Brevet US5702881 - Method and solution for organ preservation comprising retinal-derived growth ...

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METHOD AND SOLUTION FOR ORGAN stores mean that anaerobic glycolysis is the principal source

PRESERVATION COMPRISING RETINAL- of energy under traditional preservation conditions.

DERIVED GROWTH FACTOR, Therefore, glycolysis is the only source of ATP available to

CYCLODEXTRIN, anoxic cells.

MUCOPOLYSACCHARIDE AND 5 n ;s weu known that hypothermic conditions eliminate the

FLUOROCARBON effective use of oxygen by tissues. At normal physiologic

temperatures, the phospholipids making up the cell mem

RELATED APPLICATION branes are highly fluid. Under hypothermic conditions, the

This application is a continuation-in-part application of ln lipid bi-layer experiences a phase-change and becomes

U.S. patent application Ser. No. 08/033.629. filed Mar. 16. 10 gel-like wim greatiy reduced fluidity The essentially frozen

1993 now abandoned. ^pid m the cell membranes negates the use of oxygen, even

in the presence of a high oxygen-tension. The metabolic

FILED OF THE INVENTION consequence is glycolysis, which is analogous to the state of

anoxia. The hypothermic conditions used in organ preser

The present invention relates to a solution for use in 15 vation probably represent the reason why previous studies in

preserving organs and tissues and a method for organ which the oxygen-tension was raised did not uniformly

preservation. More specifically, the invention comprises a demonstrate the benefit of increased oxygenation of tissues,

method for the preservation of organs without traditional It has been estimated that approximately one third of the

hypothermia using a perfusate supplemented with an emul- energy metabolism of ceUs js expended to maintain the

sifted liquid fluorocarbon. 20 &odium pump ^ t0 preserve ceU volume. The lack of

... molecular oxygen for oxidative phosphorylation which

BACKGROUND OF THE INVENTION occurs in ischemia leads to ^ accumulation of NADH

In vitro organ preservation was originally described by &e depletion of ATP stores within the mitochondria. The

Carrel and Lindberg in 1938. Since their original studies, subsequent loss of nucleosides is probably a very important

many researchers have attempted to preserve organs prior to factor m the failure of tissues subjected to warm ischemia

transplantation. Over the subsequent years, the longest and pronged periods of cold ischemia to regenerate ATP

experimental preservation times achieved have been after restoration of the blood supply,

approximately five to seven days for kidneys. This may be The development of warm preservation technology would

due to the fact that organ preservation studies embarked JQ present the opportunity to support near normal metabolic

upon a path using hypothermic conditions very early on. In activity. In order to support the increased metabolic rate, an

1937, Bickford and Winton noted that hypothermia pro- increased oxygen-tension will be required for respiration,

longed the duration of tissue survival since hypothermia Extending the period of organ preservation in vitro further

reduces the metabolic need of an organ. At 25° C, there was cannot be achieved without increasing the metabolic activity

a 25% reduction of blood flow in the kidney and the 3J suppressed by the hypothermia and in turn supplying

creatinine clearance was reduced by 20%. Below 18° C. adequate oxygen and metabolite delivery to support this

hypothermia inhibits the tubular activity of the kidney. At 4° basal metabolism.

C, the use of oxygen is approximately 5% of that at Currently, kidney transplantation is largely dependent

normothenma. upon the availability of organs retrieved from heartbeating

A direct relationship exists between oxygen requirements 40 cadaver donors. While awaiting transplantation, kidneys

and temperature. In fact, hypothermia may exert a greater must be stored in a fashion that will result in the restoration

depression of oxidative metabolism in the kidney than in the of normal, immediate function. Therefore, the clinical pres

body as a whole and this may help to explain the success in ervation of organs is much more limited than the experi

preserving kidneys relative to other organs. A major portion mental models; approximately 48 hours for kidneys, 18

of the oxygen consumed by the kidney is used for the 45 hours for livers and 4-6 hours for hearts. There are two

process of active sodium reabsorption. and sodium reab- methods of storage for kidney—preservation by continuous

sorption is by far the most important of all the tubular hypothermic perfusion and simply hypothermic storage,

transport processes. Hypothermic storage, however, is not While a variety of perfusates have been used clinically, these

benign. It produces vasospasm and subsequent edema in an two methods of kidney storage have remained substantially

allograft Preserved organs experience glomerular endothe- 50 unchanged for the past 25 years. VIASPAN, manufactured

lial cell swelling and loss of vascular integrity along with and marketed by DuPont, represents state-of-the-art organ

tubular necrosis, which can be attributed to the hypothermia. preservation. VIASPAN, originally developed as the Uni

