Note: Descriptions are shown in the official language in which they were submitted.
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PRESERVATION SOLUTION
Field of the Invention
The present invention relates to a preservation
solution for organs and tissues or parts thereof from
humans and animals, containing dextran, glucose, electro-
lytes and a buffer.
Background Art
In coronary artery surgery (about 800 operations per
one million inhabitants a year) and in the peripheral
vascular surgery (about 100 operations per one million
inhabitants a year), use is today in most European
clinics made of so-called physiological saline solution
(0.9% NaCl) as solution for washing away blood from
blood-vessel transplants and for storing blood-vessel
transplants before inserting them in their new positions.
In coronary artery surgery use is generally made of the
vena sapena magna, i.e. the superficial vein extending
from the inside of the foot over the inner ankle and
along the inside of the lower leg and the thigh-bone to
the groin, where it joins the thigh vein (vena femora-
lis). In a coronary artery operation, first the vena
sapena magna in one leg is removed, while the breastbone
is opened and preparatory measures are taken for connec-
tion to a heart-lung machine. After removal of the vena
sapena magna, this blood vessel is flushed with saline
solution of the above-mentioned type, on the one hand to
wash away all blood from the inside of the vessel and, on
the other hand, to ensure that one has not neglected to
ligate any branch of the vein, i.e. to tie the branches
with a thread with a view to preventing leakage there-
through. Subsequently, the removed vein is placed in a
dish containing saline solution of room temperature, i.e.
20-25 C.
Then the heart-lung machine is connected and
cardioplegia is given to the heart. About 15-20-cm-long
segments are cut off from the vein in the dish and are
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sewn as a so-called aortacoronary vein bypass to the sick
coronary arteries. Before-all vessel transplants are sewn
and the blood again circulates through these, a period of
up to 2 h may have passed. For patients who are to have
one or two cardiac valves inserted as well, this period
can be still longer. Instead of storing the vessel trans-
plants in saline solution before being sewn, some sur-
geons use the patent's own blood. Blood is then drawn off
from the patient and is placed in a dish. The transplant
is then allowed to lie in this blood before being sewn to
the heart. First the temperature is 37 C but rapidly
falls to room temperature. It is considered that since
blood is the medium to which the vessel is exposed
throughout life, this must be the ideal storage medium
for a vascular transplant.
In heart surgery, the coronary artery surgery con-
stitutes about 70% of the operations on adults. It is
well-known from studying experiments on animals that when
a vascular transplant is used where the endothelium is
destructed, so-called intimal hyperplasia is released and
the transplants are occluded after some time (the vascu-
lar lumen becomes smaller and smaller and at last the
flow of blood is stopped completely). In clinical follow-
up studies, it has been found that 5 years after a coro-
nary artery operation about 30-50% of the venous trans-
plants have been occluded, and when these are studied
histologically, a pronounced intimal hyperplasia will be
discovered. This thus applies to venous transplants which
have been rinsed and stored in the above-mentioned
physiological saline solution.
The applicant's research team has intensely studied
both short-term and long-term preservation of blood ves-
sels. Regarding short-term preservation of blood vessels,
i.e. up to 2 h preservation, it has been found that a
physiological saline solution is toxic to the vascular
endothelium. If a saline solution is flushed through e.g.
the arteria iliaka of a rat, intimal hyperplasia can be
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found in the vessel after about one month. If, on the
other hand, serum is used-for rinsing correspondingly, no
intimal hyperplasia will be discovered. Thus, the use of
a physiological saline solution as preservation solution
is not favourable to the blood vessels. All the same and
in the absence of a better alternative, the clinical use
of physiological saline solution, however, continues in
most thoracic surgery centres throughout the world.
