Taxol composition for use as organ preservation and cardioplegic agents

A composition containing taxol for the ex vivo preservation or perfusion of organs for implantation in a subject requiring such implantation, or for cardioplegia, is disclosed.

FIELD OF THE INVENTION 
The present invention relates to compositions for the ex vivo preservation 
and storage of organs prior to implantation of an organ into a subject in 
need of such implantation. The compositions also double as effective 
cardioplegic agents for the reversible cessation of a beating heart during 
surgical procedures. Specifically, the present invention relates to organ 
preservation and storage solutions and cardioplegic agents which include 
taxol. 
DESCRIPTION OF THE PRIOR ART 
The last two decades have seen organ transplantation proceed from a rare 
research and experimental procedure into an established clinical therapy 
for the end-stage treatment of various terminal malfunctions of the heart, 
liver, pancreas, lung, intestine, and kidney. Progress in the science of 
organ transplantation has evolved to the point that such procedures are 
now rarely deemed to be newsworthy. 
As organ transplantation has become an accepted clinical practice, however, 
the need for extending the viable ex vivo preservation and storage of 
organs for implantation has become acute. Due to the success of surgeons 
in perfecting organ transplantation protocols, and the widespread use of 
immuno-suppressant drugs which minimize the host's rejection of the 
implanted organ, waiting lists for organ transplant procedures have grown 
enormously. This has caused a critical shortage of viable organs to be 
transplanted. 
Minimizing the wastage of precious organs for transplantation is therefore 
critical. Such waste often occurs due to the amount of time required to 
match the tissue type of the organ to the tissue type of the recipient, 
and also the time required to physically transport the stored organ to the 
patient's location. Due to the present inability to store excised organs 
for a prolonged period of time, the ex vivo organ often becomes nonviable 
before a suitable match can be established. By prolonging the viable 
storage time, such waste can be significantly minimized by providing 
sufficient time for the necessary tissue match and transportation to 
occur. 
Because organ transplantation has been a very active field, the prior art 
contains several references describing organ preservation and perfusion 
solutions for the ex vivo storage of organs prior to their 
transplantation. For instance, the work of the present inventor, as 
embodied in U.S. Pat. Nos. 4,798,824; 4,873,230; and 4,879,283 to Belzer 
and Southard, describes an organ preservation solution which has come to 
be referred to as the "UW Solution." (The solution was developed at the 
University of Wisconsin, Madison.) The UW Solution is sold under the 
trademark VIASPAN by the Du Pont de Nemours Company, Wilmington, Del. A 
related solution is marketed by Geneva Labs, Elkhorn, Wis., as a perfusion 
solution under the trademark BELZER'S PERFUSION SOLUTION. Each of the 
above patents is incorporated herein by reference in it entirety. 
The Belzer and Southard patents describe an organ preservation solution 
which contains hydroxy ethyl starch. In one embodiment of the solution, 
the hydroxy ethyl starch is substantially free of ethylene glycol, 
ethylene chlorohydrin, and acetone. As noted in the '283 patent, the 
solution described therein provides 72-hour preservation for the pancreas, 
48-hour preservation for the kidney, and at least 24-hour preservation for 
the liver. 
Lemasters et al. describe an organ preservation solution popularly referred 
to as the "Carolina Solution." This solution is described in U.S. Pat. No. 
5,145,771 and PCT Publication WO 95/05076, both of which are incorporated 
by reference herein in their entirety. The solution described in these two 
references can be used either as a rinsing solution to prevent 
re-perfusion injury, or as a storage solution, per se. The solution is 
described as having an operating temperature range of from 0.degree. C. to 
37.degree. C. The solution is further described as containing a balance of 
sodium, calcium, and potassium chloride salts; magnesium sulfate, 
monopotassium phosphate, and antioxidant such as allopurinol, 
vasodilators, and an ATP energy source such as glucose or fructose. 
Similar to the UW Solution, the Carolina Solution may also include 
modified hydroxy ethyl starch to provide oncotic support against 
interstitial edema. 