Hypothermia inhibits Na/K dependent ATPase and results in versity of Wisconsin (UW) solution, provides for optimized

the loss of the cell volume regulating capacity. The loss of organ preservation under hypothermic conditions. While

volume regulation is what causes the cellular swelling and 55 VIASPAN minimizes the edema and vasospasm normally

damage. An ample supply of oxygen actively diminishes the encountered during hypothermic storage, its use is limited to

amount of this swelling. The most successful early organ the current methods of hypothermic perfusion and storage,

preservation studies employed either supplementation with VIASPAN contains components which prevent hypothermic

hemoglobin or a pump-lung system Without adequate oxy- induced tissue edema, metabolites which facilitate organ

gen delivery, anoxia leads to disintegration of the smaller ^ function upon transplantation, anti-oxidants, membrane

vessels after several hours of perfusion. stabilizers, colloids, ions and salts.

Accordingly, supplying adequate oxygen delivery to the The formulation of this perfusate is designed to preserve

organ has been a major obstacle to successful organ pres- the kidney allografts by hypothermic induced depression of

ervation. The inability to supply oxygen led to the current metabolism. The clinical preservation times have not been

reliance on hypothermia. And yet, the hypothermia itself 65 extended; it is not designed as a perfusate that provides an

may represent the rate-limiting factor in organ preservation. opportunity to expand the currently limited donor pool. This

The lack of oxygen and the subsequent depletion of ATP is due to the fact that an allograft marginally damaged by

warm ischemia cannot tolerate further damage mediated by the hypothermia. Until a mechanism for organ preservation is developed which will provide for the use of an expanded donor pool, the severe shortage of organs in the U.S. cannot be met.

There is a need for the development of more efficient preservation technology. Better quality preservation will also provide the opportunity to expand the donor pool. Allografts with marginal function, secondary to warm ischemia, could theoretically be used because further damage via hypothermic preservation would be eliminated.

The current demand for organs cannot be met unless new sources of organs can be developed. And yet, the donor pool cannot be substantially expanded with existing technology because the rapid cooling of organs and the use of high potassium solutions eliminate the possibility of instituting any concordant, life-saving measures. A large and as yet untapped source of organs for transplantation are accident victims who succumb at the accident site and those who have short post-trauma survival times. These accident victims are not used as organ donors because of the ischemic damage. A period of warm ischemia greater than 60 minutes leads to the phenomenon of no-reflow, which is the failure of circulation to return to the organ. If a suitable means of organ preservation could be developed which minimizes, or to some extent repairs, the damage due to warm ischemia, the shortage of organs could be largely alleviated. The development of warm preservation technology would present the unique opportunity of supporting both organsalvage and life-saving protocols simultaneously. As stated above, with traditional techniques, patient life-saving measures and organ saving measures are mutually exclusive. The development of warm preservation technology would present the possibility for in vivo flushing which would satisfy patient survival and organ salvage protocols. By conservative estimates, this untapped pool of organ donors could provide approximately 10 times the number of organs currently being harvested in the U.S. The number of patients being maintained on hemodialysis has almost doubled in the last six years. Therefore, there is tremendous pressure in the U.S. to open new avenues of procuring transplantable organs. This building public pressure being placed on the medical community is reflected by the recent attempts at xenografting and the discussions pertaining to the sale of organs.

There is therefore an urgent need for a preservation solution useful for initial organ flushing and as a perfusate for ex vivo preservation of organs for transplantation using a warm preservation technology. This technology will minimize, or to some extent repair, the damage caused by warm ischemia, and may support the organ near normal metabolic rate. A desirable feature of using such a solution is that organ preservation ex vivo may be extended further by increasing metabolic activity suppressed by hypothermia, and by supplying adequate oxygen and metabolite delivery to support this basal metabolism.

