U.S. patent application number 11/667065 was filed with the patent office on 2009-12-17 for use of xenon as neuroprotectant in a neonatal subject.
This patent application is currently assigned to PROTEXEON LIMITED. Invention is credited to Nicholas Peter Franks, Mervyn Maze.
Application Number | 20090311340 11/667065 |
Document ID | / |
Family ID | 33042333 |
Filed Date | 2009-12-17 |
United States Patent
Application |
20090311340 |
Kind Code |
A1 |
Franks; Nicholas Peter ; et
al. |
December 17, 2009 |
USE OF XENON AS NEUROPROTECTANT IN A NEONATAL SUBJECT
Abstract
The present invention relates to the use of xenon in the
preparation of a medicament for preventing and/or alleviating one
or more anesthetic-induced neurological deficits in a neonatal
subject. The present invention further relates to combinations of
xenon and sevoflurane, and use thereof as preconditioning agents
for administration prior to hypoxic-ischaemic injury.
Inventors: |
Franks; Nicholas Peter;
(Highbury London, GB) ; Maze; Mervyn; (London,
GB) |
Correspondence
Address: |
KENYON & KENYON LLP
1500 K STREET N.W., SUITE 700
WASHINGTON
DC
20005
US
|
Assignee: |
PROTEXEON LIMITED
London
GB
|
Family ID: |
33042333 |
Appl. No.: |
11/667065 |
Filed: |
August 19, 2005 |
PCT Filed: |
August 19, 2005 |
PCT NO: |
PCT/GB2005/003253 |
371 Date: |
August 14, 2008 |
Current U.S.
Class: |
424/600 |
Current CPC
Class: |
A61P 25/04 20180101;
A61K 45/06 20130101; A61P 23/00 20180101; A61K 33/00 20130101; A61K
31/08 20130101; A61P 39/00 20180101; A61P 25/00 20180101; A61P
43/00 20180101; A61P 25/28 20180101; A61K 31/08 20130101; A61K
2300/00 20130101; A61K 33/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/600 |
International
Class: |
A61K 33/00 20060101
A61K033/00; A61P 25/00 20060101 A61P025/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 19, 2004 |
GB |
0418540.1 |
Claims
1-93. (canceled)
94. A method of treating, preventing, and/or alleviating
anesthetic-induced neurological deficits in a neonatal subject, the
method comprising administering a therapeutically effective amount
of xenon to said subject.
95. The method of claim 94, wherein the neurological deficit is
associated with neuronal apoptosis, neuronal necrosis, or neuronal
injury.
96. The method of claim 94, wherein the neurological deficit is
neurodegeneration.
97. The method of claim 94, wherein the neurological deficit is a
learning, memory, neuromotor, neurocognitive or psychocognitive
deficit.
98. The method of claim 94, wherein the anesthetic is a GABAergic
agent.
99. The method of claim 94, wherein the anesthetic is isoflurane,
sevoflurant or desflurane.
100. The method of claim 94, wherein the anesthetic is isoflurane,
the neurological deficit is neuronal injury, and the method further
comprises administering isoflurane to the subject.
101. The method of claim 94, wherein the anesthetic is sevoflurane,
the neurological deficit is associated with neuronal injury, and
the method further comprising administering sevoflurane.
102. The method of claim 94, wherein the anesthetic is desflurane,
the neurological deficit is associated with neuronal injury, and
the method further comprises administering desflurane.
103. The method of claim 94, wherein the xenon is administered via
perfusion.
104. The method of claim 94, wherein the xenon is in gaseous
form.
105. The method of claim 94, wherein the xenon is administered via
inhalation.
106. A method of providing anesthesia, neuroprotection, and/or
analgesia to a neonatal subject, said method comprising
administering xenon in combination with another anesthesia, wherein
the amount of xenon is sufficient to alleviate and/or prevent
anesthesia-induced neuronal injury.
107. The method of claim 106, wherein the anesthetic is isoflurane,
sevoflurane or desflurane.
108. The method of claim 106, wherein the xenon is in gaseous
form.
109. The method of claim 106, wherein xenon is administered via
inhalation.