The applicant's research team has also demonstrated
that also blood is not satisfactory as preservation solu-
tion. Blood of room temperature which is stored in a dish
and is not oxygenated is extremely toxic to the endothe-
lia of the blood vessels and inhibits the endothelial
function to a considerable extent. This may seem to be a
paradox, but since blood is an organ that has its normal
function only when it is moving and is continuously oxy-
genated in the lungs, it cannot function in the normal
manner. Deoxygenated, not moving blood contains, like all
other blood, white and red blood corpuscles and thrombo-
cytes. It is well known that white blood corpuscles are
activated in case of hypoxaemia (low concentration of
oxygen) and then produce toxic substances. The appli-
cant's research team has demonstrated that Ringer's
lactate, Kreb's solution and an LPD ("low potassium
dextran") solution could preserve blood vessels for 2 h,
both at refrigerator temperature (4 C) and room tempera-
ture (20 C). However, of these three solutions, only the
LPD solution contains a colloidal-osmotically active sub-
stance, viz. dextran 40, a large sugar molecule of an
average molecular weight of about 40 000 daltons. The
colloidal osmotic pressure is the "suction pressure" that
protein molecules and other bigger molecules which cannot
pass the capillary membrane exert so as to retain fluid
= in the capillaries. This LPD solution, which will be de-
fined in more detail below, thus has a colloidal osmotic
pressure which is slightly higher than that of normal
plasma. In a series of studies, other scientists have
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demonstrated that dextran 40 is favourable for preventing
thrombosis by covering the endothelium, which means that
activated white blood corpuscles cannot get stuck with
their receptors and thus invade and consequently destruct
the vessel. In long-term preservation of blood vessels,
i.e. more than 24 h, for instance 36 h, Ringer's lactate
or Kreb's solution cannot affect the blood vessel in a
sufficiently satisfactory manner. However, the LPD solu-
tion produced a better effect.
In the remaining clinical organ transplantation to-
day, two organ preservation solutions are prevalent, i.e.
the so-called University of Wisconsin solution and the
Euro-Collins solution. These solutions are so-called
intracellular preservation solutions, i.e. they have a
high potassium content and a low sodium content. The pur-
pose of this composition is that the cells should be
allowed to "swim" in an intracellular environment. The
applicant's research team has, however, after extensive
studies demonstrated that in respect of blood vessels,
the high potassium content of these intracellular solu-
tions causes a violent vascular spasm. Therefore there is
no logic in using preservation solutions of intracellular
electrolyte composition when storing vascular trans-
plants.
In heart surgery, a continuously increased use of
so-called homotransplants, i.e. from one individual to
another of the same species, has recently become common.
This means that blood vessels are removed from recently
deceased individuals, in most cases in institutes of
forensic medi.cine, and after a short-term storage, these
blood vessels are cryopreserved, i.e. they are stored in
fluid nitrogen at low temperatures. In heart transplanta-
tions it is in many cases also possible to make use of
the aortic root including the valve apparatus of the
heart that are to be removed and discarded in any case.
At present, this preparation is placed in a saline solu-
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tion until it is being taken care of the next day to be
cryopreserved.
In plastic surgery, the extent of microsurgical pro-
cedures increases, in which parts of organs, including
5 blood vessels, are moved from one place in the body to
another, i.e. autotransplantations. Also in this part of
surgery, there is a need of a satisfactory preservation
solution for the vascular system in the organs involved,
such that when the organ is inserted, a perfect circula-
tion of the blood can be established when the flow of
blood is started.
A further problem, which has recently been discov-
ered, is that in reperfusion of a transplanted organ or
blood vessel, injuries to the cells may arise owing to
detrimental free oxygen radicals within a few seconds up
to some minutes after the implantation.
Summing up, there is thus at present no quite satis-
factory preservation solution available for organs and
tissues or parts thereof from humans and animals, espe-
cially for blood vessels, which are to be transplanted or
stored for some other purpose, for instance for medical
studies. In these fields, there is thus a great global
need of a preservation solution which does not have the
drawbacks of existing preservation solutions and which
preserve the original structures and functions of the
organ, the tissue or parts thereof to a much greater
extent and for a considerably longer period of time.
Object of the Invention
The object of the present invention is to eliminate
the above drawbacks of existing preservation solutions
for organs and tissues or parts thereof from humans and
animals.
