pentylmorpholine, perfluoro-N-hexylmorpholine and perfluoro-N-heptyl The obtained perfusion fluids had properties similar to the properties of the perfusion fluid from Preparation 1.
Præparation 6Preparation 6
De i præparartion 1 beskrevne procedurer blev gentaget 15 med den undtagelse, at man i stedet for perfluordeca- lin anvendte perfluortributylamin. Den opnåede perfusionsvæske svarede til den i præparation 1 opnåede perfusionsvæske.The procedures described in Preparation Part 1 were repeated except that perfluorotributylamine was used instead of perfluorodecline. The perfusion fluid obtained was similar to the perfusion fluid obtained in Preparation 1.
Præparation 7 20 Den i præparation 1 beskrevne procedure blev gentaget med den undtagelse, at man i stedet for at anvende en copolymer med en molekylvægt på 8350 anvendte en poly-oxyethylen-polyoxypropylen-copolymer med en molekylvægt på 15800. Den opnåede per fusionsvæske havde egen-25 skaber, der svarede til egenskaberne af perfusions væsken fra præparation 1.Preparation 7 The procedure described in Preparation 1 was repeated except that instead of using a copolymer having a molecular weight of 8350, a polyoxyethylene polyoxypropylene copolymer having a molecular weight of 15800 was used. -25 creates similar to the properties of the perfusion fluid from Preparation 1.
I det følgende er vist eksempler på komparative eksperimenter, ved hvilke man ved dyreforsøg efterviser effektiviteten af perfusionsvæskerne ifølge opfindel-30 sen ved transplantation af nyrer.In the following, examples of comparative experiments are shown, in which animal experiments demonstrate the effectiveness of the perfusion fluids of the invention in kidney transplantation.
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1717
Overlevelsesgraden eller dødeligheden ved transplantationerne blev bestemt ved at observere funktionen af den transplanterede nyTe efter at den kontralaterale normale nyre var blevet fjernet. I tabellerne ‘betyder 5 symbolet "E.F." (early function), at der kunne observeres vandladning to dage efter den kontralaterale næphrek-tomi. Symbolet "L.F." (late function) betyder, at der ikke kunne iagttages nogen vandladning to dage efter den kontralaterale nephrektomi, men at nyrens -funktion 10 derefter gradvis blev bedre, indtil der kunne iagttages en vandladning hos forsøgsdyret, som oveilevede forsøget. Symbolet "N" (necrose) angiver dødt væv i 'f lokaliserede områder omkring den transplanterede nyre inden den kontralaterale nephrektomi (eksperimentet 15 blev afbrudt). Ved symbolet "A.T.N. (A)" [acute tubular necrosis (anuria)] forstås, at dyret efter den kontra-laterale nephrektomi led af anuri, appetitløshed og hyppige opkastninger, indtil døden indtrådte som følge af akut nyreinsufficiens. Endelig betyder symbolet 20 "A.T.N. (H)" [acute tubular necrosis (Hematuria)], at forsøgsdyret døde som følge af anuri, som.medførte hæmatori. Symbolet "N.F." (no-function) angiver, at der ikke kunne iagttages nogen funktion.The survival rate or mortality of the transplants was determined by observing the function of the transplanted kidney after the contralateral normal kidney had been removed. In the tables' 5 means the symbol "E.F." (early function) that urination could be observed two days after the contralateral nasal retraction. The symbol "L.F." (late function) means that no urination could be observed two days after the contralateral nephrectomy, but that renal function 10 then gradually improved until a urination could be observed in the experimental animal that failed the experiment. The symbol "N" (necrosis) indicates dead tissue in 'localized areas around the transplanted kidney before the contralateral nephrectomy (experiment 15 was discontinued). By the symbol "A.T.N. (A)" [acute tubular necrosis (anuria)] is understood that the animal, after the contralateral nephrectomy, suffered from anuria, loss of appetite and frequent vomiting until death occurred as a result of acute renal failure. Finally, the symbol 20 "A.T.N. (H)" [acute tubular necrosis (Hematuria)] means that the test animal died as a result of anuria which caused hemorrhage. The symbol "N.F." (no-function) indicates that no function could be observed.
