U.S. patent application number 12/529623 was filed with the patent office on 2010-05-13 for transplants.
This patent application is currently assigned to Hibernation Therapeutics Limited. Invention is credited to Geoffrey Phillip Dobson.
Application Number | 20100119554 12/529623 |
Document ID | / |
Family ID | 39737688 |
Filed Date | 2010-05-13 |
United States Patent
Application |
20100119554 |
Kind Code |
A1 |
Dobson; Geoffrey Phillip |
May 13, 2010 |
TRANSPLANTS
Abstract
The present invention relates to a method of reducing injury to
cells, a tissue or organ to be explanted from a body and upon
implantation into a body by administering a composition to the
cell, tissue or organ, including: (i) a potassium channel opener or
agonist and/or an adenosine receptor agonist; and (ii) an
antiarrhythmic agent. The invention also provides a composition for
reducing injury to vasculature ex vivo including: (i) a potassium
channel opener or agonist and/or an adenosine receptor agonist; and
(ii) an antiarrhythmic agent.
Inventors: |
Dobson; Geoffrey Phillip;
(Queensland, AU) |
Correspondence
Address: |
VENABLE LLP
P.O. BOX 34385
WASHINGTON
DC
20043-9998
US
|
Assignee: |
Hibernation Therapeutics
Limited
Wulguru, Queensland
AU
|
Family ID: |
39737688 |
Appl. No.: |
12/529623 |
Filed: |
March 3, 2008 |
PCT Filed: |
March 3, 2008 |
PCT NO: |
PCT/AU2008/000289 |
371 Date: |
September 2, 2009 |
Current U.S.
Class: |
424/247.1 ;
424/682; 435/1.1; 514/46 |
Current CPC
Class: |
A61K 31/53 20130101;
A61K 31/53 20130101; A61K 31/167 20130101; A61K 31/167 20130101;
A01N 1/02 20130101; A01N 1/0226 20130101; A61K 2300/00 20130101;
A61K 45/06 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/247.1 ;
424/682; 435/1.1; 514/46 |
International
Class: |
A61K 39/08 20060101
A61K039/08; A61K 33/06 20060101 A61K033/06; A01N 1/02 20060101
A01N001/02; A61K 31/70 20060101 A61K031/70 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 2, 2007 |
AU |
2007901098 |
Claims
1. A method of inducing injury to cells, a tissue or organ to be
explanted from a body and upon implantation into a body by
administering a composition to the cell, tissue or organ,
including: (i) a potassium channel opener or agonist and/or an
adenosine receptor agonist; and (ii) antiarrhythmic agent.
2. A method according to claim 1 wherein the composition includes
(i) and (ii) in amounts effective to arrest the heart.
3. A method according to claim 1, wherein the composition further
includes at least one muscle relaxant.
4. A method according to claim 3, wherein the muscle relaxant is
selected from the group consisting of a botulinum toxin (e.g.
botox), myosin light chain kinase inhibitor, calmodulin blocker,
calcium channel blocker, nitric oxide donor, dipyridamole, beta
blocker, Na/H inhibitor, high magnesium, opioid, phosphodiesterase
inhibitors (eg. Papaverine, milrinone, theophylline, dipyridamole,
alpha-adrenergic receptor antagonists (phenoxybenzamine) and Rho
kinase inhibitors (eg HA1077 or fausdil).
5. A method according to claim 1, wherein the composition is
administered directly to the cells, tissues or organs that are
intended to be explanted, or to cells, tissues or organs that have
been explanted or to cells, tissues or organs that have been
implanted, or administered at a combination of these stages of
explanation and implantation.
6. A method according to claim 1 wherein the composition is
administered in a non-arresting concentration to a patient
following surgery.
7. A method according to claim 1 wherein the composition is
pre-mixed with the patient's blood.
8. A method of improving recovery of cells, a tissue or organ upon
implantation into a body by administering a composition including:
(i) a potassium channel opener or agonist and/or an adenosine
receptor agonist; and (ii) antiarrhythmic agent.
9. A composition for reducing injury to vasculature ex vivo
including: (i) a potassium channel opener or agonist and/or an
adenosine receptor agonist; and (ii) antiarrhythmic agent.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a method of protecting cells, a
tissue or organ of a body, particularly during surgery. It has
particular but not exclusive application in the context of coronary
artery bypass graft surgery in protecting a blood vessel during
harvesting, testing and storage as well as implantation of the
graft and its patency.
BACKGROUND OF THE INVENTION
[0002] Pioneering work in the surgical technique of anastomosis of
arteries and veins was made in the early 1900s by the French
surgeon Alexis Carrel (1873-1944). From Carrel's careful methods of
protecting the vessels during harvest and storage and delicate
anastomosis operations he laid the groundwork for the development
of vascular surgery and transplantation. In 1912, Carrel wrote: "In
operations on blood-vessels certain general rules must be followed.
These rules have been adopted with the view of eliminating the
complications which are especially liable to occur after vascular
sutures, namely, stenosis, haemorrhage, and thrombosis." One such
rule was to carefully wash the vessel with Ringer's solution and
coat it and the surrounding parts of the operating-field with
Vaseline to protect the endothelium against "coagulating blood and
the juices of tissues". Carrel knew that damage to the vascular
endothelium, the largest organ of the body, led to injury, thrombus
and poor outcomes.
[0003] One hundred years later, despite major advances in vascular
biology and pathobiology, surgeons are still debating the best way
to harvest, store and transplant arterial or venous grafts for
vascular or cardiac surgery. Globally there are over 800,000
patients who undergo coronary artery bypass graft (CABG) surgery
each year, with more than 350,000 patients in the US. On average
there are three grafts per operation or about 2.4 million
anastomoses performed globally each year, or about 1.0 million in
the USA. With the current technology, the current patency rate of
arterialisation saphenous vein (SV) grafts following CABG is 80% in
the first year, and the patency at 10 years is around 60% compared
to 85% for the left internal mammary (LIMA) grafts to the left
anterior descending coronary artery (LAD). Thus, an ongoing problem
is the early graft occlusion rate of 20% in the first year.
[0004] The reasons for a high occlusion rate in the first year may
involve vessel damage, including endothelial damage and alterations
in vascular reactivity of the vessel, which may have occurred
during: 1) the graft harvest (blunt surgical trauma), 2) stretching
the vessel, 3) high pressure testing, 4) storage of the graft 5)
surgical attachment, 6) temperature fluctuations, and 7)
ischemia-reperfusion injury during restoration of blood flow
following the anastomoses and/or during reanimation before removing
the patient off bypass. In addition, the allograft graft vessels
may have different pre-existing pathologies and wall thicknesses
(e.g atherosclerosis, fibrosis, post inflammatory changes, various
degrees of varicosis etc), which would impact on the vulnerability
to injury and stability of the graft.
[0005] A particular area of concern with current technologies is
the storage procedure and time between harvest and surgical
attachment (or re-implantation), which may for example extend to 5
hours or longer during a CABG operation. The storage procedure
includes placing the harvested vessel conduit in a solution which
may be a patient's heparinized blood, tissue culture medium, Hanks
solution or a crystalloid solution including hyperkalemic
cardioplegia. Particular attention must be paid to the storage
temperature of the solution which may effect the extent and
duration of graft ischemia during harvest and during surgical
attachment. In more difficult operations and on older patients,
surgeries and storage times may be up to 5 hours before
re-implantation.
[0006] One of the key strategies in the protection and preservation
of the transplant is to prevent the vascular endothelium from
becoming injured or activated and to preserve endothelium-smooth
muscle interactions. An injured or activated endothelium loses many
of its homeostatic or balancing functions and becomes
proinflammatory and prothrombolytic, prooxidant, profibrinolytic
and proathrogenic. Thus past methodologies have aimed to reduce
graft reactivity, patency and early failure by preserving the
functional integrity of the vascular endothelium and its
interactions between the blood or bathing solution and the smooth
muscle layer of the vessel wall. However, no therapy has proven to
be clinically successful as evidenced by the high 20% patency
failure in the first year. For example, one troubling and
continuing problem with harvested and transplanted grafts for CABG
and vascular surgery is vasospasm. Vasospasm is defined as an
exaggerated hypercontractile response or state of a vessel's smooth
muscle to various stimuli which may be precipitated by endothelial
dysfunction, shear stresses, smooth muscle calcium
hypersensitivity, increased autonomic tone (parasympathetic and
alpha-adrenergic receptors) and increased oxidative stress.
[0007] In the past 10 years, vasospasm has become particularly
challenging with a resurgence of use of the radial artery graft
after it was abandoned in the mid-1970s because of a high incidence
of vasospasm and a 35% failure rate at 2 years. Arterial grafts are
known to have inherent spasticity compared to saphenous veins,
because of a thicker layer of smooth muscle and connective tissue,
and different endothelial-smooth muscle functions. Arterial grafts
possess more pronounced endothelium-dependent relaxation properties
to acetylcholine, bradykinin, histamine, substance P and mechanical
sheer stress than saphenous veins. In addition, cooling has shown
to act as a vasodilator in human internal thoracic arteries,
saphenous veins, aorta, coronary arteries, and pulmonary
arteries.
[0008] It is not known whether protection could be elicited by a
form of artificial hibernation-like state for the graft. Natural
hibernators possess the ability to lower their metabolic energy
demand for days to months. Hibernation, like sleep, is a form of
dormancy and helps to keep the animal's metabolic supply and demand
ratio in balance. WO00/56145 (U.S. Pat. No. 6,955,814), WO04/056180
and WO04/056181 describe compositions useful to limit damage to a
cell, tissue or organ by administering them to a patient in a
clinical setting. Selective administration of adenosine A2A
receptors has also been proposed in U.S. Pat. No. 6,372,723.
SUMMARY OF THE INVENTION
[0009] The present invention is directed toward overcoming or at
least alleviating one or more of the difficulties and deficiencies
of the prior art.
[0010] In one aspect the present invention is directed to a method
of reducing injury to cells, a tissue or organ to be explanted from
a body and upon implantation into a body by administering a
composition to the cell, tissue or organ, including: [0011] (i) a
potassium channel opener or agonist and/or an adenosine receptor
agonist; and [0012] (ii) antiarrhythmic agent.
[0013] In another embodiment, the invention is directed to a method
of improving recovery of cells, a tissue or organ upon implantation
into a body by administering a composition including: [0014] (i) a
potassium channel opener or agonist and/or an adenosine receptor
agonist; and [0015] (ii) antiarrhythmic agent.
[0016] Preferably, the composition includes (i) and (ii) in amounts
effective to arrest the heart, as described below.
[0017] Preferably, the composition according to this aspect may
further include at least one muscle relaxant. The muscle relaxant
may be selected from the group consisting of botulinum toxin (e.g.
botox), myosin light chain kinase inhibitor, calmodulin blocker,
calcium channel blocker, nitric oxide donor, dipyridamole, beta
blocker, Na/H inhibitor, high magnesium, opioid, phosphodiesterase
inhibitors (eg. papaverine, milrinone, theophylline, dipyridamole,
alpha-adrenergic receptor antagonists (phenoxybenzamine) and Rho
kinase inhibitors (eg HA1077 or fausdil).
[0018] According to this aspect of the invention, the composition
may be administered directly to the cells, tissues or organs that
are intended to be explanted, or to cells, tissue or organs that
have been explanted or to cells, tissues or organs that have been
implanted, or administered at a combination of these stages of
explantation and implantation. In addition, the composition may be
administered in a non-arresting concentration to a patient
following surgery.
[0019] Preferably, the composition is pre-mixed with the patient's
blood.
[0020] Preferably, the cell, tissue or organ is a blood vessel,
such as a saphenous vein.
[0021] In another aspect the present invention provides a
composition for reducing injury to vasculature ex vivo including:
[0022] (i) a potassium channel opener or agonist and/or an
adenosine receptor agonist; and [0023] (ii) antiarrhythmic
agent.
DETAILED DESCRIPTION
[0024] In one form, the invention provides a method of reducing
injury to cells, a tissue or organ removed or explanted from the
body comprising administering a composition including: (i) a
potassium channel opener or agonist and/or an adenosine receptor
agonist; and (ii) an antiarrhythmic agent. In one embodiment, the
composition includes (i) a potassium channel opener or agonist
and/or an adenosine receptor agonist; and (ii) an antiarrhythmic
agent, in amounts below that effective to arrest a heart, as
described below. In an alternative embodiment, the composition
includes (i) a potassium channel opener or agonist and/or an
adenosine receptor agonist; and (ii) an antiarrhythmic agent in
amounts effective to arrest the heart. In the present application,
an amount effective to arrest the heart is an amount in a
composition that contacts causes the heart of a rat to arrest upon
contact. These amounts necessary to arrest a heart are readily
determinable for a given selection of a potassium channel opener or
agonist and/or an adenosine receptor agonist, and a given
antiarrhythmic agent. For example, if adenosine and lidocaine are
the selected compounds to arrest the heart, concentrations above
0.1 mM (and preferably below 20 mM) for each in the composition
that contacts the heart are effective to arrest the heart. In this
specification, higher and lower amounts of these components are
referred to as arresting and non-arresting compositions
respectively. This is further explained below.
[0025] The composition of the invention desirably reduces, at least
in part, reperfusion injury. As outlined above, reperfusion injury
is a common deleterious occurrence upon completion of a procedure.
In this embodiment, the composition may be administered as a
composition ex vivo, as a non-arresting bolus injection or
delivered continuously via an intravenous drip or by another
delivery device or route. In one form, the present invention is
used ex vivo at high arresting concentrations and then in vivo (for
example, following a surgical operation) using a (lower)
non-arresting concentration.
[0026] In another embodiment, the invention provides a method of
keeping the membrane voltage of the cells close to or near their
resting or natural state. Voltage balance is believed to be
important to promote a healthy vessel wall, including the integrity
of the smooth muscle and endothelium.
[0027] Without being bound by any theory or mode of action, the
inventor has found that the composition according to the invention
can be used to place cells, tissue and organs, in effect, toward a
state of suspended animation like a natural hibernator or to
stabilise the cells, a tissue or organ. The overall protection
provided by therapy according to the invention is thought to
involve a multi-tiered system from modulating membrane excitability
to a multitude of intracellular signalling pathways, including heat
shock and pro-survival kinase pathways. Non-binding theories of
proposed mechanisms of the composition of the invention include (i)
reduced wide swings in cell membrane voltage and ion imbalances, in
particular sodium and calcium ion loading in the cells, which may
help defend the cell's voltage when stressed; (ii) attenuation of
local and systemic inflammatory response to injury, which is
protective in itself to reduce injury as well as reduce secondary
effects such as free radical production; and (iii) protection from
entering into a hypercoagulable state, ie an anti-clotting or
anti-thrombolytic activity. Thus the present invention is trying to
maintain the vessel in homeostatic balance in the physiological
ranges representative of the uninjured state which includes but it
not limited to keeping smooth muscle relaxed and the endothelium
from becoming proinflammatory, prothrombolytic, prooxidant,
profibrinolytic and proathrogenic. It is also believed that the
composition may reduce the cell, organ, tissue and body's demand
for oxygen to varying degrees and thus reduce damage to the body's
cells, tissues or organs.
[0028] Damage may be caused to an organ such as a heart or a blood
vessel upon reperfusion. Damage may be caused to the vessel itself,
and also to its endothelium, upon harvesting. Once harvested,
pressure testing of the vessel, often involving substantial
stretching of the outer, medial and inner layers including the
endothelium, can cause damage. Finally, the transplanting or
implanting of the vessel and restoring flow requires substantial
impact on all three layers including the endothelium. Minimising
damage to these layers including the endothelium is desirable
because during injury the endothelium becomes activated and
dramatically alters its phenotype to become pro-inflammatory,
pro-coagulant and pro-thrombotic, pro-oxidant and pro-athrogenic.