This object is achieved by a preservation solution
of the type mentioned by way of introduction, which be-
sides contains calcium and nitroglycerine. Further fea-
tures are stated in the appended claims.
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5a
According to one aspect of the present invention,
there is provided a preservation solution for an organ,
tissue or part thereof from a human or other animal,
comprising calcium, at least one coloidosmotically active
substance, and nitroglycerine.
According to another aspect of the present
invention, there is provided use of a preservation solution
described herein for flushing and immersing an organ, tissue
or part thereof from a human or other animal, which is to be
transplanted to a human or animal.
According to still another aspect of the present
invention, there is provided a method for preserving an
organ, tissue or part thereof from a human or other animal,
comprising flushing and immersing an organ, tissue or part
thereof with and in a preservation solution described herein
and storing at a temperature of 0.5-8 C for at most 36 h for
long-term preservation, or at a temperature of 4-24 C for at
most 2 h for short-term preservation.
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The present invention also relates to use of the
preservation solution, and-to a method for preserving
organs and tissues or parts thereof from humans and ani-
mals in the preservation solution.
Description of the Drawings
Fig. 1 shows comparative tests between preservation
solutions containing the combination of calcium and
nitroglycerine and these ingredients separately, and
Fig. 2 shows the effect of calcium on muscle cells
and endothelial cells in blood vessels.
Description of the Invention
The applicant's research team has, after extensive
studies and experiments, arrived at the surprising and
seemingly contradictory result that calcium has a previ-
ously not shown potent function in long-term preservation
of, above all, blood vessels and can preserve the smooth
muscle function in blood vessels for up to 36 h.
It has for a long time been considered that calcium
should not be present in preservation solutions for
transplants. The reason for this is that in case of
ischaemia, i.e. localised lack of oxygen in a tissue, the
intracellular calcium concentration rises, and therefore
one did not want to add further calcium so as to make
this available to the cells. The applicant's research
team has, however, carried out comparative studies show-
ing that long-term preservation of blood vessels in cal-
cium-free solutions is destructive to the blood vessel
involved. The presence of calcium is necessary in the
solution, among other things for maintaining the integ-
rity of the vascular endothelium. The vascular endothe-
lium contains hydrocarbon chains which are bound to
proteins in the cell membranes, and these constitute a
so-called layer of glycocalyx. This is a thin layer of a
mucous substance (composed of sugars) which is considered
to enclose the cell membranes and which is important to
the properties of immunity of the cell and makes the cell
wall permeable. These hydrocarbon chains are bound to-
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gether by, among other things, calcium. If the vascular
endothelial cells are subjected to calcium-free solutions
for a long time, a disintegration of the important layer
of glycocalyx is assumed to take place, and the function
of the endothelium thus cannot be perfect. Evidence of
these discoveries has been obtained through electron
microscope studies of cell membranes in blood vessels
that have been stored in preservation solutions with and
without calcium.
Moreover the applicant's research team has found
that the endothelial function in blood vessels can be
maintained in a surprising manner by adding nitro-
glycerine to the preservation solution. Nitroglycerine
probably constitutes a substrate for nitrogen oxide, N0,
i.e. the endogenous substance that is produced in the
endothelium and that constitutes one of the so-called
endothelium-dependent relaxation.factors (EDRF).
In a comparative experiment with LPD solutions con-
taining calcium and nitroglycerine, respectively, and an
LPD solution containing both calcium and nitroglycerine,
it has been found that a still better preservation of the
EDRF function could be achieved in the presence of nitro-
glycerine in the solution.
As shown in Fig. 1, the combination of calcium and
nitroglycerine in a transplantation solution for storing
a rat aorta gives better results in respect of contrac-
tion than if the solution does not contain either calcium
or nitroglycerine. The use of calcium in the absence of
nitroglycerine in a preservation solution according to
the present invention yields, in itself, a much better
effect than that achieved by using prior art preservation
= solutions. In combination with nitroglycerine, a still
better effect is obtained. Even if the effect of calcium
= and nitroglycerine in combination is not considerably
higher than the effect of calcium in the absence of
nitroglycerine, every improvement of the effect in this
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field is very important for the subsequent transplanta-
tion to be as successful as possible.