Optimal PFC-koncentration i opbevaringsvæsken 25 Det er afgørende nødvendigt at bestemme den PFC-koncentration, som er mest egnet til perfusion, hvilket skyldes, at en forøgelse i PFC-koncentrationen i en PFC-emulsion vil bevirke en forøgelse af den oxygenbærende kapacitet af emulsionen, mens viskositen sam-30 tidig forøges væsentligt ved lave temperaturer. Med henblik på at bestemme den optimale PFC-koncentration under perfusionen fremstillede man et antal perfusionsvæsker med varierende PFC-koncentrationer, idet man som primær saltopløsning anvendte en modificeret Collin's op-35 løsning (M.C.S.) [The Lancet, 2, 1219 - 1222 (1969)], som hævdes at være den mest favorable opløsning til hypo- DK 154253Β Λ 18 termisk opbevaring af organer, og som har en elektro-lytsammensætning, der minder om elektrolytsammensætningen i den intracellulære væske. Man satte forskellige mængder af de fluorcarbon-emulsioner, som 5 opnåedes i præparation 2, til M.C.S., således at de endelige PFC-koncentrationer i perfusionsvæskerne blev 5, 7,5, 10, 12,5 og 15% (w/v). Man gennemførte forsøg på at opbevare hundenyrer til transplantation ved perfusion ved hjælp af de ovennævnte væsker ved temperatu-10 rer på 5 - 8 °C og ved perfusionsstrømningshastigheder på 15 - 18 ml/g/time i 24 timer under oxygenering med en oxygenholdig gas (95% 0^, 5% CO2) med en strømningshastighed på 300 - 500 ml/minut. Idet man anvendte 5 hunde pr. gruppe, gennemførte man en autotransplanta-15 tion af de opbevarede nyrer. På den syvende dag efter autotransplantationen blev den kontralaterale normale nyre hos hver hund udtaget, og man noterede de efterfølgende overlevelsesdage for hver hund. Hvis en hund overlevede i 4 uger eller derover, skønnede man, at over-20 leveisen var positiv, eftersom niveauerne for såvel BUN (blod-urinstof-nitrogen) som creatin forblev normale hos hunden, ligesom der observeredes vandladning. Ud fra de opnåede data bestemtes den optimale PFC-koncentra-tion i perfusatet.Optimal PFC concentration in the storage liquid 25 It is imperative to determine the PFC concentration most suitable for perfusion, because an increase in the PFC concentration in a PFC emulsion will cause an increase in the oxygen carrying capacity of the emulsion. while at the same time the viscosity is substantially increased at low temperatures. In order to determine the optimal PFC concentration during perfusion, a number of perfusion fluids with varying PFC concentrations were prepared, using as a primary saline solution a modified Collin's solution (MCS) [The Lancet, 2, 1219-1222 (1969 )], which is claimed to be the most favorable solution for hypo- thermal storage of organs and having an electrolyte composition similar to the electrolyte composition in the intracellular fluid. Various amounts of the fluorocarbon emulsions obtained in Preparation 2 were added to M.C.S. such that the final PFC concentrations in the perfusion fluids were 5, 7.5, 10, 12.5 and 15% (w / v). Attempts were made to store dog kidneys for transplantation by perfusion using the aforementioned fluids at temperatures of 5 - 8 ° C and at perfusion flow rates of 15 - 18 ml / g / h for 24 hours under oxygenation with an oxygen-containing gas ( 95% (0.5% CO 2) at a flow rate of 300 - 500 ml / minute. Using 5 dogs per day. group, an autotransplantation of the stored kidneys was performed. On the seventh day after autotransplantation, the contralateral normal kidney of each dog was sampled and subsequent survival days were recorded for each dog. If a dog survived for 4 weeks or more, survival was estimated to be positive, as both BUN (blood urea nitrogen) and creatine levels remained normal in the dog, as was urination. From the obtained data, the optimal PFC concentration in the perfusate was determined.