110. The method of claim 106, wherein the xenon is administered via
perfusion.
111. The method of claim 106, further comprising administering a
GABAergic agent.
112. A method of protecting against hypoxic injury comprising:
administering xenon in combination with an anesthetic.
113. The method of claim 112, wherein the anesthetic is
sevoflurane, isoflurane or desflurane.
114. The method of claim 112, wherein the anesthetic is sevoflurane
and the xenon and the sevoflurane are administered prior to the
hypoxic-ischaemic injury.
115. The method of claim 112, wherein the anesthetic is sevoflurane
and the xexon and the sevoflurane are administered at least about
two hours prior to the hypoxic-ischaemic injury.
116. The method of claim 114, wherein the xenon and the sevoflurane
are administered from about 2 to about 24 hours prior to the
hypoxic-ischaemic injury.
117. The method of claim 112, wherein the xenon and sevoflurane are
administered to the mother prior to delivery, during delivery,
prior to a cesarean section, during a cesarean section, or suitable
combinations thereof.
118. A method of treating, alleviating, and/or preventing neuronal
necrosis in a subject, said method comprising administering a
therapeutically effective amount of xenon to the patient.
Description
[0001] The present invention relates to the field of anesthetics.
More specifically, the invention relates to anesthetic agents
suitable for use in newborn and/or fetal subjects.
BACKGROUND TO THE INVENTION
[0002] Xenon's anesthetic properties have been known to the medical
profession for over 50 years (Cullen and Gross, 1951). However,
despite some impressive displays of clinical efficacy in patients
(Luttropp et al., 1994; Lynch et al., 2000), everyday use of xenon
anesthesia has failed to materialize. This is largely associated
with the significant cost involved in production of xenon through
fractional distillation of liquid air, and hence the relatively
small percentage of the total refined quantity of xenon available
for anesthesia (Hanne Marx et al., 2001). Consequently, use of
xenon is likely to be restricted to special areas where there is an
appreciable cost-benefit advantage. One such area may be neonatal
anesthesia, where xenon may lack harmful side effects seen with
other commonly used neonatal anesthetics e.g. nitrous oxide
(Layzer, 1978; Amos et al., 1982; Jevtovic-Todorovic et al.,
1998).
[0003] It is well documented in the art that neonatal insults cause
long lasting effects (Anand and Scalzo, 2000; Balduini et al.,
2000; Jevtovic-Todorovic et al., 2003). Therefore it is sensible to
adopt a degree of caution when using drugs in the neonate which
could potentially alter neurodevelopment (such as alcohol,
phencyclidine, ketamine, N.sub.2O, isoflurane, benzodiazepines,
barbiturates and anticonvulsants (Olney et al., 2002d) by causing
apoptotic neurodegeneration). This is especially true given that
often only a single exposure is required, even at anesthetic doses
(Ikonomidou et al., 2001; Young et al., 2003).
Normal Neurodevelopment
[0004] Normal neurodevelopment is a carefully regulated (Butler,
1999) sequence of events including proliferation, differentiation,
migration and synaptogenesis. Glutamate is thought to have a role
in all of these processes (Ikonomidou and Lechoslaw, 2002), for
example high concentrations of glutamate at migration target zones
suggests a role as a neuronnal chemooattractant (Behar et al.,
1999) along with the NMDA receptor used to detect it (Komuro and
Rakie, 1993). The intriguing finding of specific NMDA receptor
subtypes (e.g. NR2B and NR2D) in different anatomjcal regions may
shed light on the precise nature of migration control (Behar et
al., 1999). From work by the same group, it is also apparent that
different species employ different mediators in migration
control--currently either GABA (rats) or glutamate (mice) (Behar et
al., 2001).