Morphological pictures concerning the satisfactory
and unexpected effect of calcium are shown in Fig. 2. The
pictures show on the one hand smooth muscular cells from
blood vessels and, on the other hand, endothelial cells
from blood vessels that have been stored for a long time
with or without calcium. In case of preservation without
calcium (a: smooth muscular cell and c: endothelial
cell), there appears an enormous swelling of the nuclei
and the cytoplasm in both types of cells. In case of
preservation with calcium, normal structures of the cells
appear (b: smooth muscular cell and c: endothelial cell).
Nitroglycerine has.also been found to have an impor-
tant effect in the reperfusion of the blood vessel or the
organ after implantation. Cell injuries may in fact arise
within a few seconds up to a minute or two after the im-
plantation by the forming of free oxygen radicals. The
nitroglycerine has a so-called scavenging effect on these
radicals and must be present directly in the implantation
operation to reduce this problem. This requirement is
satisfied by the nitroglycerine being included, together
with calcium, in the preservation solution according to
the present invention, in which it also exerts its
favourable combination effect on the endothelium.
The expression "organs and tissues or parts thereof
from humans and animals" as used throughout the patent
application concerns in its widest sense any type of
organ and tissue structure that has been obtained from
animals and humans and that preferably can be trans-
planted to humans and animals by autotransplantation,
syngeneic transplantation, allotransplantation and xeno-
transplantation (for instance organs from pig and monkey
to humans). The preservation solution according to the
invention is particularly suited for blood vessels or
parts thereof and lungs. By the term "blood vessel" is
thus meant veins, arteries and capillaries including the
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aortic root with valve apparatus and the pulmonary root
with valve apparatus (so-called homografts). Extensive
studies of the preservation of other parts of the body
than blood vessels, lungs and kidneys have not yet been
carried out, but there is at present nothing that indi-
cates that such organs would be affected detrimentally by
preservation in the solution according to the invention.
However, for the preservation of hearts, specific concen-
trations of electrolytes in the preservation solution are
necessary, which will be evident from the following.
The term "transplant" used throughout the patent
application, also called graft, concerns organs and tis-
sues or parts thereof according to the above definition
that are to be transplanted in any of the manners as men-
tioned.
The term "preservation solution" used throughout the
patent application concerns the solution or liquid in
which the above-mentioned organs and tissues or parts
thereof are to be stored, for instance before transplan-
tation. The preservation solution is, as mentioned above,
also intended to be used for preservation without subse-
quent transplantation, for instance for different kinds
of examinations and studies.
In a preferred embodiment, the inventive preserva-
tion solution contains, in addition to calcium and nitro-
glycerine, also about 5-15, preferably about 7-12% by
weight low-molecular dextran having an average molecular
weight of about 1 000 daltons (e.g. dextran 1), about 3-
8% by weight high-molecular dextran (e.g. dextran 40,
dextran 60, dextran 70 or higher, such as dextran 120),
about 0.1-2.6% glucose, buffer (e.g. phosphate, THAM or
hydrogen carbonate), about 4-25 mM potassium, about 1-16
mM magnesium, about 50-150 mM sodium and about 50-150 mM
chloride. 5% by weight dextran 1 theoretically results in
an osmolarity of about 50 milliosmoles. When this is
added to the solution, the concentration of electrolytes
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(for instance sodium and chlorine) must be reduced so as
to prevent the solution from becoming hyperosmolar.