25 Eksperimentet gennemførtes på sædvanlig vis i overensstemmelse med den efterfølgende procedurerækkefølge: 19The experiment was carried out as usual in accordance with the following procedure sequence: 19
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Anæstesi: Natriumpentobarbital (30 mg/kg i.v. injektion) Endotratracheal indgivelse af halothan og oxygenAnesthesia: Sodium pentobarbital (30 mg / kg i.v. injection) Endotratracheal administration of halothane and oxygen
Indsnit langs midten af bugenIncision along the center of the abdomen
Kirurgisk isolation af venstre nyre Cannulation (ureter, nyrearterie og vene) Første udvaskning med kold M.C.S. eller kold finger's opløsningSurgical Isolation of the Left Kidney Cannulation (Ureter, Renal Artery and Vein) First Leaching with Cold M.C.S. or cold finger's solution
Tilslutning til perfusionskredsløb.Connection to perfusion circuits.
Perfusionel opbevaring med oxygeneret per-fusat ved 3 - 8 °C i 24 timer elleT 72 timer.Perfusion storage with oxygenated perfusate at 3 - 8 ° C for 24 hours or 72 hours.
VV
Udvaskning (M.C.S. eller Ringer's lactatopløsning) iLeaching (M.C.S. or Ringer's lactate solution) i
Autotransplantation i femoral fossa iAutotransplantation into femoral fossa i
Forsinket kontralateral .nephrektomi TABEL 2 20 DK 1542538Delayed contralateral .nephrectomy TABLE 2 20 DK 1542538
Virkning af PFC-koncentrationen i perfusatet på overlevelsesgradenEffect of PFC concentration in the perfusate on survival rate
Hund Perfusater Overlevelsestid Overlevelses- bemærk- nr. (dage) grad ninger 1 ^ levende E.F.Dog Perfusates Survival Time Survival Note (Days) Degrees 1 ^ Living E.F.
2 N.2 N.
3 M.C.S. 5 2/5 A.T.N.Ca: ^ levende E.F.3 M.C.S. 5 2/5 A.T.N.Ca: ^ living E.F.
5 4 A.T.N.CA! 6 - N· η levende L.F.5 4 A.T.N.CA! 6 - N · η living L.F.
8 5« PFC 5 3/5 A.T.N.(A) i M.C.S.8 5 «PFC 5 3/5 A.T.N. (A) in M.C.S.
^ levende E.F.^ living E.F.
2Q levende L.F.2Q live L.F.
11 levende E.F.11 living E.F.
12 levende E.F.12 living E.F.
13 7,5¾ PFC levende 4/5 E.F.13 7.5¾ PFC live 4/5 E.F.
i M.C.S.in M.C.S.
14 7 A.T.N.CA) 15 levende E.F.14 7 A.T.N.CA) 15 live E.F.
16 levende E.F.16 live E.F.
17 5 A.T.N.CA) 18 10¾ PFC levende , E.F.17 5 A.T.N.CA) 18 10¾ PFC live, E.F.
4/5 i M.C.S.4/5 in M.C.S.
19 levende E.F.19 living E.F.
20 . levende E.F.20. living E.F.
2121
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21 levende E.F.21 living E.F.
22 levende E.F.22 living E.F.
23 12,5¾ PFC levende 4/5 E.F.23 12.5¾ PFC live 4/5 E.F.
i M.C.5.in M.C.5.
24 '5 A.T.N.(A) 25 lev end e E.F.24 '5 A.T.N. (A) 25 lev than e E.F.
26 b' A.T.N,. (H) 27 levende E.F.(H) 28 15¾ PFC 4 A.T.N.(A) i M.C.S.26 b 'A.T.N ,. (H) 27 live E.F. (H) 28 15¾ PFC 4 A.T.N. (A) in M.C.S.
29 levende 2/5 E.F.(H) 30 7 A.T.N.(A) * levende: Overlevede mere end 4 uger.29 live 2/5 E.F. (H) 30 7 A.T.N. (A) * live: Survived more than 4 weeks.