Five major changes that can lead to impaired vessel reactivity,
spasm and loss of patency are: 1) platelet adhesion, platelet
aggregation and platelet activation at the site of injury.
Activation of the endothelium leads to growth factor releases and
cytokines (Cytokine 1, 6 & 8), P selection which in turn
activates leukocytes. Platelet activation also leads to tissue
factor generation and thrombin and contributes to thrombus
generation and clot formation, 2) leukocyte activation is the
hallmark of the inflammatory process in response to endothelial
injury-recruitment and vessel wall invasion is driven by cytokines
and chemokines, inflammation to the outer adventitia layer of the
vessel also plays a role, 3) activation of the coagulation cascade
and thrombin generation arises from endothelial injury and exposure
of tissue factor in the vessel wall to the circulating blood.
Inhibiting tissue factor markedly reduces the thrombogenic response
to injury, and therefore intimal hyperplasia, 4) smooth muscle
cells in the medial layer migrate as a result of growth factors,
cytokines, extracellular matrix proteins and cell surface
receptors, and 5) smooth muscle cells begin to proliferate which
leads to the narrowing of the vessel's lumen. In rat vascular
tissue, for example, the percentage of dividing smooth muscle cells
increases from a basal 0.06% to 10-30% (a 500 fold increase) per
day following a vascular insult. The magnitude of the inflammatory
and thrombotic responses and vessel reactivity and therefore
patency depends on the severity of the injury and the degree of
subendothelial exposure. These events are just the opposite of what
is required in an implanted vessel.
[0029] Damage to the graft itself can be exacerbated by the
presence of depolarising potassium in cardioplegia as depolarising
potassium is a known and potent vasoconstrictor of isolated
vessels, which causes the grafts to constrict, often leading to
spasm, and then possibly further damage and to loss of
patency/closure. A particularly vulnerable time is when the grafted
vessel is first reperfused with a cardioplegic solution and the
temperature of that reperfusion and during reanimation. In this
way, the invention seeks to reduce or avoid the use of depolarising
potassium used during these cardiac surgical procedures for
cardioplegic induction, maintenance and reanimation.
[0030] In addition to the critical "window" between harvest,
storage and surgical implantation, which may extend to 5 hours,
another equally critical "window" for the protection and
preservation of the transplanted graft is the first 6 months
following the surgery. When a vessel is damaged during harvest,
storage and surgical implantation, these effects can lead
remodelling of the injured layers including intimal proliferation
following surgery and thereby reduced patency and possibly graft
failure. Thus there are two windows of opportunity. Damage to the
vessel during the first window can profoundly influence the outcome
in the weeks to months following surgery.
[0031] In one form, the invention provides a method for reducing
electrical disturbance of a cell, tissue or organ comprising
administering an effective amount of a composition comprising an
effective amount of (i) a potassium channel opener or agonist
and/or an adenosine receptor agonist; and (ii) an antiarrhythmic
agent, and of one or more of an anti-adrenergic, a calcium
antagonist, an opioid, an NO donor, a sodium hydrogen exchange
inhibitor and a muscle relaxant (particularly a smooth muscle
relaxant). Also provided is a method for reducing damage to an
organ or tissue following ischaemia comprising administering an
effective amount of a composition comprising an effective amount of
a local anaesthetic and of one or more of a potassium channel
opener, an adenosine receptor agonist, an anti-adrenergic, a
calcium antagonist, an opioid, an NO donor, a sodium hydrogen
exchange inhibitor and a smooth muscle relaxant.
[0032] The invention also provides a method for preconditioning a
tissue or organ prior to ischaemia or reperfusion comprising
administering an effective amount of a composition comprising an
effective amount of (i) a potassium channel opener or agonist
and/or an adenosine receptor agonist; and (ii) an antiarrhythmic
agent and of one or more of an anti-adrenergic, a calcium
antagonist, an opioid, an NO donor, a sodium hydrogen exchange
inhibitor and a smooth muscle relaxant.
[0033] The invention also provides a method for reducing
inflammation in a tissue or organ comprising administering an
effective amount of a composition comprising an effective amount of
(i) a potassium channel opener or agonist and/or an adenosine
receptor agonist; and (ii) an antiarrhythmic agent and of one or
more of an anti-adrenergic, a calcium antagonist, an opioid, an NO
donor, a sodium hydrogen exchange inhibitor and a smooth muscle
relaxant.
[0034] The invention may also be used to provide a method for
reducing damage to cells, organs and tissues before, during and
following surgery comprising administering an effective amount of a
composition comprising an effective amount of (i) a potassium
channel opener or agonist and/or an adenosine receptor agonist; and
(ii) an antiarrhythmic agent and of one or more of an
anti-adrenergic, a calcium antagonist, an opioid, an NO donor, a
sodium hydrogen exchange inhibitor and a smooth muscle
relaxant.
[0035] The invention is in one embodiment particularly directed to
improved methods for preserving transplant grafts ex vivo, such as
vasculature grafts. In particular, the invention is directed to
protecting grafts during harvesting. It is also directed to
protecting grafts during storage pending implantation (which may be
for a quite short period or a period of hours or longer). It is
also directed to protecting grafts during pressure testing, or
other testing, of the graft prior to implantation (eg inflation of
the graft in the presence of a composition of the invention). The
invention is also directed to reducing damage to grafts upon
implantation and recovery. The invention may be used at each of
these steps. It is believed that this is achieved by reducing the
degree of constriction of the grafts, and consequential reduced
vessel wall and endothelial damage, as well as providing a
non-depolarising potassium environment for implantation (closer to
normal physiological levels). The invention is also directed to
reducing the patency failure rate post-operatively of those grafts,
thus providing longer-term success of bypass procedures. This can
be achieved by relaxing the vasculature smooth muscle using a
composition of the invention, and optionally further including
long-acting smooth muscle relaxants to the grafts prior to and/or
upon implantation. Thus, in one aspect of the invention, the
composition reduces the energy demand of the vessel including the
smooth muscle layer by placing it in a hibernating-like state.
[0036] A major difference between a composition of the invention
(such as a composition which includes (i) a potassium channel
opener or agonist and/or an adenosine receptor agonist; and (ii) an
antiarrhythmic agent) compared with adenosine alone is that the
relaxation profile is not dependent on an intact endothelium (as it
is with adenosine). The effect of the invention on denuded
vasculature rings is as if the endothelium had not been removed.
One clinical significance of this surprising result is improved
storage of a subject's harvested conduit arteries or veins for
prepared for bypass surgery (or other vascular surgery) which are
known to suffer varying degrees of endothelial damage. During
dissection, storage or anastomoses, the luminal endothelial layer,
the outer adventitial layer and the medial smooth muscle layer are
vulnerable to damage and spasm (uncontrolled contraction). The
invention protects the vessel wall including the endothelium and
relaxes vascular smooth muscle and thereby reducing vascular spasms
during vessel dissection, storage or anastomoses. It can also
provide better protection against short-term and long-term
restenosis. The mechanism of action as to how greater relaxation
occurs in the presence of a composition according to the invention,
such as adenosine and lidocaine, over the summed effects of
adenosine alone and lidocaine alone has not been fully
determined.
[0037] Accordingly, the present invention provides a method of
reducing injury to cells, a tissue or organ to be removed or
explanted from a body, upon implantation into a body and during
recovery in a body by administering a composition to the cells,
tissue or organ, including (i) a potassium channel opener or
agonist and/or an adenosine receptor agonist; and (ii) an
antiarrhythmic agent, where the endothelium of the tissue or organ
is damaged or is non-functional.
[0038] The invention applies to protecting, preserving or
stabilising key organs or tissue ex vivo. In one embodiment, the
organ or tissue is blood vessels, such as those used a grafts in a
cardiac pulmonary bypass procedure.
[0039] In another form, the invention provides a composition for
protecting cells, tissues or organs to be removed, the composition
including (i) a potassium channel opener or agonist and/or an
adenosine receptor agonist; and (ii) an antiarrhythmic agent.
Analogous to the theory explained above, it is believed that the
composition reduces metabolic activity of the tissue, thus reducing
its susceptibility to damage (for example, ischaemic damage) upon
surgical excision. The composition may be administered systemically
or at the site of surgery to the cell, tissue or organ, and the
explanted cell, tissue or organ may then be maintained in a bath
of, or having a continuous supply, of a composition according to
the invention. In addition, the composition may be added to the
fluid used to test explanted tissue (for example, pressure testing)
to reduce damage during those processes. In a preferred form, the
tissue is a blood vessel, such as a saphenous vein.
[0040] In another form, the composition of the invention is
administered upon implant of a cell, tissue or organ. It is
believed, again without being bound by any theory or mode of
action, that the composition reduces undesirable vasoconstriction
and/or inflammation and/or thrombolytic responses, which may
otherwise arise from the implantation.
[0041] The invention also provides a method of reducing damage to a
cell, tissue or organ which is to be explanted, has been explanted
and has been implanted, or more than one of these. In particular,
the invention has application to the harvesting, storage and
grafting of vasculature, the method including the administration of
a composition of the invention as described herein. The invention
includes bathing explanted cells, tissue or organs in a composition
including the composition of the invention. In one form suitable
for all stages of the method described above, the composition of
the invention is pre-mixed with the patient's blood (at, for
example, 1 mM concentration) for use.
[0042] For example, a patient may be administered the composition
of the invention prior to harvesting of a tissue, such as a blood
vessel, to reduce the susceptibility of the vessel to injury from
the inherently damaging process of excision. The excised tissue is
then immediately placed in a bath of a composition that includes
primarily the patient's blood, together with a composition of the
invention (for example, adenosine and lignocaine). This combination
fluid is also suitable for testing the explanted tissue. The
invention includes administering additional composition of the
invention upon grafting or implantation of the cell, tissue or
organ. This can assist in reducing reperfusion injury, as well as
reducing the inflammatory and thrombotic responses which may
otherwise be triggered upon implantation of the cell, tissue or
organ.
[0043] In another form, the invention provides for use of (i) a
potassium channel opener or agonist and/or an adenosine receptor
agonist; and (ii) an antiarrhythmic agent in the manufacture of a
medicament for administration to a patient, or to cells, a tissue
or organ removed from a body of the patient, to reduce injury or
damage as discussed above.
[0044] In various forms, the invention may further include one or
more of a muscle relaxant which could be chosen from a list of
compounds that act either presynaptically, at the nerve-muscle
junction, post-synaptically or directly act on or within smooth
muscle itself which results in relaxation, such as vasodilation.
Some muscle relaxants include a botulinum toxin (e.g. botox),
myosin light chain kinase inhibitor, calmodulin blocker, calcium
channel blocker, nitric oxide donor, dipyridamole, beta blocker,
Na/H inhibitor, high magnesium, opioid, phosphodiesterase
inhibitors (eg. papaverine, milrinone, theophylline, dipyridamole,
alpha-adrenergic receptor antagonists (phenoxybenzamine) and Rho
kinase inhibitors (eg HA1077 or fausdil).
[0045] The compositions as described above in various embodiments
of the invention may further include other components as identified
below. In some embodiments, the potassium channel opener or agonist
and/or adenosine receptor agonist is replaced by another component
such as a calcium channel blocker. The composition preferably
contains an effect amount of (i) and (ii) for a single dose to
reduce injury.
[0046] In the embodiments of the invention described above and
below, component (i) of the composition may be an adenosine
receptor agonist. While this obviously includes adenosine itself,
the "adenosine receptor agonist" may be replaced or supplemented by
a compound that has the effect of raising endogenous adenosine
levels. This may be particularly desirable where the compound
raises endogenous adenosine levels in a local environment within a
body. The effect of raising endogenous adenosine may be achieved by
a compound that inhibits cellular transport of adenosine and
therefore removal from circulation or otherwise slows its
metabolism and effectively extends its half-life (for example,
dipyridamole) and/or a compound that stimulates endogenous
adenosine production such as purine nucleoside analogue
Acadesine.TM. or AICA-riboside (5-amino-4-imidazole carboxamide
ribonucleoside)). Acadesine is also a competitive inhibitor of
adenosine deaminase (Ki=362 microMolar in calf intestinal mucosa.
Acadesine.TM. is desirably administered to produce a plasma
concentration of around 50 microM (uM) but may range from 1 microM
to 1 mM or more preferably from 20 to 200 uM. Acadesine.TM. has
shown to be safe in humans from doses given orally and/or
intravenous administration at 10, 25, 50, and 100 mg/kg body weight
doses.
[0047] In one form of the invention, the composition, and
optionally the second composition, also contains magnesium cations.
In one embodiment, the concentration of magnesium is up to about
2.5 mM and in another embodiment magnesium is present in higher
concentrations, for example up to about 20 mM. The magnesium is
present as a physiologically and pharmaceutically acceptable salt,
such as for example magnesium chloride and magnesium sulphate.
[0048] The invention described in this specification largely
relates to methods of treatment, and methods of manufacturing a
medicament for treatment involving a composition which is described
as containing these components (i) and (ii). For convenience, this
composition will be referred to in this specification as the
"composition of the invention", although there are a number of
combinations of components embodying the invention which are
compositions according to the invention. Moreover, the components
(i) and (ii) may be present in a concentration which either
arrests, or does not arrest, a heart. These two classes of
compositions are used in different ways in the invention described
in the specification, and are referred to respectively as an
"arresting" concentration of the composition and a "non-arresting"
concentration of the composition contacting the cell, tissue or
organ. In one form, the arresting composition contains adenosine
and lignocaine, each at greater than 0.1 mM (and preferably below
20 mM). In one form of the non-arresting composition, adenosine and
lignocaine are both below 0.1 mM and preferably 50 nM to 95 uM, or
more preferably from 1 uM to 90 uM, measured as the concentration
contacting the cells (ie, initially higher concentrations may be
diluted with other components before contacting cells). It will be
appreciated that the concentrations may be diluted by body fluids
or other fluids that may be administered with the composition. For
example, containers (such as vials) may be diluted 1 to 100 parts
of blood, plasma, crystalloid or blood substitute prior to
administration. Suitable methods to determine the arresting and
non-arresting concentrations are described in WO00/56145 (U.S. Pat.
No. 6,955,814) together techniques for assessing effectiveness.
[0049] In a further form, the invention provides use of (i) a
potassium channel opener or agonist and/or an adenosine receptor
agonist; and (ii) an antiarrhythmic agent, for the preparation of a
medicament for reducing injury to cells, tissues or organs of a
body. Preferably the cells, tissues or organs are ex vivo and/or
recently implanted and/or being prepared for explantation. The use
preferably includes administering the medicament in one or more of
the ways set out elsewhere in this specification.
[0050] The term "tissue" is used herein in its broadest sense and
refers to any part of the body exercising a specific function
including organs and cells (native, donor, grown or cloned) or
parts thereof, for example, cell clones, stem cells, cell lines or
organelle preparations. Other examples include circulatory organs
such as the heart, blood vessels and vasculature, respiratory
organs such as the lungs, urinary organs such as the kidneys or
bladder, digestive organs such as the stomach, liver, pancreas or
spleen, reproductive organs such as the scrotum, testis, ovaries or
uterus, neurological organs such as the brain, germ cells such as
spermatozoa or ovum and somatic cells such as skin cells, cloned
cells, stem cells, heart cells ie, myocytes, nerve cells, brain
cells or kidney cells. The tissues may come from human or animal
donors. The donor organs may also be suitable for
xenotransplantation.