[0005] Synaptogenesis (the brain growth spurt) is a period of a
rapid establishment of synapses, characterised by a high level of
physiological cell death (up to 1% (Olney et al., 2002b)). This
includes the formation of extensive corticothalamic and
thalamocortical projections (Moolar and Blakemore, 1995). Despite
the immense complexity of inter-species embryology, it has been
shown that comparisons can be made because milestones in
neurodevelopment tend to occur in the same sequence (Clancy et al.,
2001). This permits an extrapolation of the period of peak
synaptogenic activity from the 7 day old rat pup (Olney et al.,
2002a) to a 0-8 month old human being (Ikonomidou et al., 1999;
Jevtovic-Todorovic et al., 2003). However, based on analysis of
NMDA receptor subtypes, it is more probable that humans experience
an extended period of synaptogenesis--from the beginning of the
3.sup.rd trimester of pregnancy to several years old (Dobbing and
Sands, 1979; Jevtovic-Todorovic et al., 2003).
Apoptosis in the Developing Nervous System
[0006] Apoptosis, first formally described in 1972 (Kerr et al.,
1972), is an essential feature of normal neurodevelopment in
processes such as sculpturing, trimming, control of cell numbers
and cellular disposal. It is characterised as "active cell death"
comprising initiation, commitment and execution by dedicated
cellular proteins (Sloviter, 2002). The crucial role of apoptosis
is highlighted by the fact that genetic upregulation or
downregulation of apoptosis results in a lethal genotype (Yoshida
et al., 1998; Rinkenberger et al., 2000).
[0007] Control of physiological cell death (PCD) in the immature
CNS is currently thought to be governed by the neurotrophic
hypothesis--whereby neurones which fail to reach their survival
promoting synaptic targets (Sherrard and Bower, 1998) initiate a
specialised form of cell suicide secondary to withdrawal of
environmental trophic support (Young et al., 1999) (via both
neurotrophins and electrical stimulation) (Brenneman et al., 1990).
Due to the complex divergent and convergent nature of the "survival
pathway" many ligands and mechanisms are involved in maintaining
neuronal survival. The cytosol and mitochondria of neurones field a
balanced assortment of molecules which are either anti-apoptotic
(e.g. Bcl-2 and cAMP response binding protein) or pro-apoptotic
(e.g. Bad, Bax and the caspase family) which determine cell fate.
Bcl-2 and its associated peptides are thought to be particularly
important in the developing CNS (Yuan and Yanker, 2000), as
evidenced by the high levels of expression in the neonate and the
fact that experimental over-expression of Bcl-2 can both override
lack of trophic support (Garcia et al., 1992), and even prevent PCD
altogether (Miartinou et al., 1994). A variant of Bcl-2
(Bcl-X.sub.L) may have a specialised role in maintaining developing
neurones before they have found their synaptic targets (Motoyama et
al., 1995).
Neurodegeneration in Neonates
[0008] In 1999, data were published showing that use of NMDA
receptor antagonists in neonatal rats produced specific patterns of
neurodegeneration (distinct from glial cells) (Ikonomidou et al.,
1999). On electron microscopy, this neurodegeneration was identical
to apoptotic cell death, and most evident in the laterodorsal
thalamic nucleus, one of the areas of the brain implicated in
learning and memory (Goen et al., 2002). This phenomenon has since
been demonstrated in other brain regions with other drugs (Monti
and Contestabile, 2000).
[0009] Later work done by Jevtovic-Todorovic et al. showed that
neonatal rats are vulnerable to harmful side effects of anesthesia
during the synaptogenic period. They demonstrated up to a 68-fold
increase in the number of degenerating neurones above baseline in
areas such as the laterodorsal and anteroventral thalamic nuclei
(and to some extent layer II of the parietal cortex) after exposure
to anesthetic agents (Jevtovic-Todorovic et al., 2003), which
resulted in a functional neurological deficit in behaviour tests
later in life. Specifically, the GABAergic anesthetic isoflurane
(Gyulai et al., 2001), produced dose-dependent neurodegeneration in
its own right, with synergistic neurodegeneration with the
successive addition of midazolam (a double GABAergic cocktail) and
then N.sub.2O (a triple cocktail) (Jevtovic-Todorovic et al.,
2003). This process has been shown to occur with exposure to
GABAergic agents in areas other than anesthesia, such as
anticonvulsant therapy and maternal drug abuse in rats (Bittigau et
al., 2002; Farber and Olney, 2003).