U.S. Pat. No. 5,370,989 to Stern et al., assigned to Columbia University, 
describes an organ preservation solution in which the use of sodium ions, 
chloride ions, and calcium ions is specifically avoided. Of particular 
interest in this reference is the use of an analog of cyclic adenosine 
monophosphate (cAMP), specifically, dibutyryl cAMP. Other ingredients 
included in the solution described by the Stern et al. patent include 
glucose, magnesium sulfate, monopotassium phosphate, dextran, potassium 
gluconate, BHA, BHT, N-acetycystine, adenosine, nitroglycerine, a calcium 
blocker such as verapamil, heparin, and cefazolin. This patent 
specifically notes that the intended use of the solution is for the 
preservation of heart tissue. The patent does note that the same solution 
may, however, be used successfully to preserve livers, pancreases, 
kidneys, lungs, and other organs. 
A related type of solution is described by Taylor in U.S. Pat. No. 
5,405,742. This patent describes a hypothermic blood substitute which can 
be used to replace the blood in a euthermic subject. In these procedures, 
the patient's body temperature is lowered to minimize damage to the 
subject's brain and vital organs. By reducing the temperature well below 
that normally maintained by the patient, the patient's metabolic rate is 
significantly lowered. This, in turn, decreases the demands for oxygen and 
glucose of the tissues and organs of the patient. Here, rather than 
explanting the organ from a donor subject, the blood of the donor is 
purged and replaced with the hypothermic blood substitute. This solution 
is described in the Taylor reference as containing an aqueous solution of 
electrolytes including potassium ions, sodium ions, magnesium ions, and 
calcium ions; a macromolecular oncotic agent, preferably a polysaccharide 
such as dextran 40; a biological pH buffer, at least one simple sugar, 
mannitol, an impermeant anion, adenosine, and, optionally, glutathione. 
The solution may also contain a calcium blocker such as nicardipine. This 
reference describes successfully purging the blood of a test animal (a 
dog) for more than three hours, followed by reintroduction of the blood 
with complete recovery and recuperation of the test animal. 
As noted by Stern et al., cardioplegic agents are used to stop the heart 
from beating during cardiac surgery. The principles of organ preservation 
apply equally to cardioplegic agents, and therefore these two types of 
materials are related. During open heart surgical procedures, the 
patient's circulatory system is shunted to an external heart-lung machine 
which circulates and oxygenales the patient's blood. A cardioplegic agent 
is then contacted with the patient's heart to cease its beating. In this 
fashion, the patient's heart is reversibly stopped so that the surgeons 
may complete the required procedure, such as coronary artery bypass 
surgery. In excess of 200,000 such procedures are performed annually in 
the United States alone. 
The present inventors have found that the inclusion of taxol in an organ 
preservation or perfusion solution, or in a cardioplegic agent, prolongs 
the effective storage time which can be achieved using the solution. 
Taxol is a member of the taxane family of diturpenes. The structure of 
taxol and its systematic name are given below: 
##STR1## 
TAXOL=(2aR-(2a.alpha., 4.beta., 4.alpha..beta., 6.beta., 
9.alpha.(.beta.R*,.beta.S*),11.alpha., 12.alpha., 12a.alpha., 
1-2b.alpha.)) -.beta.-(Benzoylamino-.alpha.-hydroxybenzenepropanoic 
acid6,12b-bis(acetyloxy)-12-benzoyloxy) 
-2a,3,4,4a,5,6,9,10,11,12,12a,12b,-dodecahydro-4,11-dihydroxy-4a,8,13,13-t 
etramethyl- 5-oxo-7,11-methano-1H-cyclodeca(3,4)benz(1,2-b)oxet-9-yl ester. 
Taxol was first isolated from the bark of the Pacific yew tree, Taxus 
breviofoila. Taxol has known anti-leukemic and anti-tumor activity and has 
been the subject of intense study as a chemotherapeutic agent in the 
treatment of cancer. An excellent discussion of taxol and a large number 
of taxol derivatives can be found in PCT published application WO 
95/20582, assigned to the Upjohn Company, Kalamazoo, Mich. The contents of 
this patent publication is incorporated by reference herein in its 
entirety. 
The pharmaceutical use of taxol, however, suffers from two major 
disadvantages. The first is that taxol occurs quite rarely in nature. 
Moreover, the Pacific yew tree from which taxol is isolated is relatively 
rare, grows very slowly, and yields an extremely small amount of taxol per 
tree. This makes harvesting natural taxol a practical impossibility. 