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De opnåede resultater fremgår tabel 2. Som det fremgår af tabel 2, opnåedes en høj overlevelsesgrad ved en PFC-koncentration på mellem 7,5 og 12,5% (vi//v). Hvis PFC-koncentrationen overstiger 12,5¾ (\i//v) og når op 5 på 15¾ (u//v) observeres hæmatori i alle tilfælde ved genoptagelse af blodcirkulationen efter transplantio-nen. To hunde, som overlevede i mere end 2 uger, udviste tegn på hæmatori i flere dage efter den kontra-laterale nephrektomi, hvorefter deres tilstand forbed-10 redes, og de overlevede. På basis af de ovenfor angivne resultater kunne det konkluderes, at en perfusionsvæske justeret til en PFC-koncentration på 10¾ (\i//v) er den mest effektive væske til perfusionsopbevaring af en udtaget nyre.The results obtained are shown in Table 2. As can be seen in Table 2, a high survival rate was obtained at a PFC concentration of between 7.5 and 12.5% (vi // v). If the PFC concentration exceeds 12.5¾ (\ i // v) and reaches 5 of 15¾ (u // v), hemorrhage is observed in all cases upon resumption of blood circulation after transplantation. Two dogs that survived for more than 2 weeks showed signs of hemorrhage for several days following the contralateral nephrectomy, after which their condition improved and they survived. Based on the above results, it could be concluded that a perfusion fluid adjusted to a PFC concentration of 10¾ (µ in // v) is the most effective fluid for perfusion storage of a sampled kidney.
15 EKSEMPEL 1EXAMPLE 1
Primær saltopløsningPrimary saline solution
Man gennemførte eksperimenter med perfusionsopbevaring i længere perioder ved lav temperatur med henblik på at udvælge en egnet primær saltopløsning. De under-20 søgte saltopløsninger var M.C.S. (beskrevet i det foregående) Ringer's opløsning og en modificeret Ringer's opløsning (M.R.S.) fremstillet som beskrevet i præparation 1. Perfusionsvæskerne til opbevaringen blev fremstillet ved at sætte perfluorcarbon-emulsionen fra 25 præparation 2 og okseserumalbumin til en saltopløsning, således at den endelige koncentration eksempelvis bliver 10¾ (w/v) med hensyn til PFC og 6% (w/v) med hensyn til albumin. Perfusionen blev gennemført på hunde-nyrer (5 i hver gruppe) ved 5 - 8 °C i 72 timer, idet 30 man fulgte proceduren beskrevet.ovenfor. Efter transplantation og genoptagelse af blodcirkulationen foretog man undersøgelser af blodets bevægelse igennem nyren, og man iagttog nyrens farve og nuance, vandladningen og symp- 23Experiments with perfusion storage were carried out for long periods at low temperature in order to select a suitable primary saline solution. The under-20 saline solutions sought were M.C.S. (described above) Ringer's solution and a modified Ringer's solution (MRS) prepared as described in Preparation 1. The perfusion fluids for storage were prepared by adding the perfluorocarbon emulsion of Preparation 2 and bovine serum albumin to a saline solution so that the final concentration e.g. becomes 10¾ (w / v) for PFC and 6% (w / v) for albumin. The perfusion was performed on canine kidneys (5 in each group) at 5-8 ° C for 72 hours, following the procedure described above. After transplantation and resumption of blood circulation, studies of blood movement through the kidney were performed and the color and hue of the kidney, urination and symp- 23 were observed.
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tomer hidrørende fra den kontralaterale nephrektomi, som blev foretaget på den syvende dag efter transplantationen. Perfusatet (med et totalt volumen på :Z000 ml) blev ikke udskiftet under de 72 timers perfusion, men der-5 imod recirkuleret med en hastighed på 15 - 18 ml/g/time.tonsils arising from the contralateral nephrectomy performed on the seventh day after transplantation. The perfusate (with a total volume of: Z000 ml) was not replaced during the 72 hour perfusion but, on the other hand, was recycled at a rate of 15 - 18 ml / g / hour.
Med undtagelse af de tilfælde, hvor der som primær saltopløsning anvendtes M.R.S., var transplantations-resultaterne særdeles ufavorable, og stort set alle forsøgsdyrene døde på grund af akut nyreinsufficiens i lø-10 bet af de første 10 dage efter den kontralaterale nephrektomi. Når man. derimod anvendte M.R.S. som primær saltopløsning, blev samtlige forsøgsdyr raske uden komplikationer.With the exception of the cases where M.R.S. was used as the primary saline solution, the transplant results were highly unfavorable and virtually all test animals died due to acute renal failure during the first 10 days after the contralateral nephrectomy. When you. in contrast, M.R.S. as primary saline solution, all test animals recovered without complications.