[0010] A clinical manifestation of this type of neurodegeneration
is detected in 1 to 2 infants per 1000 livebirths as Fetal Alcohol
Syndrome (FAS) (Moore and Persaud, 1998)--characterised by abnormal
facial features, microencephaly and mental retardation (Olney et
al., 2002c). It is thought that binge drinking by pregnant mothers
produces very high levels of ethanol (a dual GABAergic agent and
NMDA receptor antagonist (Farber and Olney, 2003)) in the fetal
brain, which in turn triggers the type of neurodegeneration
discussed above (Ikonomidou et al., 2000). It is worth noting that
this mechanism of action closely resembles that of current
anesthetic procedures.
[0011] The present invention seeks to provide an anesthetic agent
suitable for use in the newborn that is safe, efficacious, and does
not have any adverse effects on neurodevelopment. More
specifically, the invention seeks to provide an anesthetic agent
for neonatal subjects that is suitable for use in combination with
other anesthetics know to adversely affect neurodevelopment. In
particular, the invention seeks to provide anesthetic combinations
for use in neonates which comprise an agent capable of preventing
or alleviating the adverse effects of known anesthetic agents such
as isoflurane and/or sevoflurane, and/or desflurane.
STATEMENT OF INVENTION
[0012] In a broad aspect, the present invention relates to the use
of xenon for treating and/or preventing and/or alleviating one or
more anesthetic-induced neurological deficits in a subject,
preferably a neonatal subject.
[0013] Various aspects of the invention are set forth in
accompanying claims and in the detailed description below.
DETAILED DESCRIPTION
[0014] A first aspect of the invention relates to the use of xenon
in the preparation of a medicament for treating and/or preventing
and/or alleviating one or more anesthetic-induced neurological
deficits in a subject, preferably a neonatal subject.
[0015] It is well documented in the art that exposure to
anesthetics, including NMDA receptor antagonists such as N.sub.2O,
ketamine and other agents such as isoflurane, triggers apoptotic
neurodegeneration during the synaptogenic phase of brain
development.
[0016] Studies have demonstrated that xenon, itself an NMDA
receptor antagonist, not only lacks the characteristic toxicity
produced by ketamine and N.sub.2O in adult rats, but also
ameliorates their toxicity. The experiments detailed herein have
investigated xenon's properties in a neonatal rat model of
neurodegeneration.
[0017] An in vivo rat model of anesthesia was used in conjunction
with both histology and immunohistochemistry to identify and
quantify apoptosis induced by various combinations of anesthetic
agents. Unlike isoflurane, xenon did not induce any apoptotic
neurodegeneration above the baseline observed in controls.
Additionally, it was found that whilst nitrous oxide enhances
isoflurane-induced apoptosis, xenon reduces the degree of
injury.
[0018] By way of summary, seven day old Sprague-Dawley rats were
exposed to 25% oxygen along with one of several gas combinations
(75% nitrogen, 75% nitrous oxide, 75% xenon, 0.75% isoflurane, 75%
nitrous oxide+0.75% isoflurane, 60% xenon+0.75% isoflurane) for 6
hours (n=3-5/group). The rats were sacrificed after anesthesia, and
their brains processed to assess the severity of apoptosis (using
caspase-3 immunohistochemistry, c-Fos immunohistochemistry, and
DeOlmos silver staining).
[0019] When administered alone, neither N.sub.2O nor xenon caused a
significant increase in caspase-3 positive cells in the hippocampus
or cortex (cortical values: 22.5.+-.5.9 and 19.7.+-.9.6
respectively vs 19.3.+-.6.4 in controls; p>0.05). In contrast,
isoflurane alone significantly increased the number of degenerating
neurones in both regions (76.5.+-.11.4; p<0.01). Similarly,
sevoflurane alone caused a significant increase in degenerating
neurons from a control value of 20.0.+-.2 positive cells to 42.+-.2
positive cells when 1.5% sevoflurane was adminstered. When combined
with isoflurane, N.sub.2O considerably enhanced isoflurane-induced
apoptosis (232.0'19.9; p<0.001 vs air) while xenon reduced the
injury (26.7.+-.3.9; p>0.05 vs air).