The second drawback to the use of taxol is its extremely limited aqueous 
solubility. For instance, Shively, U.S. Pat. No. 5,407,683, describes an 
oil-based solution of taxol or a tumor-active analog of taxol. Due to the 
water-insolubility of taxol, the taxol is first dissolved within an oil, 
and the oil-based taxol solution is then used in the formation of an 
oil-in-water emulsion. The oil-in-water emulsion is then used as a 
delivery vehicle in the administration of taxol. 
The Shively patent also notes that taxol has conventionally been 
administered in formulations using cremophors. Taxol has a relatively high 
solubility in these polyoxyethylated castor oils, but several cremophors 
themselves are sufficiently toxic to preclude their use as 
pharmaceutically-acceptable carriers. Also, taxol is insufficiently 
soluble in soybean oil to use this vehicle in the parenteral 
administration of taxol. Shively describes dissolving taxol in oils from 
marine organisms which have a dipole moment of between about 0.5 Debyes 
and about 2.0 Debyes. However, no mention is made in this reference of the 
use of taxol in an organ preservation or cardioplegic composition. The 
same can be said for all of the references described herein. 
Kingston et al., U.S. Pat. No. 5,352,805, describes derivatives of taxol 
which have increased water solubility. Specifically, Kingston et al. 
describe sulfonated 2'-acryloyltaxol and sulfonated 2'-O-acyl acid taxol 
derivatives. As compared to taxol itself, these derivatives have improved 
water solubility and increased stability. Additionally, the bioactivity of 
the original taxol is maintained. The Kingston et al. reference is 
incorporated herein in its entirety for its teaching of water soluble 
derivatives of taxol. 
Nicolaou et al., U.S. Pat. No. 5,422,364, also describes taxol derivatives 
which have increased water solubility as compared to taxol itself. Here, 
an alkaline-sensitive pro-taxol is described. The pro-taxol compositions 
include 2' and 7-O-ester derivatives of taxol, as well as 2' and 
7-O-carbonate derivatives. At physiological pH, these pro-taxols are 
hydrolyzed to render the native taxol structure. The Nicolaou et al. 
reference is also incorporated herein by reference in its entirety. 
Liposome-encapsulated taxol is described in U.S. Pat. No. 5,424,073 to 
Rahman et al., incorporated herein by reference in its entirety. Rather 
than altering the substituents on the taxol skeleton, this reference 
describes encapsulating native taxol within a liposome. This ameliorates 
many of the solubility problems of taxol. Additionally, the authors note 
that liposome encapsulation improves taxol stability, while lessening the 
chance of anaphylactoid reactions and cardio toxicity. 
The production of taxol is described in references to Smith (U.S. Pat. No. 
5,344,775) and Strobel et al. (U.S. Pat. No. 5,451,392). The Smith patent 
describes the synthesis of a wide range of taxanes in a culture of cells 
taken from the pseudocallus of Pacific yew trees. The various taxanes, 
including taxol, can be isolated from pseudocallus cells which have been 
cultured on a support culture medium. 
The Strobel et al. reference describes the formation of taxol by contacting 
a sterilized yew tree stock with a reactor solution. The solution contains 
a reducing agent, an energy source, a buffer to maintain the pH within a 
defined range, a steroid inhibitor, and a taxol precursor. The natural 
metabolic action of the yew tree stock functions to synthesize the 
formation of taxol from the taxol precursor. Additionally, a 
radio-labelled precursor may be included in the reactor solution to yield 
a radio-labelled taxol. 
None of the above references, taken alone, or in any combination, is seen 
as describing the use of taxol in an organ preservation solution. 
SUMMARY OF THE INVENTION 
In light of the above discussion, the present invention is drawn to a 
composition for the ex vivo preservation and storage of organs intended 
for implantation into a subject requiring such implantation, the 
composition comprising a physiologically-acceptable ex vivo storage 
solution containing taxol and calcium. 
The present invention is further directed to a composition for the ex vivo 
preservation and storage of organs intended for implantation into a 
subject requiring such implantation which comprises a 
physiologically-acceptable ex vivo storage solution having a solution 
osmolality of about 320 mOsm/liter and including taxol, calcium, and a 
lactobionate salt. The solution also contains about 3 to 8 weight percent 
hydroxy ethyl starch having a molecular weight of from about 150,000 to 
about 350,000 Daltons, and wherein the starch is substantially free of 
ethylene glycol, ethylene chlorohydrin, sodium chloride, and acetone. 