Fluorocarbons are fluorine substituted hydrocarbons that have been used in medical applications as imaging agents and as blood substitutes. U.S. Pat No. 3,975,512 to Long discloses fluorocarbons, including brominated fluorocarbons, for use as a contrast enhancement medium in radiological imaging. Brominated fluorocarbons and other fluorocarbons are known to be safe, biocompatible substances when appropriately used in medical applications.

It is additionally known that oxygen, and gases in general, are highly soluble in some fluorocarbons. This characteristic

has permitted investigators to develop emulsified fluorocarbons as blood substitutes.

Injectable fluorocarbon emulsions act as a solvent for oxygen. They dissolve oxygen at higher tensions and release

5 this oxygen as the partial pressure decreases. Carbon dioxide is handled in a similar manner. Oxygenation of the fluorocarbon when used intravascularly occurs naturally through the lungs. For other applications, the fluorocarbon can be oxygenated prior to use.

10 Thus, fluorocarbons represent a safe, biocompatible source of oxygen which can support basal metabolism There is a need for further development of fluorocarbon technology for medical applications.

15 SUMMARY OF THE INVENTION

The present invention is directed to a preservation solution useful for the initial flushing and storage of organs intended for transplantation, and a method of preservation

20 technology and transplantation useful at temperatures between 18° C. and 37° C. In accordance with one aspect of the present invention, there is provided a method for preserving an organ prior to transplantation, comprising the steps of flushing an organ with a preservation solution, and thereafter storing said organ at a temperature between about 18° C. and about 37° C. for at least 15 minutes. The preservation solution comprises a mammalian cell culture medium comprising one or more serum proteins, growth factors, mucopolysaccharides, emulsified liquid

^ fluorocarbons, and cyclodextrin. The fluorocarbon comprises between about 1% and 50% v/v of the preservation solution. The flushing step can be performed in situ or ex vivo or the flushing and storing can be performed while the organ remains in situ or ex vivo. The method of the present

35 invention preferably comprises the additional step of continuously perfusing the organ with the preservation solution.

In accordance with another aspect of the present invention, there is provided a preservation solution which supports the preservation of organs without hypothermia, comprising a mammalian cell culture medium comprising one or more serum proteins, growth factors, mucopolysaccharides, emulsified liquid fluorocarbons, and cyclodextrin. The fluorocarbon comprises between about 1% and 50% v/v of the preservation solution. In a preferred

45 embodiment, the mammalian cell culture medium further comprises magnesium, amino acids, glucose, and salts. In preferred embodiments, the serum protein is albumin, the growth factor is bovine-retinal derived growth factor, and the fluorocarbon is perfluorooctylbromide.

so The preservation solution preferably comprises a solution having an osmolality of from about 330 to about 600 mOsM, and more preferably from about 340 to about 450 mOsM. The fluorocarbon preferably comprises between about 5% to about 40% v/v of the preservation solution, and more

55 preferably between about 15% to about 25% v/v. The fluorocarbon emulsion preferably has a particle size of less than 0.5 microns.

Another aspect of the present invention provides a method for organ transplantation, comprising the steps of identifying

60 an organ to be transplanted, flushing the organ with a preservation solution, continuously perfusing the organ with the preservation solution at a temperature between about 18° C. and about 37° C. for at least 15 minutes, and thereafter transplanting the organ into a recipient. The preservation

65 solution comprises a mammalian cell culture medium comprising one or more serum proteins, growth factors, mucopolysaccharides, emulsified liquid fluorocarbons. and 5

cyclodextrin. The fluorocarbon comprises between about 1% and 50% v/v of the preservation solution, preferably between about 5% to about 40% v/v of the preservation solution, and more preferably between about 15% and about 25% v/v.

The flushing step is performed in situ or ex vivo. Preferably, both the flushing step and the perfusing step are performed while the organ remains in situ or ex vivo.

In a preferred embodiment, the mammalian cell culture medium further comprises magnesium, amino acids, glucose, one or more polysaccharides and salts. The preservation solution comprises a solution having an osmolality of from about 330 to about 600 mOsM, preferably from about 340 to about 450 mOsM.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 graphically depicts renal function following ex vivo preservation with the preservation solution of the present invention and compares the results with those of a control without the preservation solution.