EKSEMPEL 2 15 Indvirkning af perfusafcets albuminkoncentratioon på hundenyre_EXAMPLE 2 15 Effect of Perfus Effect Albumin Concentration on Dog Kidney
De i dette forsøg anvendte perfusater bestod -af 10% emulsion af PFC i en modificeret Ringer's opløsning, der fremstilledes ved anvendelse af den i præparation 6 20 fremstillede PFC-emulsion, og som indeholdt forskellige koncentrationer af humant albumin. Nyrerne blev per-fuseret i 72 timer ved 5 - 8 °C under oxygenering, idet man anvendte den i eksempel 1 anvendte oxygenholdige gas med en strømningshastighed på 300 - 500 ml/minut.The perfusates used in this experiment consisted of 10% emulsion of PFC in a modified Ringer's solution prepared using the PFC emulsion prepared in Preparation 6 which contained various concentrations of human albumin. The kidneys were perfused for 72 hours at 5-8 ° C under oxygenation using the oxygen-containing gas used in Example 1 at a flow rate of 300-500 ml / minute.
25 Derefter foretog man en autotransplantation som beskrevet ovenfor. Strømningshastigheden under perfusionen blev holdt på mellem 15 og 18 ml/g/time. Den kontralate-rale nephrektomi blev gennemført en uge efter autotransplantationen .Then, an autotransplant was performed as described above. The flow rate during perfusion was maintained at between 15 and 18 ml / g / h. The contralateral nephrectomy was performed one week after autotransplantation.
30 De opnåede resultater fremgår af tabel 3.30 The results obtained are shown in Table 3.
TABEL 3 24TABLE 3 24
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Hund Koncentration Vægtstigning af Efter kontralateral nr. af albumin nyren under per- nephrektomi_ (¾) fusionen (%) Funktion Overlevelse 1 73,5 Inaktiv Døde 2 0 49,4 N.F. Døde 3 53,1 L.F. Overlevede ____(2 dage)__ 4 38,4 L.F. Overlevede (2 dage) 5 2 52,9 N.F. Døde 6 49,8 N.F. Døde 7 37,6 N.F. Døde 8 4 20,3 E.F. Overlevede 9 41,6 E.F. Overlevede 10 13,7 E.F. Overlevede 11 6 30,6 E.F. Overlevede .12 34,.4 E.F. Overlevede 13 11,9 E.F. Overlevede 14 8 22,8 E.F. Overlevede 15 30,5 N.F. DødeDog Concentration Weight gain of After contralateral No. of albumin kidney during pernephrectomy_ (¾) fusion (%) Function Survival 1 73.5 Inactive Dead 2 0 49.4 N.F. Dead 3 53.1 L.F. Survived ____ (2 days) __ 4 38.4 L.F. Survived (2 days) 5 2 52.9 N.F. Dead 6 49.8 N.F. Dead 7 37.6 N.F. Dead 8 4 20.3 E.F. Survived 9 41.6 E.F. Survived 10 13.7 E.F. Survived 11 6 30.6 E.F. Survived .12 34, .4 E.F. Survived 13 11.9 E.F. Survived 14 8 22.8 E.F. Survived 15 30.5 N.F. Died
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25 EKSEMPEL 3EXAMPLE 3
Langvarig perfusion ved lav temperatur af nyre til trans-plantation_______Long-term low-temperature kidney perfusion for transplantation _______
Man gennemførte transplantationsforsøg med hundenyre 5 ved en metode svarende til den ovenfor beskrevne. Hver nyre blev opbevaret i ten uge ved perfusion ved 5-8 °C og ved en strømningshastighed på 15-18 mg/g/time under oxygenering med en oxygenholdig gas ved en stirømningshastighed på 300-500 ml/minut. Perfusionsvæsken frem-10 stilledes ved anvendelse af M.R.S. fra præparation 1 som primær saltopløsning, hvortil der satte.s okseserum-albumin samt PFC-emulsionen fra præparation 2 eller en PFC-emulsion fremstillet på samme måde som i præparation 2 med den undtagelse, at der anvendtes perfluor-15 decalin i stedet for