The composition according to the present invention may also contain further 
ingredients, among them: about 5 weight percent hydroxy ethyl starch 
having a molecular of from about 200,000 to about 300,000 Daltons, from 
about 1 to about 100 nM taxol, about 25 mM potassium phosphate, about 3 mM 
glutathione, about 5 mM adenosine, about 10 mM glucose, about 10 mM HEPES 
buffer, about 5 mM magnesium gluconate, about 1.5 mM calcium chloride, 
about 105 mM sodium gluconate, 200,000 units penicillin, 40 units insulin, 
and wherein the solution has a pH of from 7.4 to 7.5. 
The present invention is also drawn to improvements in ex vivo storage 
solutions and cardioplegic agents. In particular, in a solution for the ex 
vivo preservation and storage of organs intended for implantation into a 
subject requiring such implantation, the present invention is drawn to the 
improvement comprising adding taxol and calcium to the ex vivo organ 
preservation and storage solution. Preferably, the taxol is present in an 
amount ranging from about 1 to about 100 nM, and the calcium is present in 
an amount of from about 0.5 to about 1.5 .mu.M. 
In a similar vein, the present invention is also drawn to a cardioplegic 
composition which comprises a physiologically-acceptable cardioplegic 
solution containing taxol and calcium. 
It is a principal aim of the present invention to provide a solution for 
the ex vivo storage and preservation of organs which contains taxol or one 
or more taxol derivatives. 
Another aim of the present invention is to provide an organ preservation 
solution containing taxol or a taxol derivative which extends the 
effective ex vivo storage period of explanted organs prior to 
transplantation into a subject requiring such transplantation. 
Yet a further aim of the present invention is to provide an organ 
preservation and perfusion solution containing taxol or taxol derivatives 
which minimizes tissue damage to organs stored ex vivo prior to their 
transplantation. 
A still further aim of the present invention is the effective use of taxol 
derivatives having increased water solubility in a solution for the ex 
vivo storage and maintenance of explanted organs prior to their 
transplantation into a subject requiring such transplantation. 
Still another aim of the invention is to provide a cardioplegic agent 
containing taxol or one or more taxol derivatives. 
A further aim of the present invention is to provide a cardioplegic agent 
containing taxol which has been modified to increase its aqueous 
solubility, as by incorporation in a liposome. 
The principal advantage of the present organ preservation solution is that 
it extends the viable ex vivo storage time for organs to be transplanted, 
thereby minimizing wastage of the life-saving organs. Likewise, when used 
as a cardioplegic agent or perfusate, the present composition minimizes 
tissue damage upon resuscitation of the heart or re-perfusion of the 
stored organ. 
These and other aims, objects, and advantages of the present invention will 
become apparent upon a complete reading of the Detailed Description, 
drawing FIGURES, and claims, below.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is an ex vivo organ preservation and perfusion 
solution and cardioplegic agent containing taxol or one or more taxol 
derivatives, and, preferably, calcium. For sake of clarity and brevity, 
the term "organ preservation solution" as used hereinbelow shall be deemed 
to be synonymous with "organ storage solution," "organ perfusion 
solution," and "cardioplegic agent." This convention does not limit the 
invention disclosed and claimed herein in any fashion, but is merely a 
means to concisely describe the preferred embodiment of the present 
invention. The following discussion applies with equal validity to all of 
these types of solutions. 
Additionally, as used herein, the term "taxol" shall refer to taxol and any 
pharmaceutically-equivalent derivative or analog thereof. Included among 
such equivalents are physiologically-tolerated taxol salts, esters, alkyl 
and acyl derivatives, liposome-encapsulated derivatives, oil-in-water 
emulsions of taxol, and the like. Also encompassed within the term "taxol" 
as used herein are pharmaceutically-equivalent taxol derivatives which 
have been modified to have improved aqueous solubility as compared to 
native taxol. 