[0020] These data suggest that xenon, unlike other anesthetics that
exhibit NMDA receptor blockade, does not enhance apoptotic
neurodegeneration in the neonatal rat. In fact, xenon appears to
protect against isoflurane-induced apoptosis.
[0021] As used herein, the term "neonatal subject" refers to a
newborn subject. Preferably the neonatal subject is a mammal in the
first four weeks after birth. More preferably, the neonatal subject
is a mammal in the first two weeks, more preferably still, the
first week after birth.
[0022] Even more preferably, the neonatal subject is a human.
[0023] In one preferred embodiment, the neonatal subject is a
subject which is undergoing, or requires, fetal surgery.
[0024] In one preferred embodiment, the neonatal subject is a
subject having a life-threatening condition requiring emergent or
elective surgery later in life
[0025] In another preferred embodiment, the neonatal subject
receives xenon indirectly as part of an anesthetic or analgesic
regimen administered to the mother during labour, or during
cesarean section.
[0026] Preferably, the medicament is for preventing and/or
alleviating one or more anesthetic-induced neurological deficits in
a subject, preferably a neonatal subject.
[0027] As used herein, the term "preventing and/or alleviating
neurological deficits" refers to reducing the severity of one or
more neurological deficits as compared to a subject having
undergone treatment with an anesthetic in the absence of xenon.
[0028] Preferably, the neurological deficit is a learning, memory,
neuromotor, neurocognitive and/or psychocognitive deficit.
[0029] In an even more preferred embodiment, the neurological
deficit may be a neuromotor or neurocognitive, deficit. As used
herein the term "neuromotor deficit" is given its meaning as
understood by the skilled artisan so as to include deficits in
strength, balance and mobility. Similarly, the term "neurocognitive
deficit" is given its meaning as understood by the skilled artisan
so as to include deficits in learning and memory. Such
neurocognitive deficits may typically be assessed by
well-established criteria such as the short-story module of the
Randt Memory Test [Randt C, Brown E. Administration manual: Randt
Memory Test. New York: Life Sciences, 1983], the Digit Span subtest
and Digit Symbol subtest of the Wechsler Adult Intelligence
Scale-Revised [Wechsler D. The Wechsler Adult Intelligence
Scale-Revised (WAIS-R). San Antonio, Tex.: Psychological
Corporation, 1981.], the Benton Revised Visual Retention Test
[Benton A L, Hansher K. Multilingual aphasia examination. Iowa
City: University of Iowa Press, 1978] and the Trail Making Test
(Part B) [Reitan R M. Validity of the Trail Making Test as an
indicator of organic brain damage. Percept Mot Skills 1958;
8:271-6]. Other suitable neuromotor and neurocognitive tests are
described in Combs D, D'Alecy L: Motor performance in rats exposed
to severe forebrain ischemia: Effect of fasting and 1,3-butanediol.
Stroke 1987; 18: 503-511 and Gionet T, Thomas J, Warner D, Goodlett
C, Wasserman E, West J: Forebrain ischemia induces selective
behavioral impairments associated with hippocampal injury in rats.
Stroke 1991; 22: 1040-1047).
[0030] In one preferred embodiment, the neurological deficit is
neurodegeneration.
[0031] As used herein, the term "neurodegeneration" refers to cell
shrinkage, chromatin-clumping with margination and formation of
membrane-enclosed apoptotic bodies; on application of caspase 3
antibody the neurodegenerating neurones stair black on application
of 3,3'-diamino-benzidine (dab).
[0032] In another preferred embodiment, the neurological deficit is
associated with neuronal apoptosis.
[0033] In another preferred embodiment, the neurological deficit is
associated Ash neuronal necrosis.
[0034] In another preferred embodiment, the neurological deficit is
a learning, memory, neuromotor or psychocognitive deficit.
[0035] A second aspect of the invention relates to the use of xenon
in the preparation of a medicament for treating and/or alleviating
and/or preventing anesthetic-induced neurodegeneration in a
subject, preferably a neonatal subject.