[0051] The term "organ" is used herein in its broadest sense and
refers to any part of the body exercising a specific function
including tissues and cells (native, donor, grown or cloned) or
parts thereof, for example, endothelium, epithelium, blood brain
barrier, cell lines or organelle preparations. Other examples
include circulatory organs such as the blood vessels, heart,
respiratory organs such as the lungs, urinary organs such as the
kidneys or bladder, digestive organs such as the stomach, liver,
pancreas or spleen, reproductive organs such as the scrotum,
testis, ovaries or uterus, neurological organs such as the brain,
germ cells such as spermatozoa or ovum and somatic cells such as
skin cells, cloned cells, stem cells, heart cells i.e., myocytes,
nerve cells, brain cells or kidney cells.
[0052] It will also be understood that the term "comprises" (or its
grammatical variants) as used in this specification is equivalent
to the term "includes" and should not be taken as excluding the
presence of other elements or features.
[0053] Potassium channel openers are agents which act on potassium
channels to open them through a gating mechanism. This results in
efflux of potassium across the membrane along its electrochemical
gradient which is usually from inside to outside of the cell. Thus
potassium channels are targets for the actions of transmitters,
hormones, or drugs that modulate cellular function. It will be
appreciated that the potassium: channel openers include the
potassium channel agonists which also stimulate the activity of the
potassium channel with the same result. It will also be appreciated
that there are diverse classes of compounds which open or modulate
different potassium channels; for example, some channels are
voltage dependent, some rectifier potassium channels are sensitive
to ATP depletion, adenosine and opioids, others are activated by
fatty acids, and other channels are modulated by ions such as
sodium and calcium (ie. channels which respond to changes in
cellular sodium and calcium). More recently, two pore potassium
channels have been discovered and thought to function as background
channels involved in the modulation of the resting membrane
potential.
[0054] Potassium channel openers may be selected from the group
consisting of: nicorandil, diazoxide, minoxidil, pinacidil,
aprikalim, cromokulim and derivative U-89232, P-1075 (a selective
plasma membrane KATP channel opener), emakalim, YM-934,
(+)-7,8-dihydro-6,6-dimethyl-7-hydroxy-8-(2-oxo-1-piperidinyl)-6H-pyrano[-
2,3f]benz-2,1,3-oxadiazole (NIP-121), RO316930, RWJ29009,
SDZPCO400, rimakalim, symakalim, YM099,
2-(7,8-dihydro-6,6-dimethyl-6H-[1,4]oxazino[2,3-f][2,1,3]benzoxadiazol-8--
yl) pyridine N-oxide,
9-(3-cyanophenyl)-3,4,6,7,9,10-hexahydro-1,8-(2H,5H)-acridinedione
(ZM244085),
[(9R)-9-(4-fluoro-3-125iodophenyl)-2,3,5,9-tetrahydro-4H-pyrano[3,4-b]thi-
eno[2,3-e]pyridin-8(7H)-one-1,1-dioxide] ([125]A-312110),
(-)-N-(2-ethoxyphenyl)-N'-(1,2,3-trimethylpropyl)-2-nitroethene-1,1-diami-
ne (Bay X 9228), N-(4-benzoyl
phenyl)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamine (ZD6169),
ZD6169 (KATP opener) and ZD0947 (KATP opener), WAY-133537 and a
novel dihydropyridine potassium channel opener, A-278637. In
addition, potassium channel openers can be selected from
BK-activators (also called BK-openers or BK(Ca)-type potassium
channel openers or large-conductance calcium-activated potassium
channel openers) such as benzimidazolone derivatives NS004
(5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidaz-
ole-2-one), NS1619
(1,3-dihydro-1,2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2-
H-benzimidazol-2-one), NS1608
(N-(3-(trifluoromethyl)phenyl)-N'-(2-hydroxy-5-chlorophenyl)urea),
BMS-204352, retigabine (also GABA agonist). There are also
intermediate (eg. benzoxazoles, chlorzoxazone and zoxazolamine) and
small-conductance calcium-activated potassium channel openers.
Other compounds that are believed to open KATP channels include
Levosimendan, glyceryl trinitrate (GTN), and hydrogen sulphide gas
(H.sub.2S) or the H.sub.2S donors (eg sodium hydrosulphide,
NaHS).
[0055] In addition, potassium channel openers may act as indirect
calcium antagonists ie they act to reduce calcium entry into the
cell by shortening the cardiac action potential duration through
the acceleration of phase 3 repolarisation, and thus shorten the
plateau phase. Reduced calcium entry is thought to involve L-type
calcium channels, but other calcium channels may also be
involved.
[0056] Adenosine (6-amino-9-.beta.-D-ribofuranosyl-9H-purine) is
particularly preferred as the potassium channel opener. Adenosine
is capable of opening the potassium channel, hyperpolarising the
cell, depressing metabolic function, protecting endothelial cells,
enhancing preconditioning of tissue and protecting from ischaemia
or damage. Adenosine is also an indirect calcium antagonist,
vasodilator, antiarrhythmic, antiadrenergic, free radical
scavenger, arresting agent, anti-inflammatory agent (attenuates
neutrophil activation), metabolic agent and possible nitric oxide
donor. More recently, adenosine is known to inhibit several steps
which can lead to slowing of the blood clotting process. In
addition, elevated levels of adenosine in the brain has been shown
to cause sleep and may be involved in different forms of dormancy.
An adenosine analogue, 2-chloro-adenosine, may be used.
[0057] Suitable adenosine receptor agonists may be selected from:
N.sup.6-cyclopentyladenosine (CPA), N-ethylcarboxamido adenosine
(NECA), 2-[p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamido
adenosine (CGS-21680), 2-chloroadenosine,
N.sup.6-[2-(3,5-demethoxyphenyl)-2-(2-methoxyphenyl]ethyladenosine,
2-chloro-N.sup.6-cyclopentyladenosine (CCPA),
N-(4-aminobenzyl)-9-[5-(methylcarbonyl)-beta-D-robofu
ranosyl]-adenine (AB-MECA),
([IS-[1a,2b,3b,4a(S*)]]-4-[7-[[2-(3-chloro-2-thienyl)-1-methyl-propyl]ami-
no]-3H-imidazole[4,5-b]pyridyl-3-yl]cyclopentane carboxamide
(AMP579), N.sup.6--(R)-phenylisopropyladenosine (R-PLA),
aminophenylethyladenosine (APNEA) and cyclohexyladenosine (CHA).
Others include full adenosine A1 receptor agonists such as
N-[3-(R)-tetrahydrofurany]-6-aminopurine riboside (CVT-510), or
partial agonists such as CVT-2759 and allosteric enhancers such as
PD81723. Other agonists may include
N.sup.6-cyclopentyl-2-(3-phenylaminocarbonyltriazene-1-yl)
adenosine (TCPA), a very selective agonist with high affinity for
the human adenosine A1 receptor and allosteric enhancers of A1
adenosine receptor includes the 2-amino-3-napthoylthiophenes. Other
adenosine enhancers could be selected to have similar effect.
[0058] Some embodiments of the invention utilize alpha-adrenergic
receptor antagonists also known as alpha-adrenergic blocking
agents, alpha-blocking agents, or, more commonly, alpha-blockers.
Alpha-blockers include Methyldopa, Doxazosin, Clonidine,
Phenoxybenzamine (a nonselective alpha1/alpha2-adrenergic receptor
antagonist), Guanadrel, Terazosin, Prazosin, Guanfacine, Guanabenz,
Phentolamine and Reserpine. Importantly alpha-adrenergic
antagonists can also act as possible calcium channel inhibitors.
Adenosine, as mentioned above is also an antiadrenergic.
[0059] Some embodiments of the invention utilize myosin light chain
kinase inhibitors which can assist in relaxing smooth muscle. The
myosin light chain kinase inhibitor may be a microbial product
wortmannin, ML-7
(1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine.HCl),
ML-9, myosin Light Chain Kinase Inhibitor Peptide 18), calmodulin
antagonists (e.g W-7 and trifluoperazine), Rho kinase inhibitors
(eg HA1077 or fausdil) and botulotoxin (discussed elsewhere).
[0060] Some embodiments of the invention utilize phosphodiesterase
inhibitors (eg. papaverine, milrinone, theophylline, dipyridamole
(discussed elsewhere)
[0061] Some embodiments of the invention include nitric oxide
donors. Preferably, the NO donor is either 1) nitric-oxide synthase
independent (such as nitroprusside, nitroglycerine or glycerine
trinitrate (GTN), flurbiprofen or its NO-donating derivative,
HCT1026 (2-fluoro-a-methyl[1,1'-biphenyl]-4-acetic acid and
4-(nitrooxy)butyl ester) or 2) nitric-oxide synthase dependent
(such as L-arginine). In general all classes of nitric oxide donors
range from organic nitrates to nitroso compounds, guanidines and
metal-NO complexes. Thus NO donors can be those compounds, that
release NO or one of its redox congeners spontaneously and those
that require enzymatic metabolism to generate NO. Other examples of
NO donors include diazeniumdiolate, pentaerythritol tetranitrate,
polyalkyleneamine, tertiary and quaternary amino aliphatic NO donor
compounds, Also NO donors would include NO-related redox signalling
compounds to protect against oxidative stress.
[0062] Some embodiments of the invention utilise direct calcium
antagonists, the principal action of which is to reduce calcium
entry into the cell. These are selected from at least five major
classes of calcium channel blockers as explained in more detail
below. It will be appreciated that these calcium antagonists share
some effects with potassium channel openers, particularly
ATP-sensitive potassium channel openers, by inhibiting calcium
entry into the cell.
[0063] Calcium channel blockers are also called calcium antagonists
or calcium blockers. They are often used clinically to decrease
heart rate and contractility and relax blood vessels. They may be
used to treat high blood pressure, angina or discomfort caused by
ischaemia and some arrhythmias, and they share many effects with
beta-blockers, which could also be used to reduce calcium.
Beta-blockers (or beta-adrenergic blocking agents) include atenolol
(Tenormin.TM.), propranolol hydrochloride (such as Inderal.TM.),
esmolol hydrochloride (Brevibloc.TM.), metoprolol succinate (such
as Lopressor.TM. or Toprol XL.TM.), acebutolol hydrochloride
(Sectral.TM.), carteolol (such as Cartrol.TM.), penbutolol sulfate
(Levatol.TM.) and pindolol (Visken.TM.).
[0064] Five major classes of calcium channel blockers are known
with diverse chemical structures: 1. Benzothiazepines: eg
Diltiazem, 2. Dihydropyridines: eg nifedipine, Nicardipine,
nimodipine and many others, 3. Phenylalkylamines: eg Verapamil, 4.
Diarylaminopropylamine ethers: eg Bepridil, 5.
Benzimidazole-substituted tetralines: eg Mibefradil.
[0065] The traditional calcium channel blockers bind to L-type
calcium channels ("slow channels") which are abundant in cardiac
and smooth muscle which helps explain why these drugs have
selective effects on the cardiovascular system. Different classes
of L-type calcium channel blockers bind to different sites on the
alpha1-subunit, the major channel-forming subunit (alpha2, beta,
gamma, delta subunits are also present). Different sub-classes of
L-type channel are present which may contribute to tissue
selectivity. More recently, novel calcium channel blockers with
different specificities have also been developed for example,
Bepridil, is a drug with Na+ and K+ channel blocking activities in
addition to L-type calcium channel blocking activities. Another
example is Mibefradil, which has T-type calcium channel blocking
activity as well as L-type calcium channel blocking activity.
[0066] Three common calcium channel blockers are diltiazem
(Cardizem), verapamil (Calan) and Nifedipine (Procardia).
Nifedipine and related dihydropyridines do not have significant
direct effects on the atrioventricular conduction system or
sinoatrial node at normal doses, and therefore do not have direct
effects on conduction or automaticity. While other calcium channel
blockers do have negative chronotropic/dromotropic effects
(pacemaker activity/conduction velocity). For example, Verapamil
(and to a lesser extent diltiazem) decreases the rate of recovery
of the slow channel in AV conduction system and SA node, and
therefore act directly to depress SA node pacemaker activity and
slow conduction. These two drugs are frequency- and
voltage-dependent, making them more effective in cells that are
rapidly depolarizing. Verapamil is also contraindicated in
combination with beta-blockers due to the possibility of AV block
or severe depression of ventricular function. In addition,
mibefradil has negative chronotropic and dromotropic effects.
Calcium channel blockers (especially verapamil) may also be
particularly effective in treating unstable angina if underlying
mechanism involves vasospasm.
[0067] Omega conotoxin MVI IA (SNX-111) is an N type calcium
channel blocker and is reported to be 100-1000 fold more potent
than morphine as an analgesic but is not addictive. This conotoxin
is being investigated to treat intractable pain. SNX-482 a further
toxin from the venom of a carnivorous spider venom, blocks R-type
calcium channels. The compound is isolated from the venom of the
African tarantula, Hysterocrates gigas, and is the first R-type
calcium channel blocker described. The R-type calcium channel is
believed to play a role in the body's natural communication network
where it contributes to the regulation of brain function. Other
Calcium channel blockers from animal kingdom include Kurtoxin from
South African Scorpion, SNX-482 from African Tarantula, Taicatoxin
from the Australian Taipan snake, Agatoxin from the Funnel Web
Spider, Atracotoxin from the Blue Mountains Funnel Web Spider,
Conotoxin from the Marine Snail, HWTX-I from the Chinese bird
spider, Grammotoxin SIA from the South American Rose Tarantula.
This list also includes derivatives of these toxins that have a
calcium antagonistic effect.
[0068] Direct ATP-sensitive potassium channel openers (eg
nicorandil, aprikalem) or indirect ATP-sensitive potassium channel
openers (eg adenosine, opioids, levosimendan, glyceryl trinitrate)
are also indirect calcium antagonists and reduce calcium entry into
the tissue. One mechanism believed for ATP-sensitive potassium
channel openers also acting as calcium antagonists is shortening of
the cardiac action potential duration by accelerating phase 3
repolarisation and thus shortening the plateau phase. During the
plateau phase the net influx of calcium may be balanced by the
efflux of potassium through potassium channels. The enhanced phase
3 repolarisation may inhibit calcium entry into the cell by
blocking or inhibiting L-type calcium channels and prevent calcium
(and sodium) overload in the tissue cell.
[0069] Calcium channel blockers can be selected from nifedipine,
nicardipine, nimopidipine, nisoldipine, lercanidipine, telodipine,
angizem, altiazem, bepridil, amlopidine, felodipine, isradipine and
cavero and other racemic variations.
[0070] In a preferred form, the potassium channel opener or agonist
and/or an adenosine receptor agonist has a blood half-life of less
than one minute, preferably less than 20 seconds.
[0071] In some embodiments, the composition may include additional
potassium channel openers or agonists, for example diazoxide or
nicorandil.
[0072] The inventor has also found that the inclusion of diazoxide
with a potassium channel opener or adenosine receptor agonist and a
local anaesthetic reduces injury. Thus in another aspect, the
composition according to the invention further includes
diazoxide.
[0073] Diazoxide is a potassium channel opener and in the present
invention it is believed to preserve ion and volume regulation,
oxidative phosphorylation and mitochondrial membrane integrity
(appears concentration dependent). More recently, diazoxide has
been shown to provide cardioprotection by reducing mitochondrial
oxidant stress at reoxygenation. At present it is not known if the
protective effects of potassium channel openers are associated with
modulation of reactive oxygen species generation in mitochondria.
Preferably the concentration of the diazoxide is between about 1 to
200 uM. Typically this is as an effective amount of diazoxide. More
preferably, the concentration of diazoxide is about 10 uM.
[0074] The inventor has also found that the inclusion of nicorandil
with a potassium channel opener or adenosine receptor agonist and a
local anaesthetic reduces injury. Thus in another aspect, the
composition according to the invention further includes
nicorandil.