perfluortributylamin. Koncentra tionerne af albumin og PFC i perfusionsvæsken var henholdsvis 6 % og 10 % (w/v). Efter en uges opbevaring under perfusion blev nyrerne autotransplanteret som beskrevet ovenfor. Den kontralaterale nephrektomi blev 20 gennemført en uge efter autotransplantationen, og symp tomerne observeredes. Resultaterne er angivet i tabel 4. Plasmaniveauerne for creatinin og BUN hos de overlevende dyr blev normale i løbet af 3 uger.Transplantation experiments with dog kidney 5 were performed by a method similar to that described above. Each kidney was stored for ten weeks by perfusion at 5-8 ° C and at a flow rate of 15-18 mg / g / hour under oxygenation with an oxygen-containing gas at a flow rate of 300-500 ml / minute. The perfusion liquid was prepared using M.R.S. from Preparation 1 as the primary saline solution to which added bovine serum albumin as well as the PFC emulsion from Preparation 2 or a PFC emulsion prepared in the same manner as in Preparation 2 except perfluorodecalin was used instead of perfluorotributylamine . The concentrations of albumin and PFC in the perfusion fluid were 6% and 10% (w / v), respectively. After one week of storage under perfusion, the kidneys were autotransplanted as described above. The contralateral nephrectomy was performed one week after autotransplantation and the symp- tomers were observed. The results are given in Table 4. Plasma levels of creatinine and BUN in the surviving animals were normal within 3 weeks.
TABEL 4 26TABLE 4 26
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Eksempel Perfusat Vægtstigning af nyren Efter kontralateral nr under perfusionen nephrektomi_ ___(¾)__Funktion Overlevelse 1 34,3 E.F. Overlevede 2 M.R.S. 46,9 N.F. Døde _albumin ___ 3 6% (\i//v) 20,1 L.F. OverlevedeExample Perfusate Renal weight gain After contralateral nos during perfusion nephrectomy_ ___ (¾) __ Function Survival 1 34.3 E.F. Survived 2 M.R.S. 46.9 N.F. Dead _albumin ___ 3 6% (\ i // v) 20.1 L.F. survived
Perfluor-__(2 dage)_ 4 tributyl- 18,7 L.F. Døde amin 5 10¾ (w/v) 33,5 E.F. Overlevede 6 _19,3__E.F.__Overlevede 7 M.R.S. 40,1 L.F. Døde , albumin__(3 dage)_ _8 _ Perfluor- 40j9_ E . F.__Overlevede 9 decalin —_23,5__E.F.__Overlevede 10 10¾ (w/v) 21,6 N.F. Døde L__ 27 EKSEMPEL 5Perfluoro -__ (2 days) _ 4 tributyl- 18.7 L.F. Dead amines 5 10¾ (w / v) 33.5 E.F. Survived 6 _19,3__E.F .__ Survived 7 M.R.S. 40.1 L.F. Dead, albumin __ (3 days) _ _8 _ Perfluoro- 40j9_ E. F .__ Survived 9 decalin —_23.5__E.F .__ Survived 10 10¾ (w / v) 21.6 N.F. Dead L__ 27 EXAMPLE 5
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Under anvendelse af hu.ndenyrer gennemførte man transplantationsforsøg. på lignende måde som beskrevet i det foregående . Hver nyre 'blev opbevaret ved 18 - 22 °C 5 i 12 eller 24 timer ved perfusion med en strømnings hastighed på 40 - 50 ml/g/time under oxygenering med en blandet gas (95¾. 0^,, 5% C02), der gennemblffi.st.es med en hastighed på 300 - 500 ml/minut. Perfusionsvæsken blev fremstillet ved afvendelse af M.R.S. fra præpara-10 tion 1 som primær saltopløsning, hvortil der sattes den i præparation 2 fremstillede PFC-emulsion ag okseserumalbumin. Koncentrationerne af PFC og albumin i per fusionsvæsken var henholdsvis 10¾ og 6% (w/v). Efter 12 eller 24 timers opbevaring bl©v nyrer-15 ne autotransplanteret- Den kontralaterale nephrek- tomi blev genenmført