[0036] A third aspect of the invention relates to the use of xenon
in the preparation of a medicament for treating and/or alleviating
and/or preventing anesthetic-induced neuronal apoptosis in a
subject, preferably a neonatal subject.
[0037] A third aspect of the invention relates to the use of xenon
in the preparation of a medicament for preventing and/or
alleviating anesthetic-induced neuronal injury in a subject,
preferably a neonatal subject.
[0038] For all of the above aspects, preferably the anesthetic is a
volatile anesthetic agent. Examples of volatile anesthetics include
isoflurane, sevoflurane and desflurane.
[0039] For all of the above aspects, preferably the anesthetic is
either a GABAergic agent such as isoflurane, sevoflurane or
desflurane, or an NMDA receptor antagonist anesthetic (eg ketamine
or nitrous oxide).
[0040] More preferably, the anesthetic is isoflurane, sevoflurane,
or desflurane.
[0041] Isoflurane is a halogenated volatile anesthetic which
induces and maintains general anesthesia by depression of the
central nervous system and resultant loss of consciousness.
Throughout maintenance of anesthesia, a high proportion of the
isoflurane is eliminated by the lungs. When administration is
stopped, the bulk of the remaining isoflurane is eliminated
unchanged from the lungs. As solubility of isoflurane is low,
recovery from isoflurane anesthesia in man is rapid.
[0042] As isoflurane has a mild pungency, inhalation is usually
preceded by the choice of a short-acting barbiturate, or other
intravenous induction agent, to prevent coughing. Isoflurane can
induce increased salivation and coughing in small children upon
administration. Adverse reactions encountered with isoflurane are
similar to those observed with other halogenated anesthetics and
include hypotension, respiratory depression and arrhythmias. Other
minor side-effects encountered while using isoflurane are an
increase in the white blood cell count (even in the absence of
surgical stress) and also shivering, nausea and vomiting during the
post-operative period. There have also been rare reports of mild,
moderate and severe (some fatal) post-operative hepatic
dysfunction. The causal relationship for this is unknown.
[0043] Isoflurane causes an increase in cerebral blood flow at
deeper levels of anesthesia; this may give rise to an increase in
cerebral spinal fluid pressure. Where appropriate, this can be
prevented or reversed by hyperventilating the patient before or
during anesthesia. As with other halogenated anesthetics,
isoflurane must be used with caution in patients with increased
intracranial pressure.
[0044] Isoflurane is a powerful systemic and coronary arterial
dilator. The effect on systemic arterial pressure is easily
controlled in the normal healthy patient and has been used
specifically as a means of inducing hypotension. However, the
phenomenon of "coronary steal" means that isoflurane should be used
with caution in patients with coronary artery disease. In
particular, patients with subendocardial ischaemia might be
anticipated to be more susceptible.
[0045] Sevoflurane, a fluorinated methyl-isopropyl ether is
relatively pleasant and non-pungent and is used to cause general
anesthesia before and during surgery. It is administered by
inhalation. As it has a blood/gas partition coefficient of only
0.6, onset and recovery times are fast.
[0046] The dose of sevoflurane required varies from patient to
patient, depending on age, physical condition, interactions with
other medicines and the type of surgery being performed. Side
effects include bradycardia, hypotension, tachycardia, agitation,
laryngospasm, airway obstruction, cough, dizziness, drowsiness,
increased amount of saliva, nausea, shivering, vomiting, fever,
hypothermia, movement, headache. As sevoflurane is metabolized very
slowly in the human body there is a high risk of renal toxicity.
When used in children sevoflurane has been known to cause increased
agitation.
[0047] In the context of the present invention, xenon may be
administered to the subject simultaneously, in combination,
sequentially or separately with the anesthetic agent.
[0048] As used herein, "simultaneously" is used to mean that the
xenon is administered concurrently with the anesthetic agent,
whereas the term "in combination" is used to mean the xenon is
administered, if not simultaneously, then "sequentially" within a
timeframe in which the xenon and the anesthetic both exhibit a
therapeutic effect, i.e. they are both are available to act
therapeutically within the same time-frame. Thus, administration
"sequentially" may permit the xenon to be administered within 5
minutes, 10 minutes or a matter of hours before or after the
anesthetic.