[0075] Nicorandil is a potassium channel opener and nitric oxide
donor which can protect tissues and the microvascular integrity
including endothelium from ischemia and reperfusion damage. Thus it
can exert benefits through the dual action of opening KATP channels
and a nitrate-like effect. Nicorandil can also reduce hypertension
by causing blood vessels to dilate which allows the heart to work
more easily by reducing both preload and afterload. It is also
believed to have anti-inflammatory and anti-proliferative
properties which can further attenuates ischemia/reperfusion
injury.
[0076] The composition according to the invention also includes an
antiarrhythmic agent. Antiarrhythmic agents are a group of
pharmaceuticals that are used to suppress fast rhythms of the heart
(cardiac arrhythmias). The following table indicates the
classification of these agents.
TABLE-US-00001 Repolarisation CLASS Channel effects Time Drug
Examples IA Sodium block Prolongs Quinidine, disopyramide, Procaine
IB Sodium block Shortens Lidocaine, phenytoin, mexiletine,
Tocainide IC Sodium block Unchanged Flecainide Propafenone,
moricizine II Phase IV Unchanged Beta-blockers (depolarising
including sotalol current); Calcium channel III Repolarising
Markedly prolongs Amiodarone, Potassium Sotalol, bretylium Currents
IVA AV nodal calcium Unchanged Verapamil, block diltiazem IVB
Potassium Unchanged Adenosine, ATP channel openers
[0077] It will also be appreciated that the antiarrhythmic agent
may induce local anaesthesia (or otherwise be a local anaesthetic),
for example, mexiletine, diphenylhydantoin, prilocalne, procaine,
mepivocaine, quinidine, disopyramide and Class 1B antiarrhythmic
agents.
[0078] Preferably, the antiarrhythmic agent is a class I or class
III agent. Amiodarone is a preferred Class III antiarrhythmic
agent. More preferably, the antiarrhythmic agent blocks sodium
channels. More preferably, the antiarrhythmic agent is a class IB
antiarrhythmic agent. Class 1B antiarrhythmic agents include
lignocaine or derivatives thereof, for example, QX-314.
[0079] Preferably the class 1B antiarrhythmic agent is Lignocaine.
In this specification, the terms "lidocaine" and "lignocaine" are
used interchangeably. Lignocaine is also known to be capable of
acting as a local anaesthetic probably by blocking sodium fast
channels, depressing metabolic function, lowering free cytosolic
calcium, protecting against enzyme release from cells, possibly
protecting endothelial cells and protecting against myofilament
damage. At lower therapeutic concentrations lignocaine normally has
little effect on atrial tissue, and therefore is ineffective in
treating atrial fibrillation, atrial flutter, and supraventricular
tachycardias. Lignocaine is also a free radical scavenger, an
antiarrhythmic and has anti-inflammatory and anti-hypercoagulable
properties. It must also be appreciated that at non-anaesthetic
therapeutic concentrations, local anaesthetics like lignocaine
would not completely block the voltage-dependent sodium fast
channels, but would down-regulate channel activity and reduce
sodium entry. As anti-arrhythmic, lignocaine is believed to target
small sodium currents that normally continue through phase 2 of the
action potential and consequently shortens the action potential and
the refractory period.
[0080] As lignocaine can act by blocking sodium fast channels, it
will be appreciated that other sodium channel blockers may be used
instead of or in combination with the local anaesthetic in the
method and composition of the present invention. It will also be
appreciated that sodium channel blockers include compounds that act
to substantially block sodium channels or at least downregulate
sodium channels. Examples of suitable sodium channel blockers
include venoms such as tetrodotoxin and the drugs primaquine, QX,
HNS-32 (CAS Registry # 186086-10-2), NS-7, kappa-opioid receptor
agonist U50 488, crobenetine, pilsicamide, phenyloin, tocamide,
mexiletine, NW-1029 (a benzylamino propanamide derivative),
RS100642, riluzole, carbamazepine, flecamide, propafenone,
amiodarone, sotalol, imipramine and moricizine, or any of
derivatives thereof. Other suitable sodium channel blockers
include: Vinpocetine (ethyl apovincaminate); and Beta-carboline
derivative, nootropic beta-carboline (ambocarb, AMB). In one
aspect, the composition according to the invention consists
essentially of (i) a potassium channel opener or agonist and/or an
adenosine receptor agonist; and (ii) a local anaesthetic.
[0081] In another aspect, the composition according to the
invention may further include an opioid. The further addition of an
opioid may have similar if not improved effect on the reduction of
injury.
[0082] Opioids, also known or referred to as opioid agonists, are a
group of drugs that inhibit opium (Gr opion, poppy juice) or
morphine-like properties and are generally used clinically as
moderate to strong analgesics, in particular, to manage pain, both
peri- and post-operatively. Other pharmacological effects of
opioids include drowsiness, respiratory depression, changes in mood
and mental clouding without loss of consciousness.
[0083] Opioids are also believed to be involved as part of the
`trigger` in the process of hibernation, a form of dormancy
characterised by a fall in normal metabolic rate and normal core
body temperature. In this hibernating state, tissues are better.
preserved against damage that may otherwise be caused by diminished
oxygen or metabolic fuel supply, and also protected from ischemia
reperfusion injury.
[0084] There are three types of opioid peptides: enkephalin,
endorphin and dynorphin. Opioids act as agonists, interacting with
stereospecific and saturable binding sites, in the heart, brain and
other tissues. Three main opioid receptors have been identified and
cloned, namely mu, kappa, and delta receptors. All three receptors
have consequently been classed in the G-protein coupled receptors
family (which class includes adenosine and bradykinin receptors).
Opioid receptors are further subtyped, for example, the delta
receptor has two subtypes, delta-1 and delta-2.
[0085] Cardiovascular effects of opioids are directed within the
intact body both centrally (ie, at the cardiovascular and
respiratory centres of the hypothalamus and brainstem) and
peripherally (ie, heart myocytes and both direct and indirect
effects on the vasculature). For example, opioids have been shown
to be involved in vasodilation. Some of the action of opioids on
the heart and cardiovascular system may involve direct opioid
receptor mediated actions or indirect, dose dependent non-opioid
receptor mediated actions, such as ion channel blockade which has
been observed with antiarrhythmic actions of opioids, such as
arylacetamide drugs. It is also known that the heart is capable of
synthesising or producing the three types of opioid peptides,
namely, enkephalin, endorphin and dynorphin. However, only the
delta and kappa opioid receptors have been identified on
ventricular myocytes.
[0086] Without being bound by any mode of action, opioids are
considered to provide cardioprotective effects, by limiting
ischemic damage and reducing the incidence of arrhythmias, which
are produced to counter-act high levels of damaging agents or
compounds naturally released during ischemia. This may be mediated
via the activation of ATP sensitive potassium channels in the
sarcolemma and in the mitochondrial membrane and involved in the
opening potassium channels. Further, it is also believed that the
cardioprotective effects of opioids are mediated via the activation
of ATP sensitive potassium channels in the sarcolemma and in the
mitochondrial membrane. Thus it is believed that the opioid can be
used instead or in combination with the potassium channel opener or
adenosine receptor agonist as they are also involved in indirectly
opening potassium channels.
[0087] It will be appreciated that the opioids include compounds
(natural or synthetic) which act both directly and indirectly on
opioid receptors. Opioids also include indirect dose dependent,
non-opioid receptor mediated actions such as ion channel blockade
which have been observed with the antiarrhythmic actions of
opioids.
[0088] Accordingly, the opioid may be selected from enkephalins,
endorphins and dynorphins. Preferably the opioid is an enkephalin
which targets delta, kappa and/or mu receptors. More preferably the
opioid is a delta opioid receptor agonist. Even more preferably the
opioid is selected from delta-1-opioid receptor agonists and
delta-2-opioid receptor agonists. [D-Pen 2, 5]enkephalin (DPDPE),
is a particularly preferred delta-1-opioid receptor agonist.
[0089] The inclusion of a compound for minimizing or reducing the
uptake of water by a cell in a tissue with a potassium channel
opener or adenosine receptor agonist and a local anaesthetic
assists in reducing injury to a body, such as a composition
comprising sucrose, adenosine and lignocaine. Sucrose reduces water
shifts as an impermeant. Impermeant agents such as sucrose,
lactobionate and raffinose are too large to enter the cells and
hence remain in the extracellular spaces within the tissue and
resulting osmotic forces prevent cell swelling that would otherwise
damage the tissue, which would occur particularly during storage of
the tissue.
[0090] Thus in a further aspect, the composition according to the
invention may further include at least one compound for minimizing
or reducing the uptake of water by a cell in the cell, tissue or
organ. Accordingly, these compounds are involved in the control or
regulation of osmosis. One consequence is that a compound for
minimizing or reducing the uptake of water by a cell in the tissue
reduces cell swelling that is associated with oedema, such as
oedema that can occur during ischemic injury.
[0091] Compounds for minimizing or reducing the uptake of water by
a cell in a tissue are typically impermeants or receptor
antagonists or agonists. An impermeant according to the present
invention may be selected from one or more of the group consisting
of: sucrose, pentastarch, hydroxyethyl starch, raffinose,
trehalose, mannitol, gluconate, lactobionate, and colloids.
Colloids include albumin, hetastarch, polyethylene glycol (PEG),
Dextran 40, Dextran 60, Dextran 30 or other sizes of dextrans.
Other compounds that could be selected for osmotic purposes include
those from the major classes of osmolytes found in the animal
kingdom including polyhydric alcohols (polyols) and sugars, other
amino acids and amino-acid derivatives, and methylated ammonium and
sulfonium compounds. Thus volume expanders may be colloid-based or
crystalloid-based.
[0092] Preferably, the concentration of the compound for minimizing
or reducing the uptake of water by the cells in the tissue is
between about 5 to 500 mM. Typically this is an effective amount
for reducing the uptake of water by the cells in the tissue. More
preferably, the concentration of the compound for reducing the
uptake of water by the cells in the tissue is between about 20 and
100 mM. Even more preferably the concentration of the compound for
reducing the uptake of water by the cells in the tissue is about 70
mM.
[0093] In a further embodiment, the composition according to the
invention may include more than one compound for minimizing or
reducing the uptake of water by the cells in the tissue. For
example, a combination of impermeants (raffinose, sucrose and
pentastarch) may be included in the composition or even a
combination of colloids, and fuel substrates may be included in the
composition.
[0094] The composition according to the invention may be hypo, iso
or hyper osmotic.
[0095] Cell swelling can also result from an inflammatory response
which may be important during organ retrieval, preservation and
surgical grafting. Substance P, an important pro-inflammatory
neuropeptide is known to lead to cell oedema and therefore
antagonists of substance P may reduce cell swelling. Indeed
antagonists of substance P, (-specific neurokinin-1) receptor
(NK-1) have been shown to reduce inflammatory liver damage, i.e.,
oedema formation, neutrophil infiltration, hepatocyte apoptosis,
and necrosis. Two such NK-1 antagonists include CP-96,345 or
R2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)-methyl)-1-azabicyclo[-
2.2.2.)-octan-3-amine (CP-96,345)] and L-733,060 or
[(2S,3S).sub.3-([3,5-bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidi-
ne]. R116301 or
[(2R-trans)-4-[1-[3,5-bis(trifluoromethyl)benzoyl]-2-(phenylmethyl)-4-pip-
eridinyl]-N-(2,6-dimethylphenyl)-1-acetamide
(S)-Hydroxybutanedioate] is another specific, active neurokinin-1
(NK(1)) receptor antagonist with subnanomolar affinity for the
human NK(1) receptor (K(i): 0.45 nM) and over 200-fold selectivity
toward NK(2) and NK(3) receptors. Antagonists of neurokinin
receptors 2 (NK-2) that may also reduce cell swelling include
SR48968 and NK-3 include SR142801 and SB-222200. Blockade of
mitochondrial permeability transition and reducing the membrane
potential of the inner mitochondrial membrane potential using
cyclosporin A has also been shown to decrease ischemia-induced cell
swelling in isolated brain slices. In addition glutamate-receptor
antagonists (AP5/CNQX) and reactive oxygen species scavengers
(ascorbate, Trolox(R), dimethylthiourea, tempol(R)) also showed
reduction of cell swelling. Thus, the compound for minimizing or
reducing the uptake of water by a cell in a tissue can also be
selected from any one of these compounds.
[0096] It will also be appreciated that the following energy
substrates can also act as impermeants. Suitable energy substrate
can be selected from one or more from the group consisting of:
glucose and other sugars, pyruvate, lactate, glutamate, glutamine,
aspartate, arginine, ectoine, taurine, N-acetyl-beta-lysine,
alanine, proline, beta-hydroxy butyrate and other amino acids and
amino acid derivatives, trehalose, floridoside, glycerol and other
polyhydric alcohols (polyols), sorbitol, myo-innositol, pinitol,
insulin, alpha-keto glutarate, malate, succinate, triglycerides and
derivatives, fatty acids and carnitine and derivatives. In one
embodiment, the at least one compound for minimizing or reducing
the uptake of water by the cells in the tissue is an energy
substrate. The energy substrate helps with recovering metabolism.
The energy substrate can be selected from one or more from the
group consisting of: glucose and other sugars, pyruvate, lactate,
glutamate, glutamine, aspartate, arginine, ectoine, taurine,
N-acetyl-beta-lysine, alanine, proline and other amino acids and
amino acid derivatives, trehalose, floridoside, glycerol and other
polyhydric alcohols (polyols), sorbitol, myo-innositol, pinitol,
insulin, alpha-keto glutarate, malate, succinate, triglycerides and
derivatives, fatty acids and carnitine and derivatives. Given that
energy substrates are sources of reducing equivalents for energy
transformations and the production of ATP in a cell, tissue or
organ of the body, it will be appreciated that a direct supply of
the energy reducing equivalents could be used as substrates for
energy production. For example, a supply of either one or more or
different ratios of reduced and oxidized forms of nicotinamide
adenine dinucleotide (e.g. NAD or NADP and NADH or NADPH) or flavin
adenine dinucleotides (FADH or FAD) could be directly used to
supply bond energy for sustaining ATP production in times of
stress. Preferably, beta-hydroxy butyrate is added to the
composition of the invention for protecting, or reducing injury to,
cells, a tissue or organ.
[0097] In addition to providing energy substrates to the whole
body, organ, tissue or cell, improvements in metabolising these
substrates may occur in the presence of hydrogen sulphide
(H.sub.2S) or H.sub.2S donors (eg NaHS). The presence of hydrogen
sulphide (H.sub.2S) or H.sub.2S donors (eg NaHS) may help
metabolise these energy substrates by lowering energy demand during
arrest, protect and preserve the whole body, organ, tissue or cell
during periods of metabolic imbalance such ischemia, reperfusion
and surgery. Concentrations of Hydrogen sulfide above 1 microM
(10.sup.-6 M) concentration can be a metabolic poison that inhibits
respiration at Respiratory Complex IV, which is part of the
mitochondrial respiratory chain that couples metabolising the high
energy reducing equivalents from energy substrates to energy (ATP)
generation and oxygen consumption. However, it has been observed at
lower concentrations, below 10.sup.-6 M (eg 10-.sup.10 to
10.sup.-9M), hydrogen sulfide may reduce the energy demand of the
whole body, organ, tissue or cell which may result in arrest,
protection and preservation. In other words, very low levels of
sulfide down-regulate mitochondria, reduce O.sub.2 consumption and
actually increase "Respiratory Control" whereby mitochondria
consume less O.sub.2 without collapsing the electrochemical
gradient across the inner mitochondrial membrane. Thus there are
observations that a small amount of sulfide, either directly or
indirectly, may close proton leak channels and better couple
mitochondrial respiration to ATP production more tightly, and this
effect may improve the metabolism of high energy reducing
equivalents from energy substrates. There is also the possibility
that a sulphur cycle exists between the cell cytosol and
mitochondria in mammals, including humans, providing the sulphur
concentration is low. The presence of a vestige sulphur cycle would
be consistent with current ideas on the evolutionary origin of
mitochondria and their appearance in eukaryote cells from a
symbiosis between a sulfide-producing host cell and a
sulfide-oxidizing bacterial symbiont. Thus, hydrogen sulphide
(H.sub.2S) or H.sub.2S donors (eg NaHS) may be energy substrates
themselves in addition to improving the metabolism of other energy
substrates. Accordingly, in one form, the invention provides a
composition as described above further including hydrogen sulphide
or a hydrogen sulfide donor.