In a further preferred embodiment of the invention,
the preservation solution contains, in addition to cal-
5 cium and nitroglycerine, also a Perfadex solution, which
is commercially available. The Perfadex solution contains
50 g/l dextran 40 of an average molecular weight of 40
000 daltons, 5 mM glucose, phosphate buffer which gives a
phosphate content of 0.8 mM, 6 mM potassium, 0.8 mM mag-
10 nesium, 138 mM sodium, 142 mM chlorine and 0.8 mM sul-
phate and an addition of THAM buffer such that a pH of
about 7.4 is obtained.
All the above-mentioned contents are based on the
final preservation solution.
Regarding the preservation of hearts in the preser-
vation solution according to the present invention, the
potassium cohtent must be increased to about 16-25, pref-
erably to 20 mM, and the magnesium concentration to about
12-16 mM. The concentration of sodium then decreases such
that the osmolarity of the solution does not exceed about
340 milliosmoles per litre. When storing other organs and
tissues, the potassium concentration usually is about 4-6
mM and the magnesium concentration usually is about 1-4
mM, based on the final preservation solution.
The Perfadex*solution, which is one type of the
above-mentioned LPD solution, has previously been found
to function as lona-term preservation liquid for trans-
plants, especially kidneys, before transplantation.
Perfadex*is a so-called extracellular solution, i.e.
having a sodium and potassium concentration which is
about the same as that in plasma. The phosphate buffer
and the THAM buffer therein have a great buffering capac-
ity and give the solution a pH of 7.4. The glucose func-
tions as substrate in the metabolism, and dextran 40
gives the solution a colloidosmotic pressure correspond-
ing to that of normal plasma.
*Trade-mark
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All greater dextran molecules, such as from dextran
40 and upwards, suffer from the drawback that when admin-
istered, they may cause anaphylactic reactions. Only
small amounts of these molecules suffice to cause these
fatal reactions. Low-molecular dextran, for instance
*
dextran 1, also called Promiten, is therefore adminis-
tered to patients in these contexts with a view to coun-
teracting this reaction. The risk of such anaphylactic
reactions is eliminated by the preservation solution
according to the invention containing low-molecular
dextran.
*
However, the Perfadex solution does not in itself
have satisfactory properties in respect of maintaining
the smooth muscle function and the endothelial function
in blood vessels.
The addition of calcium and nitroglycerine to
Perfadex results, however, in a significant improvement
of the properties of the preservation solution in respect
of maintaining these functions in long-term preservation.
In the preservation solution according to the
present invention, calcium is present in a concentration
of 0.3-1.5 mM, preferably about 0.7 mM, based on the
final preservation solution. Calcium can be added sepa-
rately during the preparation of the preservation solu-
tion in the form of a solution, for instance an aqueous
solution, of calcium or is added in solid state, such as
a salt, preferably CaC12, the negatively charged ion in
the salt being such as not to detrimentally affect the
properties of the preservation solution.
In the preservation solution according to the pres-
ent invention, nitroglycerine is present in a concentra-
tion of 10-9-10-" M, preferably about 10-5-10-6 M, based on
the final solution. Nitroglycerine can be added sepa-
rately during the preparation of the solution, either in
the form of a solution or in solid state. A usable alter-
native to nitroglycerine is a preparation called Nipride,
whose active ingredient is nitroprusside, e.g. in the
*Trade-mark
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form of nitroprusside sodium. Another alternative to
nitroglycerine is papaverine.
According to the present invention, also heparin can
optionally be added to the preservation solution in a
concentration of 1-12 IE/ml, preferably 10 IE/ml, based
on the final preservation solution. Heparin is used for
the purpose of preventing coagulants from forming on the
inside of the vascular transplant in case it should be
impossible to wash away all blood when removing the
organ. Moreover, extensive studies have shown that hepa-
rin is not toxic to the endothelial function. As an
alternative to common heparin, a so-called low-molecular
heparin, preferably fragmin (Dalteparin), can be used.
Besides, antibiotics can optionally be added to the
preservation solution according to the present invention.
An example of usable antibiotics is benzyl penicillin in
a concentration of about 120 mg/1.