[0049] In one particularly preferred embodiment, the xenon is
administered to the subject prior to the volatile anesthetic agent.
Studies have indicated that xenon is capable of changing the
vulnerability of the subject to all kinds of injury of an apoptotic
or necrotic variety.
[0050] In one preferred embodiment the xenon is administered before
hypoxic-ischaemic injury or any other injury which is
apoptosis-dependent (i.e. in which apoptosis is the pathway to cell
death) or necrosis-dependent (i.e. in which necrosis is the pathway
to cell death), i.e. the xenon functions as a preconditioning
agent.
[0051] In another particularly preferred embodiment, the xenon is
administered after the volatile anesthetic agent. Thus, in one
preferred embodiment the xenon is administered after
hypoxic-ischaemic injury or any other injury which is
apoptosis-dependent (i.e. in which apoptosis is the pathway to cell
death) or necrosis-dependent (i.e. in which necrosis is the pathway
to cell death).
[0052] In contrast to "in combination" or "sequentially",
"separately" is used herein to mean that the gap between
administering the xenon and exposing the subject to anesthetic
agent is significant i.e. the xenon may no longer be present in the
bloodstream in a therapeutically effective amount when the subject
is exposed to the anesthetic agent, or the anesthetic may no longer
be present in the bloodstream in a therapeutically effective amount
when the subject is exposed to the xenon.
[0053] More preferably, the xenon is administered sequentially or
simultaneously with the anesthetic agent, more preferably
simultaneously.
[0054] More preferably, the xenon is administered prior to, or
simultaneously with, the anesthetic agent, more preferably
simultaneously.
[0055] In one preferred embodiment of the invention, the xenon is
administered in a therapeutically effective amount.
[0056] In another preferred embodiment, the xenon is administered
in a sub-therapeutically effective amount. In this context, the
term "sub-therapeutically effective amount" means an amount which
is lower than that typically required to produce anesthesia.
Generally, an atmosphere of about 70% xenon is sufficient to induce
or maintain anesthesia. Accordingly, a sub-therapeutic amount of
xenon corresponds to less than about 70% xenon.
[0057] Even more preferably, the combination of Xenon and
anesthetic agent has a synergistic effect, i.e., the combination is
synergistic.
[0058] Another aspect of the invention relates to the use of (i)
xenon, and (ii) an anesthetic selected from isoflurane, sevoflurane
and desflurane, in the preparation of a medicament for alleviating
and/or preventing isoflurane-induced and/or sevoflurane-induced
and/or desflurane-induced neuronal injury in a subject, preferably
a neonatal subject.
[0059] Another aspect of the invention relates to the use of (i)
xenon, and (ii) isoflurane, in the preparation of a medicament for
alleviating and/or preventing isoflurane-induced neuronal injury in
a subject, preferably a neonatal subject.
[0060] Another aspect of the invention relates to the use of (i)
xenon, and (ii) sevoflurane, in the preparation of a medicament for
alleviating and/or preventing sevoflurane-induced neuronal injury
in a subject, preferably a neonatal subject.
[0061] Another aspect of the invention relates to the use of (i)
xenon, and (ii) desflurane, in the preparation of a medicament for
alleviating and/or preventing desflurane-induced neuronal injury in
a subject, preferably a neonatal subject.
[0062] Yet another aspect of the invention relates to the use of
xenon in the preparation of a medicament for alleviating and/or
preventing isoflurane-induced and/or sevoflurane-induced and/or
desflurane-induced neuronal injury in a subject, preferably a
neonatal subject.
[0063] Yet another aspect of the invention relates to the use of
(i) xenon, and (ii) an anesthetic selected from isoflurane,
sevoflurane and desflurane, in the preparation of a medicament for
providing anesthesia in a subject, preferably a neonatal subject,
wherein the amount of xenon is sufficient to alleviate or prevent
anesthetic-induced injury.