[0098] The inventor has also found that the inclusion of a compound
for inhibiting transport of sodium and hydrogen ions across a
plasma membrane of a cell in the tissue with a potassium channel
opener or adenosine receptor agonist and a local anaesthetic
assists in reducing injury.
[0099] Thus in another aspect, the composition according to the
invention further includes a compound for inhibiting transport of
sodium and hydrogen ions across a plasma membrane of a cell in the
tissue.
[0100] The compound for inhibiting transport of sodium and hydrogen
across the membrane of the cell in the tissue is also referred to
as a sodium hydrogen exchange inhibitor. The sodium hydrogen
exchange inhibitor reduces sodium and calcium entering the
cell.
[0101] Preferably the compound for inhibiting transport of sodium
and hydrogen across the membrane of the cell in the tissue may be
selected from one or more of the group consisting of Amiloride,
EIPA(5-(N-entyl-N-isopropyl)-amiloride), cariporide (HOE-642),
eniporide, Triamterene (2,4,7-triamino-6-phenylteride), EMD 84021,
EMD 94309, EMD 96785, EMD 85131, HOE 694. B11 B-513 and T-162559
are other inhibitors of the isoform 1 of the Na.sup.+/H.sup.+
exchanger.
[0102] Preferably, the sodium hydrogen exchange inhibitor is
Amiloride (N-amidino-3,5-diamino-6-chloropyrzine-2-carboximide
hydrochloride dihydrate). Amiloride inhibits the sodium proton
exchanger (Na.sup.+/H.sup.+ exchanger also often abbreviated NHE-1)
and reduces calcium entering the cell. During ischemia excess cell
protons (or hydrogen ions) are believed to be exchanged for sodium
via the Na.sup.+/H.sup.+ exchanger.
[0103] Preferably, the concentration of the compound for inhibiting
transport of sodium and hydrogen across the membrane of the cell in
the tissue is between about 1.0 nM to 1.0 mM. More preferably, the
concentration of the compound for inhibiting transport of sodium
and hydrogen across the membrane of the cell in the tissue is about
20 uM.
[0104] The inventor has also found that the inclusion of
antioxidant with a potassium channel opener or adenosine receptor
agonist and a local anaesthetic. Thus in another aspect, the
composition of the present invention may further include an
antioxidant.
[0105] Antioxidants are commonly enzymes or other organic
substances that are capable of counteracting the damaging effects
of oxidation in the tissue. The antioxidant component of the
composition according to the present invention may be selected from
one or more of the group consisting of: allopurinol, carnosine,
histidine, Coenzyme Q 10, n-acetyl-cysteine, superoxide dismutase
(SOD), glutathione reductase (GR), glutathione peroxidase (GP)
modulators and regulators, catalase and the other metalloenzymes,
NADPH and AND(P)H oxidase inhibitors, glutathione, U-74006F,
vitamin E, Trolox (soluble form of vitamin E), other tocopherols
(gamma and alpha, beta, delta), tocotrienols, ascorbic acid,
Vitamin C, Beta-Carotene (plant form of vitamin A), selenium, Gamma
Linoleic Acid (GLA), alpha-lipoic acid, uric acid (urate),
curcumin, bilirubin, proanthocyanidins, epigallocatechin gallate,
Lutein, lycopene, bioflavonoids, polyphenols, trolox(R),
dimethylthiourea, tempol(R), carotenoids, coenzyme Q, melatonin,
flavonoids, polyphenols, aminoindoles, probucol and nitecapone,
21-aminosteroids or lazaroids, sulphydryl-containing compounds
(thiazolidine, Ebselen, dithiolethiones), and N-acetylcysteine.
Other antioxidants include the ACE inhibitors (captopril,
enalapril, lisinopril) which are used for the treatment of arterial
hypertension and cardiac failure on patients with myocardial
infarction. ACE inhibitors exert their beneficial effects on the
reoxygenated myocardium by scavenging reactive oxygen species.
Other antioxidants that could also be used include
beta-mercaptopropionylglycine, O-phenanthroline, dithiocarbamate,
selegilize and desferrioxamine (Desferal), an iron chelator, has
been used in experimental infarction models, where it exerted some
level of antioxidant protection. Spin trapping agents such as
5'-5-dimethyl-1-pyrrolione-N-oxide (DMPO) and
(a-4-pyridyl-1-oxide)-N-t-butylnitrone (POBN) also act as
antioxidants. Other antioxidants include: nitrone radical scavenger
alpha-phenyl-tert-N-butyl nitrone (PBN) and derivatives PBN
(including disulphur derivatives); N-2-mercaptopropionyl glycine
(MPG) a specific scavenger of the OH free radical; lipooxygenase
inhibitor nordihydroguaretic acid (NDGA); Alpha Lipoic Acid;
Chondroitin Sulfate; L-Cysteine; oxypurinol and Zinc.
[0106] Preferably, the antioxidant is allopurinol
(1H-Pyrazolo[3,4-a]pyrimidine-4-ol). Allopurinol is a competitive
inhibitor of the reactive oxygen species generating enzyme xanthine
oxidase. Allopurinol's antioxidative properties may help preserve
myocardial and endothelial functions by reducing oxidative stress,
mitochondrial damage, apoptosis and cell death. Preferably, the
concentration of the antioxidant is between about 1 nM to 100
uM.
[0107] The inventor has also found that the inclusion of particular
amounts of calcium and magnesium ions with a potassium channel
opener or adenosine receptor agonist and a local anaesthetic
reduces injury. The effect of the particular amounts of calcium and
magnesium ions is to control the amount of ions within the
intracellular environment. Calcium ions tend to be depleted,
exported or otherwise removed from the intracellular environment
and magnesium ions tend to be increased or otherwise restored to
the levels typically found in a viable, functioning cell.
[0108] Thus in another aspect, the composition according to the
invention further includes a source of magnesium in an amount for
increasing the amount of magnesium in a cell in body tissue.
Preferably the magnesium is present at a concentration of between
0.5 mM to 20 mM, more preferably about 2.5 mM.
[0109] In addition, typical buffers or carriers (which are
discussed in more detail below) in which the composition of the
invention is administered typically contain calcium at
concentrations of around 1 mM as the total absence of calcium has
been found to be detrimental to the cell, tissue or organ. In one
form, the invention also includes using carriers with low calcium
(such as for example less than 0.5 mM) so as to decrease the amount
of calcium within a cell in body tissue, which may otherwise build
up during surgery or storage of cells, a tissue or organ. As
described in the present invention, elevated magnesium and low
calcium has been associated with protection during ischemia and
reoxygenation of an organ. The action is believed to be due to
decreased calcium loading. Preferably the calcium present is at a
concentration of between 0.1 mM to 0.8 mM, more preferably about
0.3 mM.
[0110] In one embodiment, the composition includes elevated
magnesium ions. Magnesium sulphate and magnesium chloride is a
suitable source. In another embodiment, the composition includes a
cellular transport enzyme inhibitor, such as dipyridamole, to
prevent metabolism or breakdown of components in the
composition.
[0111] In a further aspect, the invention provides a composition
including an antiarrhythmic agent and one or more of: [0112]
potassium channel opener; [0113] nitric oxide donor [0114] opioid;
[0115] at least one compound for reducing uptake of water; [0116]
sodium hydrogen exchange inhibitor; [0117] antioxidant; and [0118]
a source of magnesium in an amount for increasing the amount of
magnesium in a cell in body tissue.
[0119] The processes of inflammation and thrombosis are linked
through common mechanisms. Therefore, it is believed that
understanding of the processes of inflammation will help with
better management of thrombotic disorders including the treatment
of acute and chronic ischaemic syndromes. In the clinical and
surgical settings, a rapid response and early intervention to an
organ or tissue damaged from ischemia can involve both
anti-inflammatory and anti-clotting therapies. In addition to
protease inhibitors which attenuate the inflammatory response,
further anti-inflammatory therapies have included the
administration of aspirin, normal heparin, low-molecular-weight
heparin (LMWH), non-steroidal anti-inflammatory agents,
anti-platelet drugs and glycoprotein (GP) IIb/IIIa receptor
inhibitors, statins, angiotensin converting enzyme (ACE) inhibitor,
angiotensin blockers and antagonists of substance P. Examples of
protease inhibitors are indinavir, nelfinavir, ritonavir,
lopinavir, amprenavir or the broad-spectrum protease inhibitor
aprotinin, a low-molecular-weight heparin (LMWH) is enoxaparin,
non-steroidal anti-inflammatory agent are indomethacin, ibuprofen,
rofecoxib, naproxen or fluoxetine, an anti-platelet drug is
Clopidogrel or aspirin, a glycoprotein (GP) IIb/IIIa receptor
inhibitor is abciximab, a statin is pravastatin, an angiotensin
converting enzyme (ACE) inhibitor is captopril and an angiotensin
blocker is valsartin.
[0120] Accordingly, in another embodiment of the invention, a
selection of these agents is added to a composition according to
the invention to deliver improved management of inflammation and
clotting. Alternatively, the composition according to the invention
may be administered together with any one or more of these
agents.
[0121] In particular, protease inhibitors attenuate the systemic
inflammatory response in patients undergoing cardiac surgery with
cardiopulmonary bypass, and other patients where the inflammatory
response has been heightened such as AIDS or in the treatment of
chronic tendon injuries. Some broad spectrum protease inhibitors
such as aprotinin are also reduce blood loss and need for blood
transfusions in surgical operations such as coronary bypass.
[0122] Compounds that substantially prevent the breakdown of
adenosine in the blood such as nucleoside transport inhibitors,
such as dipyridamole could be are used as additives in the
composition of the invention. The half life of adenosine in the
blood is about 10 seconds so the presence of a medicament to
substantially prevent its breakdown will maximise the effect of the
composition of the present invention.
[0123] Dipyridamole is advantageously included in a concentration
from about 0.01 microM to about 10 mM, preferably 0.05 to 100
microM, and has major advantages with respect to cardioprotection.
Dipyridamole may supplement the actions of adenosine by inhibiting
adenosine transport and breakdown leading to increased protection
of cells, tissues and organs of the body during times of stress.
Dipyridamole may also be administered separately for example by 400
mg daily tablets to produce a plasma level of about 0.4
microgram/ml, or 0.8 microM concentration. Other antiproliferative
drugs which may optionally be included are paclitaxel (0-100
microg/mL), and tranilast (0-300 microg/mL).
[0124] In some embodiments, the composition may further include
toxins. These may include the conotoxins referred to above (such as
the N type calcium channel blockers) and toxins such as Botulinum
toxin ("botox") which promote smooth muscle relaxation. Botulinum
toxin type A is a neurotoxin protein complex produced by the
bacteria (clostridium botulinum) that can cause food poisoning
known as botulism. There are seven or more known types of C.
Botulinum toxin, but only types A (BOTOX.RTM. Cosmetic) and B
(Myobloc.RTM.) are used as medical treatments. The type A toxin
affects the nerves and when injected in small amounts into a
muscle, the muscle relaxes and reduces its metabolic activity. The
toxin is injected at multiple sites to ensure complete dispersal of
toxin through the target regions. Normally, multiple 0.1 ml
therapeutic effective injections containing 5 to 20 Upper injection
site are used for treatments with a total dose per patient not
normally exceeding about 100-150 U. At higher concentrations (above
20 U), the toxin has also been shown to directly inhibit smooth
muscle contractility as evidenced by the decreased contractile
response to ACh. Thus doses of the Botulinum toxin type A can be
between about 0.01 U/kg and about 35 U/kg. Above 35 U/kg is
approaching the toxic dose, and the lethal human dose is about
200-300 pg/kg. Botulinum toxin type A is currently used to treat
dystonia. In one form of the invention, the composition further
includes Botulinum toxin type A, preferably at 1 to 35 U/ml.
Botulinum toxin type B (NeuroBloc) may alternatively be used.
[0125] In another aspect of the invention, antibiotics such as
vancomycin, cefotaxime, and gentamicin are present in the graft
solution to minimise transmission of infection, such as during
surgical attachment and therefore loss of patency. Also, the
composition of the invention may further include cryoprotective
glycerol, trehalose, high glucose concentrations (above 200 mM) or
other additives that inhibit the intracellular water from freezing
and damaging or fracturing the cell membrane. This permits the
grafts to be stored below freezing until use.
[0126] The composition according to the present invention is highly
beneficial at about 10.degree. C. but can also be used to prevent
injury over a wider temperature range up to about 37.degree. C. The
composition according to the invention may be used at a temperature
range selected from the following: 0.degree. C. to 5.degree. C.,
5.degree. C. to 20.degree. C., 20.degree. C. to 32.degree. C. and
32.degree. C. to 38.degree. C.
[0127] The composition may be administered intravenously or be
administered both intravenously and intraperitoneally or in special
circumstances directly accessing a major artery such as the femoral
artery or aorta, for example in patients who have no pulse. In one
embodiment, the composition of the invention may be administered
intravenously and intraperineally simultaneously, the perineum
acting as, in effect, a reservoir of composition for the
bloodstream as well as acting on organs in the vicinity with which
it comes into contact.
[0128] As described herein, in particular embodiments of the
invention, the composition of the present invention protects and
preserves tissue of a body with good to excellent recoveries of
function or viability of body tissue after reperfusion. Affecting
viability of a tissue during preservation and recovery of the body
tissue, such that affected tissue remains viable or living during
those processes and is capable of returning to its function,
particularly after the tissue has been subject to shock, is
important to patient welfare.
[0129] Preferably, the invention reduces injury to affected tissue,
such that the tissue is capable of returning to its function.
Maintaining or stabilising the tissue in a viable state includes
maintaining the membrane potential of tissue cells at or around
resting level, so as to reduce sodium or calcium loading of the
cell which is a cause of injury during ischaemia and reperfusion.
Preservation is known as the act or process of preserving the
tissue or keeping from injury, destruction or decay. In this
application, the composition according to the invention acts to
minimise any potential injury, destruction or decay of cells, a
tissue or organ, particularly during surgery, especially surgery
involving excision of tissue and its implantation or grafting.
[0130] The composition of the present invention is particularly
useful in reducing injury to heart tissue during heart surgery
(open-heart or robotic heart surgery), including heart transplants,
and neonate/infant hearts. Other applications include reducing
heart damage before, during or following cardiovascular
intervention which may include a heart attack, angioplasty or
angiography. For example, the composition may be administered to
subjects who have suffered or are developing a heart attack and
used at the time of administration of blood clot-busting drugs such
as streptokinase. As the clot is dissolved, the presence of the
composition may protect the heart from further injury such as
reperfusion injury. The composition may be particularly effective
as a cardioprotectant in those portions of the heart that have been
starved of normal flow, nutrients and/or oxygen for different
periods of time. For example, the pharmaceutical composition may
also be used to treat heart ischaemia which could be pre-existing
or induced by cardiovascular intervention.