It has been found, quite unexpectedly, that the inclusion of taxol and/or 
taxol derivatives in an ex vivo organ preservation solution significantly 
increases the amount of time an explanted organ can be successfully stored 
ex vivo prior to its transplantation into a subject requiring such 
transplantation. By providing for the prolonged storage of explanted 
organs, organ wastage can be minimized. It has also been found that the 
inclusion of taxol in a cardioplegic agent (i.e., an agent which, when 
applied to an in vivo beating heart, reversibly induces heartbeat 
cessation), lessens the extent of heart tissue injury upon resumption of 
the heartbeat. 
As noted above, several basal organ preservation solutions, including the 
UW Solution, are known in the art. The present invention, an organ 
preservation solution including taxol and calcium, will perform with equal 
success using any type of basal organ preservation solution. It has been 
found that the addition of taxol to an organ preservation solution greatly 
increases its ability to maintain the viability of an explanted organ 
stored therein. It has further been found that the addition of calcium has 
a synergistic effect which increases the organ preservation capacity of a 
solution over time greater still. 
While the organ preservation solution of the present invention will 
function with equal success using any type of basal organ preservation 
solution, two basal solutions, namely the UW Solution and Belzer's 
perfusion solution are preferred. 
The UW basal solution contains hydroxy ethyl starch having a molecular of 
from about 200,000 to about 300,000 Daltons, potassium phosphate, 
glutathione, adenosine, potassium lactobionate, raffinose, magnesium 
sulfate, allopurinol, dexamethasone, penicillin, and insulin. Generally, 
the UW basal solution has a pH of from about 7.4 to 7.5. 
Specifically, the preferred UW basal solution for use in the present 
invention contains about 5 weight percent hydroxy ethyl starch having a 
molecular of from about 200,000 to about 300,000 Daltons, about 25 mM 
potassium phosphate, about 3 mM glutathione, about 5 mM adenosine, about 
100 mM potassium lactobionate, about 20 mM raffinose, about 5 mM magnesium 
sulfate, about 1.5 mM allopurinol, about 8 mg/l dexamethasone, about 
200,000 units penicillin, and about 40 units insulin. 
The Belzer basal perfusion solution also contains hydroxy ethyl starch, 
potassium phosphate, glutathione, adenosine, penicillin, and insulin, as 
in the UW basal solution. But, in contrast to the UW basal solution, the 
Belzer contains glucose, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic 
acid buffer (HEPES), magnesium gluconate, calcium chloride, and sodium 
gluconate. As in the UW basal solution, the Belzer basal solution 
preferably has a pH of from about 7.4 to about 7.5. 
Specifically, the Belzer basal solution preferably contains about 5 weight 
percent hydroxy ethyl starch having a molecular of from about 200,000 to 
about 300,000 Daltons, about 25 mM potassium phosphate, about 3 mM 
glutathione, about 5 mM adenosine, about 10 mM glucose, about 10 mM HEPES 
buffer, about 5 mM magnesium gluconate, about 1.5 mM calcium chloride, 
about 105 mM sodium gluconate, about 200,000 units penicillin, and about 
40 units insulin. 
To the basal organ preservation solution is added taxol and calcium, 
preferably in the form of calcium chloride. In the case of the Belzer 
basal solution, which already contains an adequate supply of calcium ions, 
additional calcium need not be added. For basal solutions devoid of 
calcium, however, sufficient calcium should be added, preferably in the 
form of calcium chloride, to raise the calcium ion content to 
approximately 1.5 mM. 
To introduce taxol, per se, into a solution, it is preferred that the taxol 
is first gently dissolved in dimethylsulfoxide (DMSO). Small amounts of 
the taxol-in-DMSO solution are then added, with gentle mixing, to the 
basal organ preservation solution. Because of the extreme aqueous 
insolubility of taxol, some taxol precipitating from solution is to be 
expected. Taxol and several taxol derivatives are available commercially 
from, for instance ICN Biomedicals, Inc., Aurora, Ohio, as well as other 
national and international suppliers. 
Taxol should be added to the solution to give a nominal concentration of 
approximately 100 .mu.M. At this level of saturation, the actual solution 
concentration of taxol in the basal solution ranges from approximately 1 
to about 100 nM taxol. Even at this low concentration, taxol imparts a 
remarkably improved viable cold storage time for explanted organs. 
It is preferred that the osmolality of the overall solution fall within the 
range of from approximately 300-350 mOsm/liter. It is most preferred that 
the solution have an osmolality of approximately 320 mOsm/l. 