en uge efter autotransplanta-tionen, og symptomerne 'blev iagttaget. I tabel 5 er anført resultaterne af de ovenfor beskrevne eksperimenter sammen med resultaterne af et kontrol— 20 eksperiment, ved hvilket der ikke tilsattes RFC,, og et sammenlignende eksperiment, hvori man anvendte M.C.S. som primær saltopløsning.Transplantation experiments were performed using canine kidneys. in a similar manner as described above. Each kidney was stored at 18 - 22 ° C for 12 or 24 hours by perfusion at a flow rate of 40 - 50 ml / g / hour under oxygenation with a mixed gas (95 ° C, 5% CO 2). which is effected at a rate of 300 - 500 ml / minute. The perfusion fluid was prepared by inverting M.R.S. from Preparation 1 as the primary saline solution, to which was added the PFC emulsion and bovine serum albumin prepared in Preparation 2. The concentrations of PFC and albumin in per fusion fluid were 10¾ and 6% (w / v), respectively. After 12 or 24 hours of storage, the kidneys were autotransplanted. The contralateral nephrectomy was reintroduced one week after autotransplantation and the symptoms were observed. Table 5 lists the results of the experiments described above, together with the results of a control experiment which did not add RFC, and a comparative experiment using M.C.S. as primary saline solution.
2828
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TABEL 5TABLE 5
Eksempel Perfusat Perfusion Efter kontralateral nr. (antal nephrektomi__ timer) Funktion Overlevelse 1 M.R.S. N.F. Døde albumin 2 6¾ (w/v) 12 " "Example Perfusate Perfusion After Contralateral No. (Number of Nephrectomy__ Hours) Function Survival 1 M.R.S. N. F. Dead Albumin 2 6¾ (w / v) 12 ""
3 Π II3 Π II
4 ii ii 5 N.F. Døde 6 M.C.S. 12 7 albumin " " 8 6% (w/v) PFC 10¾ (w/v) 9 M.R.S. E.F. Overlevede albumin 10 6% (w/v) 12 " " 11 PFC 10¾ " 12 (w/ν') " " 13 M.R.S. E.F. Overlevede ^ albumin N.F. Døde 6% (w/v) 24 15 PFC 10¾ E.F. Overlevede 16 (w/v) L.F. Overlevede (2 dage)4 ii ii 5 N.F. Died 6 M.C.S. 12 7 albumin "" 8 6% (w / v) PFC 10¾ (w / v) 9 M.R.S. E. F. Survived albumin 10 6% (w / v) 12 "" 11 PFC 10¾ "12 (w / ν ')" "13 MRSEF Survived ^ albumin NF Dead 6% (w / v) 24 15 PFC 10¾ EF Survived 16 (w / v) v) LF Survived (2 days)
Claims (9)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/JP1980/000011 WO1981002103A1 (en) | 1978-10-10 | 1980-01-28 | Solution for perfusion-preserving organ to be transplanted,and preserving method |
| JP8000011 | 1980-01-28 |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| DK426181A DK426181A (en) | 1981-09-25 |
| DK154253B true DK154253B (en) | 1988-10-31 |
| DK154253C DK154253C (en) | 1989-05-22 |
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ID=13706003
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK426181A DK154253C (en) | 1980-01-28 | 1981-09-25 | PERFUSATE FOR STORAGE OF BODIES FOR TRANSPLANTATION, PROCEDURE FOR PREPARING THE PERFUSATE, AND PROCEDURE FOR STORING AN ORGAN USING THE PERFUSATE |
Country Status (2)
| Country | Link |
|---|---|
| BR (1) | BR8009028A (en) |
| DK (1) | DK154253C (en) |
-
1980
- 1980-01-28 BR BR8009028A patent/BR8009028A/en unknown
-
1981
- 1981-09-25 DK DK426181A patent/DK154253C/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| DK154253C (en) | 1989-05-22 |
| BR8009028A (en) | 1981-12-01 |
| DK426181A (en) | 1981-09-25 |
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