FIG. 2 graphically depicts renal function following in situ preservation with the preservation solution of the present invention and compares the results with those of a control without the preservation solution.

DETAILED DESCRIPTION OF THE
INVENTION

The present invention is directed to a new hyperosmolar solution particularly useful as a preservation solution for initial organ flushing, and/or as a perfusate for storage of organs using a warm preservation technology (18°-37° C.) without the need for extreme hypothermia. "Hyperosmolar" is used herein to indicate that the composition has an increased osmotic concentration as compared to basal mammalian cell culture medium and normal body fluids. Thus, hyperosmolar is represented by >330 mOsM.

The preservation solution or perfusate of the present invention comprises a basal mammalian cell culture medium supplemented with an emulsified liquid fluorocarbon, wherein the fluorocarbon comprises between about 1% and about 50% v/v of the preservation medium. The preservation solution or perfusate additionally comprises components designed to support the nutritional and metabolic needs of the vascular endothelium within a graft at temperatures between about 18° C. and 37° C, thereby maintaining the integrity of the vasculature and subsequently the normal permeability of the organ. One preferred embodiment of the invention comprises a highly enriched mammalian cell culture medium, supplemented with one or more emulsified liquid fluorocarbons, growth factors, serum proteins, mucopolysaccharides, and cyclodextrin. Another preferred embodiment of the preservation solution additionally comprises magnesium, amino acids, salts, one or more polysaccharides, ions, and glucose. The perfusate of the present invention is able to preserve organs without concomitant extreme hypothermia.

While the studies described herein relate to the preservation of renal allografts, the application of the preservation solution using a warm preservation technique relates to the preservation of other organs as well. The solution of the present invention was specifically designed to potentiate the simultaneous growth of microvessel and large vessel endothelial cells, to support the integrity of the vascular endothelium within a graft, and to support more normal permeability and metabolism without the need for extreme hypothermic conditions.

The preservation solution of the present invention advantageously employs a mammalian cell culture basal medium and one or more emulsified liquid fluorocarbons, to which is added a variety of supplements including one or more serum

5 proteins such as transferrin, albumin and insulin, one or more growth factors, one or more mucopolysaccharides such as heparin and chondroitin sulfate, magnesium, cyclodextrin, pyruvate, amino acids, salts, one or more

10 polysaccharides, ions and glucose. These supplements serve to enhance the ability of the solution of the present invention to serve as a preservation solution for organs for transplantation using a warm preservation technology. For example, serum albumin acts as a source of protein and colloid;

15 glucose provides for metabolic support; cyclodextrin serves as a source of impermeant, a scavenger, and a potentiator of cell attachment factors; a very high Mg** concentration provides for microvessel metabolism support;

2q mucopolysaccharides, comprising primarily chondroitin and heparin sulfates, provide growth factor potentiation and hemostasis; and an emulsified liquid fluorocarbon supplies the necessary oxygen. The perfusate is preferably hyperosmolar, with a final osmolality after perfluorochemi

25 cal emulsion supplementation of 330-600 mOsM, preferably from about 340-450 mOsM and more preferably about 350 mOsM.

The preservation solution of the present invention has 30 been found to preserve organs without the need for extreme hypothermia, and also reduce or eliminate the common problems encountered with cold storage perfusates, namely edema, vasospasm, depletion of ATP stores, inhibition of ion 35 pumps, glycolysis, and the generation of toxic free radical intermediates following reperfusion. The preservation solution of the present invention provides for more efficacious preservation thereby presenting the potential to use nonheartbeating cadaver donors, thus expanding the potential 40 donor pool.

In addition, the present invention has direct applications for the preservation of the vasculature in any form of trauma, including infarction or aneurysm, involving ischemia or

45 hemorrhage. Likewise, localized preservation of isolated anatomy is possible. The preservation solution and method of use of the present invention can also be used in cases of controlled ischemia during surgical procedures, as well as in

50 the areas of limb salvage and organ repair.