[0131] Accordingly, in another embodiment of the invention, there
is provided a method of preserving cells, a tissue or organ of the
body, such as a blood vessel, comprising administering a
composition as described above. The composition may be administered
prior to medical intervention affecting the cells, tissue or organ
as well as, or alternatively following, any such medical
intervention. Indeed, the invention is desirably used before,
during and after the procedure so that a fluid of common
composition is used throughout to minimise stress on and/or injury
to the explanted tissue. The composition used in this embodiment of
the invention may have an arresting or a non-arresting
concentration of active components in it. In one form, the method
includes administering a non-arresting concentration of the
composition and, in another form, it has an arresting concentration
of the composition (preferably as a bolus) followed by a
non-arresting concentration of the composition.
[0132] In another embodiment, the present invention may be
administered with or contain blood or blood products or artificial
blood or oxygen binding molecules or solutions to improve the
body's oxygen transport ability and survival by helping to reduce
hypoxic and ischemic damage from blood loss. The oxygen-containing
molecules, compounds or solutions may be selected from natural or
artificial products. For example, an artificial blood-based product
is perfluorocarbon-based or other haemoglobin-based substitute.
Some of the components may be added to mimic human blood's oxygen
transport ability such Hemopure.TM., Gelenpol.TM., Oxygent.TM., and
PolyHeme.TM.. Hemopore is based on a chemically stabilized bovine
hemoglobin. Gelenpol is a polymerized hemoglobin which comprises
synthetic water-soluble polymers and modified heme proteins.
Oxygent is a perflubron emulsion for use as an intravenous oxygen
carrier to temporarily substitute for red blood cells during
surgery. Polyheme is a human hemoglobin-based solution for the
treatment of life-threatening blood loss.
[0133] It is believed that the oxygenation of the body from a
variety of ways including but not limited to oxygen gas mixture,
blood, blood products or artificial blood or oxygen binding
solutions maintains mitochondrial oxidation and this helps preserve
the myocyte and endothelium of the organ. Without being bound by
any particular mode or theory, the inventor has found that gentle
bubbling with 95% O.sub.2/5% CO.sub.2 helps maintains mitochondrial
oxidation which helps preserve the myocyte and coronary
vasculature.
[0134] In one preferred embodiment of this aspect of the present
invention with respect to whole body or organs outside the body,
the composition is aerated with a source of oxygen before and/or
during use. The source of oxygen may be an oxygen gas mixture where
oxygen is the predominant component. The oxygen may be mixed with,
for example, CO.sub.2. Preferably, the oxygen gas mixture is 95%
O.sub.2 and 5% CO.sub.2.
[0135] In another aspect of the present invention there is provided
a method for protecting cells, a tissue or organ, preferably a
blood vessel for implantation, including: [0136] providing in a
suitable container a composition according to the invention; [0137]
providing one or more nutrient molecules selected from the group
consisting of blood, blood products, artificial blood and a source
of oxygen; [0138] optionally aerating the composition with the
oxygen (for example, in the case of isolated organs) or combining
the nutrient molecules with the composition, or both; and [0139]
placing the tissue in contact with the combined composition under
conditions sufficient to reduce injury.
[0140] Preferably the oxygen source is an oxygen gas mixture.
Preferably oxygen is the predominant component. The oxygen may be
mixed with, for example CO.sub.2. More preferably, the oxygen gas
mixture is 95% O.sub.2 and 5% CO.sub.2. Preferably the composition
is aerated before and/or during contact with the tissue.
[0141] The composition according to this aspect of the invention
may be in liquid form. Liquid preparations of the pharmaceutical
composition may take the form of, for example, solutions, syrups,
or suspensions, or may be presented as a dry product for
constitution with water or other suitable vehicle. Such liquid
preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents,
emulsifying agents, non-aqueous vehicles, preservatives and energy
sources. In another form, the invention comprises a composition in
tablet form and in another form, the invention comprises an aerosol
which could be administered via oral, skin or nasal routes.
[0142] In another aspect of the invention, there is provided a
method of protecting vasculature tissue from reperfusion injury,
including inflammatory and blood clotting and coagulation effects
often experienced during reperfusion following an ischaemic event.
The method comprises administering a solution comprising a
non-arresting form of the composition according to the present
invention, optionally following a bolus of an arresting form.
[0143] The body may be a human or an animal such as a livestock
animal (eg, sheep, cow or horse), laboratory test animal (eg,
mouse, rabbit or guinea pig) or a companion animal (eg, dog or
cat), particularly an animal of economic importance. Preferably,
the body is human.
[0144] The method of the present invention involves contacting a
tissue with the composition according to the invention, for a time
and under conditions sufficient for the tissue to be
preconditioned, arrested, protected and/or preserved. The
composition may be infused or administered as a bolus intravenous,
intracoronary or any other suitable delivery route as pre-treatment
for protection during a cardiac intervention such as open heart
surgery (on-pump and off-pump), angioplasty (balloon and with
stents or other vessel devices) and with clot-busters
(anti-clotting drug or agents).
[0145] The composition may be administered intravenously or be
administered both intravenously and intraperitoneally or in special
circumstances directly accessing a major artery such as the femoral
artery or aorta or in the carotid artery or another artery during
aortic dissection to protect the brain from hypoxia or ischemia. In
one embodiment, the composition of the invention may be
administered intravenously and intraperineally simultaneously, the
perineum acting as, in effect, a reservoir of composition for the
bloodstream as well as acting on organs in the vicinity with which
it comes into contact. Moreover, where the composition contains two
or more components, these may be administered separately but
simultaneously. Substantially simultaneous delivery of the
component to the target site is desirable. This may be achieved by
pre-mixing the components for administration as one composition,
but that is not essential. The invention is directed towards the
simultaneous increase in local concentration (for example an organ
such as the heart) of the components of a composition according to
the invention (for example, where a first component is (i) a
potassium channel opener or agonist and/or an adenosine receptor
agonist; and (ii) a local anaesthetic). One preferred form of the
composition is a combination of adenosine and lignocaine.
[0146] Accordingly, the tissue may be contacted by delivering the
composition according to the invention intravenously to the tissue.
This involves using blood as a vehicle for delivery to the tissue.
In particular, the composition according to the invention may be
used for blood cardioplegia. Alternatively, the composition may be
administered directly as a bolus by a puncture (eg, by syringe)
directly to the tissue or organ, particularly useful when blood
flow to a tissue or organ is limiting. The composition for
arresting, protecting and preserving a tissue may also be
administered as an aerosol, powder, solution or paste via oral,
skin or nasal routes.
[0147] Alternatively, the composition may be administered directly
to the tissue, organ or cell or to exposed parts of the internal
body to reduce injury. In particular, the composition according to
the invention may be used for crystalloid cardioplegia.
[0148] The composition according to the invention may be delivered
according to one of or a combination of the following delivery
protocols: intermittent, continuous and one-shot.
[0149] Accordingly, in another aspect of the invention, there is
provided a composition for arresting, protecting and preserving a
cell, tissue or organ of a body upon administration of a single
dose of the composition, the composition including a primary
potassium channel opener or agonist and/or adenosine receptor
agonist and a local anaesthetic. The invention also provides a
method for arresting and protecting an tissue comprising
administering as a single dose an effective amount of that
composition.
[0150] In another aspect of the invention, there is provided a
composition for arresting, protecting and preserving a tissue by
intermittent administration of the composition, the composition
including an effective amount of a primary potassium channel opener
or agonist and/or adenosine receptor agonist and a local
anaesthetic. A suitable administration schedule is a 2 minute
induction dose every 20 minutes throughout the period. The actual
time periods can be adjusted based on observations by one skilled
in the art administering the composition, and the animal/human
model selected. The invention also provides a method for
intermittently administering a composition for arresting,
protecting and preserving a tissue.
[0151] The composition can of course also be used in continuous
infusion with both normal and injured tissues or organs, such as
heart tissue. Continuous infusion also includes static storage of
the tissue, whereby the tissue is stored in a composition according
to the invention, for example the tissue may be placed in a
suitable container and immersed in a solution according to the
invention for transporting donor tissues from a donor to
recipient.
[0152] The dose and time intervals for each delivery protocol may
be designed accordingly. For example, a composition according to
the invention may be delivered as a one-shot to the tissue to
initially arrest of the tissue. A further composition according to
the invention may then be administered continuously to maintain the
tissue in an arrested state. Yet a further composition according to
the invention may be administered continuously to reperfuse the
tissue or recover normal function.
[0153] As mentioned previously, the composition according to the
invention may be used or contact the tissue at a temperature range
selected from one of the following: from about 0.degree. C. to
about 5.degree. C., from about 5.degree. C. to about 20.degree. C.,
from about 20.degree. C. to about 32.degree. C. and from about
32.degree. C. to about 38.degree. C. It is understood that
"profound hypothermia" is used to describe a tissue at a
temperature from about 0.degree. C. to about 5.degree. C. "Moderate
hypothermia" is used to describe a tissue at a temperature from
about 5.degree. C. to about 20.degree. C. "Mild hypothermia" is
used to describe a tissue at a temperature from about 20.degree. C.
to about 32.degree. C. "Normothermia" is used to describe a tissue
at a temperature from about 32.degree. C. to about 38.degree. C.,
though the normal body temperature is around 37 to 38.degree.
C.
[0154] While it is possible for each component of the composition
to contact the tissue alone, it is preferable that the components
of the pharmaceutical composition be provided together with one or
more pharmaceutically acceptable carriers, diluents, adjuvants
and/or excipients. Each carrier, diluent, adjuvant and/or excipient
must be pharmaceutically acceptable such that they are compatible
with the components of the pharmaceutical composition and not
harmful to the subject. Preferably, the pharmaceutical composition
is prepared with liquid carriers, diluents, adjuvants and/or
excipients.
[0155] The composition according to the invention may be suitable
for administration to the tissue in liquid form, for example,
solutions, syrups or suspensions, or alternatively they may be
administered as a dry product for constitution with water or other
suitable vehicle before use. Such liquid preparations may be
prepared by conventional means.
[0156] The composition according to the invention may be suitable
for topical administration to the tissue. Such preparation may be
prepared by conventional means in the form of a cream, ointment,
jelly, solution or suspension.
[0157] The composition may also be formulated as depot
preparations. Such long acting formulations may be administered by
implantation (eg, subcutaneously or intramuscularly) or by
intramuscular injection. Thus, for example, the composition
according to the invention may be formulated with suitable
polymeric or hydrophobic materials (eg, as an emulsion in an
acceptable oil or ion exchange resins, or as sparingly soluble
derivatives, for example, as a sparingly soluble salt.
[0158] Accordingly, this aspect of the invention also provides a
method for reducing injury, which includes providing the
composition together with a pharmaceutically acceptable carrier,
diluent, adjuvant and/or excipient. A preferred pharmaceutically
acceptable carrier is a buffer having a pH of about 6 to about 9,
preferably about 7, more preferably about 7.4 and/or low
concentrations of potassium. For example, the composition has a
total potassium concentration of up to about 10 mM, more preferably
about 2 to about 8 mM, most preferably about 4 to about 6 mM.
Suitable buffers include Krebs-Henseleit which generally contains
10 mM glucose, 117 mM NaCl, 5.9 mM KCl, 25 mM NaHCO.sub.3, 1.2 mM
NaH.sub.2PO.sub.4, 1.12 mMCaCl.sub.2 (free Ca.sup.2+=1.07 mM) and
0.512 mM MgCl.sub.2 (free Mg.sup.2+=0.5 mM), Tyrodes solution which
generally contains 10 mM glucose, 126 mM NaCl, 5.4 mM KCl, 1 mM
CaCl.sub.2, 1 mM MgCl.sub.2, 0.33 mM NaH.sub.2PO.sub.4 and 10 mM
HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethane sulphonic acid],
Fremes solution, Hartmanns solution which generally contains 129
NaCl, 5 mM KCl, 2 mM CaCl.sub.2 and 29 mM lactate and
Ringers-Lactate. Other naturally occurring buffering compounds that
exist in muscle that could be also used in a suitable ionic
environment are carnosine, histidine, anserine, ophidine and
balenene, or their derivatives. One advantage of using low
potassium is that it renders the present composition less injurious
to the subject, in particular paediatric subjects such as
neonates/infants. High potassium has been linked to an accumulation
of calcium which may be associated with irregular heart beats
during recovery, heart damage and cell swelling. Neonates/infants
are even more susceptible than adults to high potassium damage
during cardiac arrest. After surgery a neonate/infant's heart may
not return to normal for many days, sometimes requiring intensive
therapy or life support.
[0159] It is also advantageous to use carriers having low
concentrations of magnesium, such as, for example up to about 2.5
mM, but it will be appreciated that high concentrations of
magnesium, for example up to about 20 mM, may be used if desired
without substantially affecting the activity of the
composition.
[0160] In another embodiment of the present invention there is
provided use of a composition according to the present invention
for reducing injury.
[0161] In the figures:
[0162] FIG. 1. Graph showing the effect of increasing the
concentrations of adenosine on the tension of intact and denuded
rat aortic rings precontracted with norepinephrine. The A
(Adenosine) concentrations comprised 10, 50, 100, 200, 300, 400 and
500 uM (final concentrations), shown in log concentrations on the X
axis.
[0163] FIG. 2. Graph showing the effect of increasing the
concentrations of lidocaine on the tension of intact and denuded
rat aortic rings precontracted with norepinephrine. The L
(Lidocaine) concentrations comprised 10, 50, 100, 200, 300, 400 and
500 uM (final concentrations), shown in log concentration on the X
axis.
[0164] FIG. 3. Graph showing the effect of increasing the
concentrations of adenosine and lidocaine (AL) on the tension of
intact and denuded rat aortic rings precontracted with
norepinephrine. The AL concentrations comprised 10 uM AL (10 uM A
and 10 uM L), 10 uM AL (10 uM A and 10 uM L), 50 uM AL (50 uM A and
50 uM L), 100 uM AL (100 uM A and 100 uM L), 200 uM AL (200 uM A
and 200 uM L), 300 uM AL (300 uM A and 300 uM L), 400 uM AL (400 uM
A and 400 uM L), and 500 uM AL (50 uM A and 500 uM L), shown in log
concentration on the X axis.
EXAMPLES
[0165] The following are provided as non-limiting examples of the
invention for the purpose of illustrating the invention.
Example 1
Effect of Adenosine, Lidocaine and Adenosine Plus Lidocaine on Rat
Aorta Muscle Tension and Relaxation
[0166] This example illustrates the different effect on intact
isolated vasculature rings of an adenosine-lidocaine solution
according to the invention, which did not lead to relaxation by
over 5% until 200 uM. Adenosine and AL (adenosine and lidocaine)
show similar effects, and about 30% greater falls in relaxation
than lidocaine alone at 400 and 500 uM concentrations when bathed
in 10 mM glucose in Krebs-Henseleit at pH 7.4 37.degree. C. under
aerobic conditions (95% O.sub.2 & 5% CO.sub.2). A major
difference between AL and adenosine alone is that the AL-induced
relaxation profile is not dependent on an intact endothelium. The
effect of AL in the denuded rings is as if the endothelium is not
removed.
[0167] Animal Preparation: Male Sprague Dawley rats (300-350 g)
were fed ad libitum and housed in a 12-hour light/dark cycle. On
the day of the experiment rats were anaesthetized using CO.sub.2
anaesthesia which has been shown to have less effect than
pentobarbital to alter the vascular synthesis of prostacyclin and
smooth muscle contractility which could interfere with the results
(Butler M M et al Lab Anim Sci. 1990 40 277-83). Animals were
treated in accordance with the Guide for the Care and Use of
Laboratory Animals published by the US national Institutes of
Health (NIH Publication No. 85-23, revised 1996). Lignocaine
hydrochloride was sourced as a 2% solution (ilium) from the local
Pharmaceutical Suppliers (Lyppard, Queensland). All other
chemicals, including adenosine (A9251 >99% purity), were
purchased from Sigma Aldrich (Castle Hill, NSW).