As illustrated by the Example, below, the presence of taxol and calcium in 
a basal organ preservation solution elicits a synergistic effect which is 
greater than the effects of either calcium alone or taxol alone. 
EXAMPLE 
The following Example is provided for illustrative purposes only, to 
provide a more complete understanding of the present invention. The 
Example does not limit the invention disclosed and claimed herein in any 
fashion. 
All of the references described below are incorporated herein by reference 
in their entirety. 
Sprague-Dawley rats weighing from 250 to 330 grams were used to isolate 
hepatocytes as described by Seglen (1976). In short, after excising the 
liver, the liver is perfused with collagenase at 37.degree. C., which 
causes the liver tissue to be quantitatively converted into a suspension 
of individual cells. 
The hepatocytes so isolated were then analyzed as described by Marsh et al 
(1990). Immediately after determining the viability of the isolate 
hepatocytes, cell samples were re-suspended in various cell preservation 
solution. Four groups of hepatocyte cold storage (4.degree. C.) samples 
were assembled in duplicate. The first sample used the University of 
Wisconsin (UW) Solution, as described above, for the storage solution. The 
second sample used the UW Solution plus 1.5 mM calcium (in the form of 
calcium chloride) as the storage solution. The third sample used the UW 
Solution plus 100 .mu.M taxol as the preservation solution. The fourth 
sample used the UW Solution plus 1.5 mM calcium (in the form of calcium 
chloride) and 100 .mu.M taxol as the storage solution. 
The suspended cells were then stored for 24 or 48 hours at 4.degree. C. in 
an O.sub.2 /CO.sub.2 atmosphere (95%/5%) in closed 125 ml polycarbonate 
Erlenmeyer flasks (25 ml of suspension per flask) with continuous shaking 
(orbital shaker, 90 RPM). Krebs Hanseleit buffer (KHB), a well known 
physiologically-buffered salt solution, was used to study the hepatocytes 
at normothermia (Marsh et al., 1990). 
To prepare the solutions containing taxol, the taxol was first slowly 
dissolved in dimethylsulfoxide (DMSO). Small volumes of the resultant 
taxol-in-DMSO solution were then added to UW Solution to give a final 
nominal concentration of 100 .mu.M. Calcium chloride was added directly to 
the UW Solution with gentle stirring. 
To assess cell death, lactate dehydrogenase (LDH) was measured in the 
hepatocytes and in the supernate. LDH was analyzed using a commercially 
available kit from Sigma Chemical Company (St. Louis, Mo.), following the 
instructions provided with the kit. 
The control hepatocytes were suspended in UW solution (4.degree. C.) having 
a concentration of 5 mg protein/ml (as calculated by the biuret method). 
The cells were stored, without agitation, in centrifuge tubes. At the end 
of 24 of 48 hours of storage, the cells were sedimented by centrifugation 
(600.times.g), re-suspended in KHB (5 mg protein/ml) at 37.degree. C., and 
incubated with shaking (90 to 100 rpm) in an atmosphere of room air. After 
120 minutes of incubation, the cells were rapidly sedimented by 
centrifugation at 13,000.times.g for 30 seconds. Free LDH in the supernate 
was measured. 
The sedimented cells were re-suspended in KHB and sonicated. Bound LDH from 
the sonicated cells was then measured. 
The percentage of free LDH equals the amount of free LDH found in the cell 
supernate relative to the total amount of cellular LDH (free+bound). 
Four different hepatocyte preparations were analyzed in duplicate. The 
results of the testing are depicted in the sole drawing FIGURE. The FIGURE 
shows the percentage of free LDH, which is proportional to cell death 
(inversely proportional to cell survival), for hepatocytes incubated in 
the UW solution alone, the UW solution plus 1.5 mM calcium, the UW 
solution plus taxol, and the UW solution plus taxol and calcium. As 
depicted in the FIGURE, the solutions rated, in order of maximum cell 
survival to minimum cell survival, as follows: UW solution plus taxol and 
calcium, the UW solution plus taxol, the UW solution plus 1.5 mM calcium, 
and the UW solution. 
The present organ preservation solution and cardioplegic agent is not 
limited to the above-described preferred embodiment, but encompasses all 
such extensions and modifications thereof as fall within the scope of the 
attached claims.