Cell culture media suitable for use in the present invention include any basal mammalian cell culture media well known to those of skill in the art, including Dulbecco's Modified

55 Eagle's medium. Ham's F-12 medium. Eagle's minimal essential medium, Medium 199, and the like, which is supplemented with the necessary components. These components include one or more serum proteins, growth factors, mucopolysaccharides, and cyclodextrin. At least one emul

60 sifted liquid fluorocarbon is then added to this supplemented basal medium to a final volume of between about 1% and 50% v/v to produce a solution useful in preserving organs for transplantation using warm preservation technology.

65 An exemplary formula of a preservation solution in accordance with the present invention is set forth below in Table I.

ENDO GRO is a bovine retinal-derived growth factor which is effective in promoting cell proliferation with low serum concentration, and is available from VEC TEC, Inc., Schenectady, N.Y. It is an anionic heparin binding glycoprotein with a molecular weight of 18,000 and an isoelectric point of 4.5.

While it is contemplated that the various components of the basal cell culture medium set forth above may be mixed in a liter of distilled water to produce the formulation, there may exist various commercial preparations that contain many of the components, in the desired constituent ranges, of the basal medium, and that those components deficient in the particular commercial preparation can be added to that preparation to produce the basal formulation described above.

Id addition to a basal cell culture medium, the preservation solution of the present invention also includes one or more emulsified liquid fluorocarbons. This fluorocarbon emulsion comprises from approximately 1% to 50% of (he preservation solution (v/v). More preferably, the emulsified fluorocarbon comprises from about 5% to about 40% of the total solution (v/v). and even more prefereably from about 15% to about 25% (v/v). Fluorocarbon compounds useful in this invention are generally able to promote gas exchange, and most of these fluorocarbons readily dissolve oxygen and 40 carbon dioxide. As is known in the art. emulsions having a particle size significantly greater than 0.4 microns tend to occlude small vessels and to collect too rapidly in the liver, spleen and other organs, enlarging them and endangering their function. Thus, the fluorocarbon emulsion of the 45 present invention preferably has a particle size of less than about 0.5 microns.

There are a number of fluorocarbons that are contemplated for medical use. These fluorocarbons include bis(Falkyl) ethanes such as C4F9CH=CH4CF9 (sometimes des50 ignated "F-44E") , i-C3F9CH=CHC6F13 ("F-i36E"). and C6F13CH=CHC6F13 ("F-66E"), cyclic fluorocarbons, such as C10F18 ('T-decalin," "perfluorodecalin" or "FDC"), F-adamantane ("FA"), F-methyladamantane ("FMA"), F-l, 3-dimethyladamantane ("FDMA"), F-di-or 55 F-trimethylbicyclo[3,3,l]nonane ("nonane"); perfluorinated amines, such as F-tripropylamine ("FTPA") and F-tributylamine ("FTBA"), F-4-methyloctahydroquinolizine ("FMOQ"), F-n-methyl-decahydroisoquinoline ("FMIQ"), F-n-methyldecahydroquinoline ("FHQ"), F-n60 cyclohexylpurrolidine ("FCHP") and F-2butyltetrahydrofuran ("FC-75" or "RM101").

Other fluorocarbons include brominated perfluorocarbons. such as 1-bromo-heptadecafluoro-octane (CgF17Br, sometimes designated perfluorooctylbromide or 65 "PFOB"), 1-bromopentadecafJuoroheptane (C^F^Br). and 1-hromotridecafluorohexane (CsF^r, sometimes known as perfluOTohexylbromide or "PFHB"). Other brominated fluorocaibons and fluorocarbon emulsions suitable for use in the present invention can be of the type described in U.S. Pat No. 3,975,512 to Long, which also describes methods of preparing fluorocarbon emulsions. Also contemplated are fluorocarbons having nonfiuorine substituents. such as perfluorooctyl chloride, perfiuorooctyl hydride, and similar compounds having different numbers of carbon atoms.