[0168] Aortic ring preparation and organ bath tension measurements:
The abdominal and thoracic cavity of anaesthetised rats were opened
and the thoracic aorta was removed and placed in a cold solution of
Krebs Henseleit (117 mM NaCl, 5.9 mM KCl, 1.2 mM Na.sub.2PO.sub.4,
0.5 mM MgCl.sub.2, 1.12 mM CaCl.sub.2, 25 mM NaHCO.sub.3) pH 7.4
with 10 mM glucose.
[0169] The aorta was carefully dissected from surrounding fat and
connective tissue and cut into short transverse segments. Aortic
rings (about 3 mm wide) were equilibrated in a standard 20 ml
volume organ bath containing Krebs Henseleit (117 mM NaCl, 5.9 mM
KCl, 1.2 mM Na.sub.2PO.sub.4, 0.5 mM MgCl.sub.2, 1.12 mM
CaCl.sub.2, 25 mM NaHCO.sub.3) pH 7.4 with 10 mM glucose and
continuously bubbled with 95% O.sub.2 and 5% CO.sub.2 at 37.degree.
C. for 1 hour (zero tension). The rings were vertically mounted on
small stainless steel stirrups and connected to an isometric force
transducer coupled to a MacLab and computer. The ring tension was
manually adjusted to 1.4 g and the rings allowed to equilibrate for
30 min. The aortic rings were then washed with freshly prepared
Krebs Henseleit buffer pH 7.4 containing 10 mM glucose and the
tension was readjusted to 1.4 g tension. Each preparation was
contracted submaximally using 60 ul of 0.1 mM Noradrenalin (0.3 uM
final concentration) (Zerkowski H R et al, 1993, Evans G R et al
1997) and those rings that failed to contract were discarded. After
stabilisation, 20 ul of 10 mM acetylcholine (10 uM final
concentration) was applied to confirm the presence or absence of an
intact endothelium in all preparations. Acetylcholine will induce
rapid relaxation of precontracted rings if the endothelium is
intact, and will have little or no effect if the endothelium is
damaged (or denuded) and the rings will remain in contracted state
(Furchgott, R F et al Nature 1980, Nagao, T et al AJP 1992). Aortic
rings were denuded by gently rubbing the intimal surface of the
vessel segment with a smooth metal probe. After noradrenalin and
acetylcholine additions, the rings were washed three times. The
aortic rings were allowed to stabilize for 20 min and the tension
adjusted to 1.4 g. Noradrenalin (0.3 uM) or in some cases
depolarising potassium chloride (65 mM) was then added and the
experiment commenced after stabilisation of tension (10-15 min).
Adenosine, lidocaine or adenosine and lidocaine (AL) were added to
the bath in a concentration-dependent manner and the change in
tension of precontracted rings was assessed. Preliminary
experiments showed that noradrenalin (0.3 uM) increased tension and
plateaued after 10 min and remained at this level over the 60 min,
the time course of each experiment. Adenosine alone and lidocaine
alone concentration-response curves for intact and denuded aortic
rings were obtained by adding 10, 50, 100, 200, 300, 400 and 500 uM
(final concentrations). The AL concentration-response curves were
obtained by adding 5 uM each of A+L (10 uM total), 25 uM each of
A+L (50 uM total), 50 uM each of A+L (100 uM total), 100 uM each of
A+L (200 uM total), 150 uM each of A+L (300 uM total), 200 uM each
of A+L (400 uM total), 250 uM each of A+L (500 uM total).
[0170] Various compositions according to the invention as described
above are illustrated as follows: [0171] 1 mM A and 1 mM L, [0172]
2 mM A and 2 mM L (compare to 0.5 mM each) [0173]
AL+NG-nitro-L-arginine methyl ester (L-NAME, 100 uM for 30 min) in
denuded rings and intact rings. Involvement of NO is also assessed.
At the plateau of the precontraction, A, L or AL are added in the
bath and relaxation recorded. [0174] AL+L-arginine (0.5 mM)
precursor of endothelium-derived NO(NO donor) to confirm a role for
eNOS (endothelial NO synthase) in adenosine endothelium-dependent
relaxation. [0175] AL+8-sulfophenyltheophyline (SPT) (non-specific
Ado blocker) (100 uM) [0176] AL+Pertussus Toxin [0177]
AL+glibenclamide (30 uM) (non-specific blocker of KATP channels
[0178] AL+mitoKATP channel blocker [0179] AL+papaverine (0.5 mg/ml)
[0180] AL+GTN (0.5 mg/ml) [0181] AL+16 mM MgCl2 (can induce
relaxation due to Ca entry inhibition) [0182] AL+opioid agonist
[0183] AL+blood anti-coagulant (eg. heparin) [0184] AL+naloxone
(0.1 mM) (incubated for 20 min before adding AL) [0185]
AL+amilioride [0186] AL+BoTox [0187] AL+Ca activated K channel
blocker (0.1 mM tetraethylammonium, TEA-Sigma Sahin A S et al 2005,
also Langton P D et al AJP 260H927-34, 1991)) [0188]
AL+ultra-short-acting beta-blocker, esmolol [0189] AL+60 mM KCL
[0190] AL+Ca activated channel blocker (0.1 mM tetraethylammonium,
TEA-Sigma Sahin A S et al 2005) [0191] AL+AMP579 (AMP579 is a mixed
adenosine agonist with both A1 and A2 effects) [0192]
AL+5-hydroxydecanoate (5HD) (10 uM) specific blocker of
mitochondrial K.sub.ATP channels
Results:
[0193] Effect of increasing the concentration of adenosine,
lidocaine and adenosine+lidocaine on the tension of intact and
denuded are shown in FIGS. 1 to 3 respectively (n=6). Table 1 below
shows tensions of intact and denuded isolated rat aortic rings
using 0.3 uM norepinephrine followed by different concentrations of
adenosine, lidocaine and adenosine-lidocaine (AL). Values are
expressed grams (.+-.S.E.M). Table 2 (below) shows extent of
relaxation of intact and denuded isolated rat aortic rings using
different concentrations of adenosine, lidocaine and
adenosine-lidocaine (AL). Values are expressed as a percentage
(.+-.S.E.M) of precontracted norepinephrine (0.3 uM) tensions (see
Table 1 for precontracted tensions).
TABLE-US-00002 TABLE 1 Tensions of intact and denuded isolated rat
aortic rings using 0.3 uM norepinephrine followed by different
concentrations of adenosine, lidocaine and adenosine-lidocaine
(AL). Values are expressed grams (.+-.S.E.M). Type of aortic rings
Precontracted Drug concentrations (.mu.M) Drugs (n = 6) Tension (g)
10 60 160 360 660 1060 1560 Adenosine Intact 3.68 .+-. 0.11 3.65
.+-. 0.12 3.54 .+-. 0.11 3.30 .+-. 0.09 2.81 .+-. 0.11 2.23 .+-.
0.14 1.78 .+-. 0.13 1.43 .+-. 0.08 Denuded 3.55 .+-. 0.08 3.56 .+-.
0.08 3.55 .+-. 0.09 3.51 .+-. 0.09 3.39 .+-. 0.11 3.12 .+-. 0.14
2.73 .+-. 0.17 2.23 .+-. 0.17 Lidocaine Intact 3.58 .+-. 0.07 3.57
.+-. 0.08 3.50 .+-. 0.08 3.44 .+-. 0.07 3.28 .+-. 0.07 3.02 .+-.
0.07 2.73 .+-. 0.13 2.59 .+-. 0.14 Denuded 3.33 .+-. 0.13 3.32 .+-.
0.12 3.31 .+-. 0.12 3.24 .+-. 0.12 3.11 .+-. 0.12 2.92 .+-. 0.12
2.75 .+-. 0.12 2.53 .+-. 0.13 AL Intact 3.78 .+-. 0.24 3.68 .+-.
0.25 3.63 .+-. 0.24 3.53 .+-. 0.25 3.02 .+-. 0.23 2.35 .+-. 0.21
1.76 .+-. 0.16 1.36 .+-. 0.09 Denuded 3.71 .+-. 0.19 3.70 .+-. 0.19
3.66 .+-. 0.19 3.53 .+-. 0.21 3.13 .+-. 0.19 2.51 .+-. 0.17 1.99
.+-. 0.15 1.55 .+-. 0.11
TABLE-US-00003 TABLE 2 Extent of relaxation of intact and denuded
isolated rat aortic rings using different concentrations of
adenosine, lidocaine and adenosine-lidocaine (AL). Values are
expressed as a percentage (.+-. S.E.M) of precontracted
norepinephrine (0.3 uM) tensions (see Table 1 for precontracted
tensions). Drug concentrations (.mu.M) Drugs Type of rings 10 60
160 360 660 1060 1560 Adenosine Intact (n = 6) 99.0 .+-. 0.3 96.2
.+-. 0.7 89.7 .+-. 1.5 76.3 .+-. 3.1 60.7 .+-. 4.2 48.5 .+-. 3.9
38.9 .+-. 2.8 Denuded 100.3 .+-. 0.2 100.0 .+-. 0.4 98.9 .+-. 0.5
95.2 .+-. 1.1 87.6 .+-. 2.3 76.6 .+-. 3.4 62.4 .+-. 4.2 (n = 6)
Lidocaine Intact (n = 6) 99.7 .+-. 0.2 97.8 .+-. 0.7 96.2 .+-. 0.6
91.7 .+-. 0.5 84.4 .+-. 1.2 76.2 .+-. 3.5 72.2 .+-. 3.5 Denuded
100.0 .+-. 0.4 99.4 .+-. 0.4 96.8 .+-. 0.7 93.4 .+-. 0.6 87.60.8
82.4 .+-. 1.0 75.8 .+-. 2.0 (n = 6) AL Intact (n = 6) 97.2 .+-. 0.5
95.8 .+-. 1.3 93.1 .+-. 1.9 79.7 .+-. 2.3 61.8 .+-. 2.8 46.3 .+-.
2.3 36.2 .+-. 1.3 Denuded 99.8 .+-. 0.1 98.5 .+-. 0.5 95.0 .+-. 1.2
84.4 .+-. 2.0 67.5 .+-. 2.5 53.4 .+-. 2.3 42.0 .+-. 1.3 (n = 6)
Example 1A
Effect of Increasing the Concentrations of Adenosine (10, 50, 100,
200, 300, 400 and 500 uM) on the Tension of Intact and Denuded Rat
Aortic Rings Precontracted with Noradrenalin (FIG. 1).
[0194] The effect of increasing the concentrations of adenosine on
the tension of intact and denuded rat aortic rings precontracted
with norepinephrine are shown in FIG. 1a and Table 1. In intact rat
aortic rings, the mean tension in the precontracted state was
3.68.+-.0.11 g (n=6). Expressed as a percent of baseline (3.68 g),
the tension values were 99, 96, 89, 76, 61, 48 and 39% for 10, 50,
100, 200, 300, 400 and 500 uM adenosine respectively (Table 1).
Muscle tension did not begin to decrease by over 5% relative to the
precontracted state until 100 uM adenosine with a relaxation of
10%. At 200, 300, 400 and 500 uM adenosine concentrations, the
percent tension decrease was 24, 39, 52 and 61% respectively
relative to the baseline norepinephrine precontracted state (Table
2). In denuded aortic rings, the mean tension of the precontracted
state was 3.55.+-.0.11 g (n=6). A very different response was found
when the endothelium had been removed (FIG. 1). Expressed as a
percent of baseline, tension values were 100, 100, 99, 95, 88, 77
and 63% for 10, 50, 100, 200, 300, 400 and 500 uM adenosine
respectively (Table 1). In denuded rings the tension did not begin
to decrease by over 5% until 300 uM, 400 uM and 500 uM adenosine
with a relaxation of 12, 23 and 37% respectively relative to the
norepinephrine precontracted state (Table 2).
[0195] It can be seen from this data that: [0196] In isolated
intact aortic rings, there was no change in adenosine induced
relaxations at 10 and 50 uM and only 4% relaxation was found at 100
uM relative to the baseline Nor-adrenalin precontracted state when
bathed in 10 mM glucose in Krebs-Henseleit at pH 7.4 37.degree. C.
under aerobic conditions (95% O.sub.2 & 5% CO.sub.2). [0197]
Adenosine induced relaxations in intact aortic rings above 5%
relative to the baseline Nor-adrenalin precontracted state were
only observed at 200 uM (11% relaxation), 300 uM (21% relaxation),
400 uM (34% relaxation) and 500 uM (48% relaxation). [0198] The
maximum relaxation in intact aortic rings was found to be at 500 uM
adenosine and 48% relative to the baseline Nor-adrenalin
precontracted state. [0199] The effect of removing the endothelium
was to reduce adenosine's ability to relax the aortic rings at
concentrations above 200 uM. The percent adenosine-relaxations in
denuded vs intact aortic rings at 200, 300, 400 and 500 uM were 4%
vs 11% 5 vs 21, 12% vs 34% and 21 vs 48% respectively. Relaxations
over 10% in denuded aortic rings only occurred at 400 uM and
relaxation was 35% of the relaxation of the intact aortic rings at
the same concentration. Similarly at 500 uM the denuded ring
relaxed to 44% of the intact ring at 500 uM. [0200] It is concluded
that 1) adenosine can relax intact aortic rings at 200, 300, 400
and 500 uM concentrations, and 2) this relaxation effect is
dependent on an intact endothelium. It is concluded that an intact
endothelium is a key factor to explain adenosine's ability to relax
isolated rat aortic rings precontracted with Nor-adrenalin under
aerobic conditions. [0201] Without being bound by any theory or
mode of action, one explanation for the endothelium-dependent
effect of adenosine is that the endothelium produces a metabolite
or factor which relaxes aortic smooth muscle. A candidate for part
of the relaxation is nitric oxide, which is normally produced by
the healthy endothelium and is known to relax smooth muscle.
Example 1B
Effect of Increasing the Concentrations of Lidocaine on the Tension
of Intact and Denuded Rat Aortic Rings Precontracted with
Noradrenalin (FIG. 2)
[0202] The effect of increasing the concentrations of lidocaine on
the tension of intact and denuded rat aortic rings precontracted
with norepinephrine are shown in FIG. 2 and Table 1. The mean
tension for intact rings in the precontracted state 3.58.+-.0.07 g
(n=6) (Table 1). Expressed as a percent of baseline, tension values
were 100, 96, 96, 91, 84, 76 and 72% for 10, 50, 100, 200, 300, 400
and 500 uM lidocaine respectively (Table 1). Muscle tension did not
begin to decrease by over 5% relative to the precontracted state
until 200 uM lidocaine with a relaxation of 9% and at 300, 400 and
500 uM lidocaine concentrations, the percent tension decrease was
16, 24 and 28% respectively relative to baseline. This tension
relaxation profile for lidocaine in intact rings was similar to
adenosine alone in denuded rings (ie for adenosine denuded rings
the percent fall in tension were 12, 23 and 37% for 300, 400 and
500 uM). In denuded rings the mean tension for the precontracted
state was 3.32.+-.0.13 g (n=6) (Table 1). In direct contrast to
adenosine, there was little or no difference in lidocaine's
tension-relaxation response when the endothelium had been removed
(FIG. 1b). Expressed as a percent of the baseline, tension values
were 100, 99, 98, 93, 88, 83 and 76% for 10, 50, 100, 200, 300, 400
and 500 uM adenosine respectively (Table 1). Thus, tension did not
begin to decrease by over 5% relative to the precontracted state
until 200 uM lidocaine where there was 7% relaxation (Table 2). The
% relaxation for 300, 400 and 500 uM were 12, 17 and 24%
respectively (Table 2). At 500 uM lidocaine, a denuded endothelium
led to no significant difference in fall in relaxation compared to
the intact endothelium, demonstrating that lidocaine's ability to
relax the vessel was endothelium independent.