All ingredient in the preservation solution accord-
ing to the invention, also the optional ones, can be
added separately and in any order whatever. In a pre-
ferred embodiment, calcium, nitroglycerine and optionally
heparin and/or an antibiotic are added to a ready-mixed
solution, e.g. Perfadex, containing the remaining ingre-
dients for producing the preservation solution.
When used, the pH of the preservation solution
should be kept in a range of about 7.4-7.6. Any buffer
whatever that yields the necessary pH and that does not
detrimentally affect the function of the preservation
solution can be used.
The inventive preservation solution is ideal for its
purposes by containing the following ingredients and
having the following functions:
1) extracellular composition of electrolytes includ-
ing calcium, i.e. no electrolytes causing vascular spasm,
2) an effective buffer system that can keep a pH of
about 7.4-7.6,
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3) colloidosmotically active substances (e.g. high-
molecular dextran), which-can give the solution a col-
loidosmotic pressure corresponding to that of plasma,
i.e. 25 mm Hg or higher, when necessary,
4) a low-molecular, but cell-membrane-impermeable
substance (e.g. 5-15% dextran 1), which can give the
solution an osmolarity in the range of 50-150 mil-
liosmoles,
5) an efficiently vasodilating substance, e.g.
nitroglycerine, nitroprusside or papaverine,
6) a coagel-inhibiting additive, e.g. heparin or
fragmin (Dalteparin),
7) glucose for the metabolism during the term of
preservation,
8) optionally an antibiotic which is not tissue-
toxic in long-term preservation, and
9) increased potassium and magnesium contents for
the storing of hearts.
In relation to prior art preservation solutions for
organs and/or tissues which, for instance, are to be
transplanted, the preservation solution according to the
present invention thus contains two new active ingredi-
ents which each separately, and especially in combina-
tion, in a surprising manner favourably affect the organs
and/or tissues during preservation. The effects of these
two ingredients, i.e. calcium and nitroglycerine, are not
previously known in this context, and make the inventive
preservation solution a globally seen very promising
preparation.
The inventive preservation solution can be held in
any conventional container that is suitable in the art.
The invention further relates to a method for pre-
serving organs and tissues or parts thereof from humans
and animals in a preservation solution according to the
invention, the organ and tissue or parts thereof being
flushed with and immersed in the preservation solution,
and the temperature of the preservation solution being
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adjusted in the range of 4-24 C for a time of at most
about 2 h for short-term preservation, or at a tempera-
ture in the range of 0.5-8 C for at most 36 h for long-
term preservation.
The organ, tissue or parts thereof from humans and
animals that have been removed from the donor involved
should, if possible, be flushed in situ and/or as soon as
possible after that be placed in the preservation solu-
tion for minimising detrimental effects, if any.
The optimal storing temperature for the inventive
preservation solution is completely dependent on the
planned storing time. In case of e.g. short-term preser-
vation of blood vessels, i.e. up to 2 h, the optimal tem-
perature is room temperature. Too low temperatures are
not optimal for the endothelium, but it resists down to
4 C fairly well. After about 2 h reperfusion in vivo
after the transplant has been fixed, the endothelial
function has been restored completely. When decreasing
the temperature to 1 C, the function of the endothelium
will be deteriorated and is not restored after 2 h, but
after 24 h. For long-term preservation, it is thus an ab-
solute requirement that the temperature be low. 0.5-8 C,
preferably 4 C, has been found to be most advantageous.
Different organs have a specific optimal storing tempera-
ture when stored up to 36 h.
In the EU alone, having more than 400 million in-
habitants, for instance more than about 300 000 coronary
bypass operations are performed each year, and in Sweden
about 7 000 a year. In the USA, this type of operations
is the most common operation in all categories. A preser-
vation solution according to the present invention
should, for instance, also be available for taking care
of homotransplants in institutes of forensic medicine. As
mentioned above, there is also a great need of storing
blood vessels in a satisfactory preservation solution
until they can be cryopreserved the next working day. In
peripheral vascular surgery and in plastic and recon-
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struction surgery, there is also a need of having such a
preservation solution.
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