Additional fluorocarbons contemplated in accordance with this invention include perfluoroalkylated ethers or polyethers, such as ... (CF3) 2CFO(CF2CF2)3OCF(CF3), (CF3)CFO(CF2CF2)F, (CF3) 2CF0(CF2CF2)2F, (C6F13)20. Further, fluorocarbonhydrocarbon compounds, such as, for example compounds having the general formula C„F2n+1C„F2n+1, C„F2n+ iOC„.F2n+1, or C„F2n+1CF=CHC„.F2„.+1, where n and n' are the same or different and are from about 1 to about 10 (so long as the compound is a liquid at room temperature). Such

compounds, for example, include C8

[table][merged small][merged small][merged small]

-,H, and

... It will be appreciated that esters, thioethers, and other variously modified mixed fluorocarbon-hydrocarbon compounds are also encompassed within the broad definition of "fluorocarbon" materials suitable for use in the present invention. Mixtures of fluorocarbons are also contemplated. Additional "fluorocarbons" not listed here, but having those properties described 25 in this disclosure are additionally contemplated.

Preparation of fluorocarbon emulsions is well known and is described, for example, in U.S. Pat. No. 5,080,885. Such an emulsion was formerly sold by Alpha Therapeutics (Boston. Mass.) under the trademark FLUOSOL and is being developed by Alliance Pharmaceutical Corp. (San Diego, Calif.) under the trademark OXYGENT.

Particularly preferred fluorocarbon compounds for use in the present invention are the bromineted perfluorocarbons. Perfluorooctylbromide, or PFOB, is particularly preferred.

An emulsified liquid fluorocarbon is added to the supplemented cell culture medium to produce the preservation solution of the present invention. Such an emulsion can be prepared using the following components: PFOB, egg yolk 40 phospholipid (EYP), NaH-jPO,,, Na2HP04, CaNajEDTA, d-a Tocopherol, and NaCl. A particularly preferred formulation for a fluorocarbon emulsion is set forth in Table II below.

The following examples further illustrate the use of the liquid fluorocarbon emulsion preservation solution of the present invention as a perfusate for organ preservation using a warm preservation technology. As used herein, "flush" describes the initial administration to or rinsing of an organ using the solution to remove existing blood from the blood vessels. By "perfusion" is meant the continuous administration of perfusate to the organ. Although the examples described herein disclose the use of a perfluorooctylbromide (PFOB) emulsion, the preferred fluorocarbon emulsion, other oxygen carrying fluorocarbons in an emulsified form may be used as well.

EST VITRO STUDIES

The following studies were performed to evaluate the efficacy of the perfusate of the present invention in preserving organs when compared with existing preservation methods and perfusate without perfluorochemical.

EXAMPLE 1 Pulsatile Preservation Characteristics

Canine kidneys were dissected using a midline incision. The renal arteries were identified and the kidneys were isolated with the vasculature intact The renal arteries were then cannulated. After cannulation. the renal arteries were flushed with approximately 250 cc of the perfusate described in Formula 1, Table I. One set of kidneys was flushed with perfusate supplemented with 20% PFOB v/v (as a 90% emulsion) and another set was flushed with perfusate containing no PFOB. After flushing, the kidneys were placed on a modified Waters MOX-100 preservation system and pumped at 25° C. and 32° C. for up to 18 hours. Apulse rate of 60/minute was maintained with systolic pressures below 70 mmHg. A control set of kidneys were preserved using VIASPAN at 4° C, which represent state-of-the-art organ preservation. Results

a. Flow Characteristics

Mean pressures, pH and vascular flow rate data are listed below in Table IV:

TABLE IV

b. Physical Appearance

The kidneys preserved with perfusate containing 20% PFOB experienced negligible weight gain during the period of preservation. The kidneys maintained normal turgor and displayed no discoloration. Kidneys preserved in either VIASPAN at 4° C. or in the perfusate without perfluorochemical supplementation experienced discoloration over time and had a net weight gain of approximately 20% and 10%, respectively.

c. Histology

Blinded histological studies revealed that all the kidneys were well preserved. There was no detectable difference between the kidneys preserved in perfusate with PFOB supplementation and those kidneys maintained in perfusate without the PFOB. The histologic evaluations revealed normal glomeruli and tubules. The control kidneys preserved with VIASPAN displayed normal glomeruli with focal dilated tubules along with focal edema.

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Brevets

Method and solution for organ preservation comprising retinal-derived growth ...