[0203] It can be seen from this data that: [0204] In intact aortic
rings, there was no change in lidocaine-induced relaxations at 10
and 50 uM and only 5% relaxation was found at 100 uM relative to
the baseline Nor-adrenalin precontracted state when bathed in 10 mM
glucose in Krebs-Henseleit at pH 7.4 37.degree. C. under aerobic
conditions (95% O.sub.2 & 5% CO.sub.2). This was similar to the
effect of adenosine alone on intact aortic ring preparation over
the same concentration range (20 to 100 uM). [0205] Lidocaine
induced relaxations in intact aortic rings of 10% and above
relative to the baseline Nor-adrenalin precontracted state were
observed at 200 uM (10% relaxation), 300 uM (18% relaxation), 400
uM (24% relaxation) and 500 uM (34% relaxation). This was similar
to adenosine alone intact aortic ring preparation over the same
concentrations (200-500 uM) however the percent relaxations at 300,
400 and 500 uM were less in the lidocaine vs adenosine group ie the
lido group generated 14, 30 and 30% of the relaxations of intact
adenosine alone at 300, 400 and 500 uM respectively. [0206] The
effect of removing the endothelium had little effect on lidocaine's
ability to relax the denuded aortic rings implying that lidocaine's
effect to relax was mostly via its vascular smooth muscle effect.
The percent lidocaine-relaxation in denuded vs intact aortic rings
relative to the baseline Noradrenalin contracted state at 200, 300,
400 and 500 uM were 7% vs 10%, 14% vs 18%, 19% vs 24% and 29% vs
34% respectively. [0207] It is concluded that 1) lidocaine relaxes
intact isolated aortic rings at higher concentrations (200, 300,
400 and 500 uM), and 2) this effect does not appear to be
endothelium dependent. This is in direct contrast to adenosine's
effect to relax isolated aortic rings precontracted with
Nor-adrenalin under aerobic conditions.
Example 1C
Effect of Increasing the Concentrations of Adenosine and Lidocaine
Solution (Eg 10 uM Adenosine and 10 uM Lidocaine=10 uM Conc al, 50
uM Adenosine and 50 uM Lidocaine=50 uM AL and 500 uM Adenosine and
500 uM Lidocaine=500 uM AL) on the Tension of Intact and Denuded
Rat Aortic Rings Precontracted with Noradrenalin
[0208] The effect of increasing the concentrations of adenosine and
lidocaine on the tension of intact and denuded rat aortic rings
precontracted with norepinephrine is shown in FIG. 3 and Table 1.
In the intact rings, the mean tension in the precontracted state
was 3.78.+-.0.24 g (n=6). Expressed as a percent of the baseline
(3.78 g), tension values were 97, 96, 93, 80, 62, 47 and 36% for
10, 50, 100, 200, 300, 400 and 500 uM adenosine-lidocaine
respectively (Table 1). Muscle tension did not begin to decrease by
over 5% until 100 uM adenosine-lidocaine with a relaxation of 7%.
At 200, 300, 400 and 500 uM adenosine-lidocaine concentrations, the
percent tension decrease was 20, 38, 53 and 64% respectively
relative to the baseline norepinephrine state (Table 2). In denuded
rings, the mean tension in the precontracted state was 3.71.+-.0.19
g (n=6). Expressed as a percent of the baseline, tension values
were 100, 99, 95, 86, 68, 54 and 42% for 10, 50, 100, 200, 300, 400
and 500 uM adenosine-lidocaine respectively. Thus, tension did not
begin to decrease by over 5% until 200 uM adenosine-lidocaine with
a relaxation of 14% (Table 2). At 300, 400 and 500 uM
adenosine-lidocaine concentrations, the percent tension decrease
was 32, 46 and 58% respectively relative to the baseline (Table 2).
The profile of increasing the concentration of AL on relaxation of
denuded isolated rat aortic rings was the same as if the intact
endothelium was present. No significant differences were found in
the AL relaxation profiles between intact and denuded rings when
bathed in 10 mM glucose in Krebs-Henseleit at pH 7.4 and 37.degree.
C. under aerobic conditions (95% O.sub.2 & 5% CO.sub.2).
[0209] It can be seen from this data that: [0210] for the intact
endothelium: [0211] In intact aortic rings, there was little or no
change in AL-induced relaxations at 10 and 50 uM and only 4%
relaxation was found at 100 uM relative to the baseline
Nor-adrenalin precontracted state when bathed in 10 mM glucose in
Krebs-Henseleit at pH 7.4 37.degree. C. under aerobic conditions
(95% O.sub.2 & 5% CO.sub.2). This was similar to the effect of
adenosine alone and lidocaine alone on intact aortic ring
preparation over the same concentration range (10 to 100 uM).
[0212] AL solution did not begin to reduce tension in intact aortic
rings relative to the baseline precontracted Noradrenalin state
until 200, 300, 400 and 500 uM adenosine-lidocaine concentrations
with 8, 18, 36 and 48% falls in tension respectively. These
concentrations and falls in tension were similar to adenosine alone
(% fall in tension for 200, 300, 400 and 500 uM adenosine were 11,
21, 34 and 48%), and greater than lidocaine alone at 400 and 500 uM
(lidocaine relaxations were 10%, 18%, 24% and 34% of baseline at
200, 300, 400 and 500 uM). In summary, adenosine-lidocaine had a
similar percentage effect to reduce tension (or increase
relaxation) as adenosine alone on aortic rings with intact
endothelium and about 30% greater falls in tension than lidocaine
alone at 400 and 500 uM concentrations for intact and denuded
lidocaine preparations. [0213] In denuded aortic rings, there was
no difference in the AL-solution induced percentage fall in tension
or increased relaxation compared to intact aortic rings when bathed
in 10 mM glucose in Krebs-Henseleit at pH 7.4 37.degree. C. under
aerobic conditions (95% O.sub.2 & 5% CO.sub.2). The profile of
relaxation was surprising and the same as if the intact endothelium
was present.
Example 2
The Effect of Lowering Body Temperature on the Different
Resuscitation Strategies
[0214] The above example is repeated at 35, 33, 20, and 4.degree.
C. The formulations are equilibrated with air or aerated or have an
oxygen containing perfluorocarbon based, or haemoglobin based
substitute present or blood, a blood product or artificial blood.
Components may be added to mimic human blood's oxygen transport
ability such as Hemopure.TM., Gelenpol.TM., Oxygent.TM.,
PolyHeme.TM.
Example 3
Treatment During Surgery
[0215] The compositions and methods of the invention can also be
used during periods of reduced metabolic activity to reduce damage,
such as cell quiescence (medically induced or otherwise). Cardiac
surgery is one example. In this example, a known hyperkalemic
cardioplegic is used, and the composition of the present invention
is administered to reduce tissue damage during the operation.
[0216] More specifically, the invention is illustrated by an AL
graft storage solution with and without magnesium, dipyridamole and
Botulinum Toxin Type A on saphenous vein patency during and
following coronary bypass graft surgery. As coronary artery bypass
surgery enters its fourth decade of use, an ongoing problem is the
early graft occlusion rate of 20% in the first year, or about
480,000 grafts per annum worldwide.
[0217] The example's objectives are to (1) examine the antispasm
and protective effects of adenosine and lidocaine storage solution
mixed with a patient's heparinized blood, with and without
magnesium and dipyridamole, on saphenous vein grafts and compare to
graft storage in a patient's heparinized blood alone (2) compare
the sections of the saphenous vein using histologic analysis
immediately after harvesting and before and during 30, 60, 120 and
180 min storage time, and (3) compare the rate of restenosis at 1
month, 6 months and 12 months using non-invasive multi-slice
computed tomography (CT) angiography method for visualising and
assessing coronary arteries (total vessel diameter, lumen diameter,
and wall thickness).
[0218] All drugs are obtained from hospital supply houses and
approved for clinical use. Adenosine and lidocaine ("AL") are added
to 100 ml of the patient's heparinized blood to yield 1 mM final
concentrations of each drug. In the magnesium-containing versions,
magnesium sulphate is added to a final concentration of 16 mM, and
in the dipyridamole-containing versions, the drug is added at 150
uM final concentration. Dipyridamole is a phosphodiesterase
inhibitor and a weak antiplatelet agent and known to inhibit the
growth of vascular smooth muscle cells, especially venous smooth
muscle cells. Kim et al showed that approximately 90% inhibition
was achieved at dipyridamole concentrations of 75 microg/mL
(0.075/506.times.1000=0.150 mM). It appears that antiproliferative
effects of dipyridamole are sustained for 48 hr after drug exposure
of only 15 min.
[0219] In Botulinum Toxin experiments diluted amounts of the
Botulinum Toxin Type A are added to the AL graft blood storage
solution (10 U/ml and 20 U/ml blood ex vivo). The concentration was
based on preclinical studies using human saphenous vein rings and
rat aortic rings which showed a dose dependent relaxation of
vascular smooth muscle. The grafts are kept in the AL storage
solution. However before surgical attachment the grafts are removed
and washed three times with AL/blood solution alone to remove any
residual toxin adhering to the inner wall of the vessel. The
temperature of the graft solution will be at 37.degree. C. but may
gradually drift down to the temperature of the operating room
(22.degree. C.). Special note of the temperature is made because
cooling acts as a vasodilator.
[0220] Different groups of patients exemplify different embodiments
of the invention as follows:
Group 1: Heparinized blood alone; Group 2: Adenosine and lidocaine
(AL) alone (1 mM each)
Group 3: AL+Magnesium Sulphate (16 mM)
[0221] Group 4: AL+dipyridamole (150 uM)
Group 5: AL+Botulinum Toxin Type A (20 U/ml)
[0222] Group 6: AL+Magnesium+dipyridamole+Botulinum Toxin Type
A
[0223] Sapenous vein harvesting and histological analysis: Patients
are anaesthetized using the `Standard` anaesthetic technique for
cardiac surgery. The surgical procedure is the Standard Surgical
Technique used by surgeons. Patients have their left or right leg
greater saphenous vein harvested by means of the open technique
with a traditional longitudinal incision. This is performed by a
surgeon's assistant preparing the legs circumferentially and making
an incision from the groin to the knee and, if necessary, to below
the knee, exposing the entire vein using a continuous incision. The
vein is dissected with a combination of Metzenbaum scissors and
electrocautery and the vein branches clipped proximally and
distally. Once fully dissected, the vein is removed and placed in
one of the six storage solutions in 100 ml of the patients
heparinized blood. Hemostasis is carried out using electrocautery
and surgical clips. The wound is closed in a one- or two-layer
fashion with absorbable sutures. The skin layer is closed with
surgical staples. The leg is then immediately wrapped with a
sterile elastic bandage and remained wrapped for up to 72
hours.
[0224] The graft vessel is connected to a pressurized syringe
system and tested for any leaks and then any leakages are repaired
using sutures or surgical clips. Pressure testing is carried out by
connecting the vessel to a syringe system and injecting the
appropriate storage solution for each group into the vessel to a
pressure of 180 mmHg with one end clamped. After pressure testing
the segments or conduits are selected for up to four bypass
grafts.
[0225] Immediately on harvesting a 0.5 cm segment of the vessel is
taken for histological analysis before pressure testing, after
pressure testing, and after 30 min and 60 min in the storage
solution, and if possible 120 min and 180 min after first placing
in storage. The segments are labelled proximal, distal, and centre
and placed immediately in a 10% buffered formalin vial. The
specimens are fixed and sectioned, processed using light
microscopy, and stained with Movat's pentachrome. The slides are
evaluated for changes in the intima, media, and adventitia.
Cardioplegia Protocol
[0226] The cardioplegia protocol used is as follows.
1) Composition of cardioplegia solution: Induction cardioplegia 20
mM K+ solution (final): BAXTER (Code AHK5524). Each 500 ml
Contains: Sodium Chloride BP 4.5 g, Potassium Chloride BP 3 g,
Magnesium Chloride BP 2.6 g, Lidocaine HCl BP 250 mg. Before use
Sodium Bicarbonate (25 mmol/500 ml) and monosodium Aspartate (14
mmol/500 ml) is added. .about.pH 3.7 .about.Osmolality 547 mOsm 2)
Maintenance cardioplegia 9 mM K.sup.+ solution (final): BAXTER
(Code AHK5525). Each 500 ml Contains: Sodium Chloride BP 4.5 g,
Potassium Chloride BP 1 g, Magnesium Chloride BP 2.6 g. Before use
Sodium Bicarbonate (25 mmol/500 ml) and monosodium Aspartate (14
mmol/500 ml) is added. .about.pH 3.7 .about.Osmolality 547 mOsm
[0227] During reanimation, the arrest solution is same as K+
maintenance but the myocardial heart temperature during induction,
maintenance and terminal shot are 32 to 38.degree. C. The heart
remains arrested at this time.
[0228] Hemodynamic and Metabolic Parameters: Routine hemodynamic
parameters are measured before, during and following surgery.
Echocardiography is used to assess left ventricular wall function.
Time to arrest, volumes of cardioplegia administered, myocardial
temperature, coronary vascular resistance, incidence of reanimation
during arrest, incidence of arrhythmias during arrest, and arterial
and coronary sinus lactate, blood gases, pH and ions are measured
at 15 min intervals during arrest. After cross-clamp removal, time
to first beat, need to electrically cardioconvert, incidence of
reperfusion arrhythmias are recorded. CKMB and Troponin I levels
are measured before, during and 4, 6 and 12 hours after
reanimation. In addition, the use of intra-operative inotropes,
blood loss, blood products, pre- and post-operative wall motion and
contractility, ejection fraction, ICU and ward stay, incidence of
atrial arrhythmias and other post-operative arrhythmias are
recorded. Systemic inflammatory markers Interleukin 6 and 8 are
also measured during and following surgery.
[0229] Assessment of graft patency at 1 month. 6 month and 12
months post-operatively
[0230] The rate of restenosis in the grafted vessels can be
assessed at 1 month, 6 months and 12 months using non-invasive
multi-slice computed tomography (CT) angiography method for
visualising and assessing coronary arteries (total vessel diameter,
lumen diameter, and wall thickness). Non-invasive examinations are
carried out using a scanner with 16 detector rows (Brilliance 16,
Philips Medical Systems, Cleveland, Ohio, USA). Contrast flow rate
is adapted according to a test bolus acquisition. Pitch settings
are modified according to mean heart rate. Coronary lumen diameter
is measured using electronic callipers. Hoffmann et al showed that
CT angiography using a 16 row multislice detector had very
excellent sensitivity (96%) and very good specificity (84%)
compared with the invasive "gold standard" for coronary angiography
diagnosis of coronary artery disease (Hoffman M H et al
Evidence-Based Medicine 2006; 11:24)
[0231] Of course, a similar technique could be used but using the
newer endoscopic harvesting technique (e.g using Guidant's Vaso
View graft harvest system) instead of the more conventional open
technique of saphenous graft harvesting described above. Similarly,
one could use arterial grafts (eg left or right radial artery)
instead of saphenous vein grafts or other artery or venous grafts.
It could also be carried out on patients undergoing "on-pump" using
cardioplegia as described above or it could be carried out using
"off-pump" on the beating heart.
[0232] It will be understood that the invention disclosed and
defined in this specification extends to all alternative
combinations of two or more of the individual features mentioned or
evident from the text or drawings. All of these different
combinations constitute various alternative aspects of the
invention.
* * * * *