[0064] Another aspect of the invention relates to the use of xenon
and isoflurane in the preparation of a medicament for providing
anesthesia in a subject, preferably a neonatal subject, wherein the
amount of Xenon is sufficient to alleviate or prevent
isoflurane-induced neuronal injury.
[0065] Another aspect of the invention relates to the use of xenon
and sevoflurane in the preparation of a medicament for providing
anesthesia in a subject, preferably a neonatal subject, wherein the
amount of xenon is sufficient to alleviate or prevent
sevoflurane-induced neuronal injury.
[0066] Another aspect of the invention relates to the use of xenon
and desflurane in the preparation of a medicament for providing
anesthesia in a subject, preferably a neonatal subject, wherein the
amount of xenon is sufficient to alleviate or prevent
desflurane-induced neuronal injury.
[0067] For all of the above aspects, preferably the xenon is
administered in combination with a pharmaceutically acceptable
diluent, excipient and/or carrier.
[0068] Examples of such suitable excipients for the various
different forms of pharmaceutical compositions described herein may
be found in the "Handbook of Pharmaceutical Excipients", 2.sup.nd
Edition, (1994), Edited by A Wade and P J Weller.
[0069] Acceptable carriers or diluents for therapeutic use are well
known in the pharmaceutical art, and are described, for example, in
Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R.
Gennaro edit. 1985). Examples of suitable carriers include lactose,
starch, glucose, methyl cellulose, magnesium stearate, mannitol,
sorbitol and the like. Examples of suitable diluents include
ethanol, glycerol and water.
[0070] The choice of pharmaceutical carrier, excipient or diluent
can be selected with regard to the intended route of administration
and standard pharmaceutical practice. The pharmaceutical
compositions may comprise as, or in addition to, the carrier,
excipient or diluent any suitable binder(s), lubricant(s),
suspending agent(s), coating agent(s), solubilising agent(s).
[0071] Examples of suitable binders include starch, gelatin,
natural sugars such a, glucose, anhydrous lactose, free-flow
lactose, beta-lactose, corn sweeteners, natural and synthetic gums,
such as acacia, tragacanth or sodium alginate, carboxymethyl
cellulose and polyethylene glycol.
[0072] Examples of suitable lubricants include sodium oleate,
sodium stearate, magnesium stearate, sodium benzoate, sodium
acetate, sodium chloride and the like.
[0073] Preservatives, stabilizers and dyes may be provided in the
pharmaceutical composition. Examples of preservatives include
sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
Antioxidants and suspending agents may be also used.
[0074] The xenon may also be administered in combination with
another pharmaceutically active agent. The agent may be any
suitable pharmaceutically active agent including anesthetic or
sedative agents which promote GABAergic activity. Examples of such
GABAergic agents include propofol and benzodiazapines.
[0075] The xenon may also be administered in combination with other
active ingredients such as L-type calcium channel blockers, N-type
calcium channel blockers, substance P antagonists, sodium channel
blockers, purinergic receptor blockers, or combinations
thereof.
[0076] The xenon may be administered by any suitable delivery
mechanism, or two or more suitable delivery mechanisms.
[0077] In one particularly preferred embodiment, the xenon is
administered by perfusion. In the context of the present invention,
the term "perfusion" refers to the introduction of an oxygen/xenon
mixture into, and the removal of carbon dioxide from, a patient
using a specialised heart-lung machine. In general terms, the
heart-lung machine replaces the function of the heart and lungs and
provides a bloodless, motionless surgical field for the surgeon.
The perfusionist ventilates the patient's blood to control the
level of oxygen and carbon dioxide. In the context of the present
invention, the perfusionist also introduces xenon into the
patient's blood. The perfusionist then propels the blood back into
the arterial system to provide nutrient blood flow to all the
patient's vital organs and tissues during heart surgery.
[0078] In one preferred embodiment, the medicament is in gaseous
form.
[0079] In another highly preferred embodiment, the xenon is
administered by inhalation.
[0080] In one preferred embodiment, the medicament further
comprises oxygen, nitrogen or mixtures thereof, more particularly
air.
[0081] In another preferred embodiment, the medicament further
comprises helium, NO, CO, CO.sub.2, N.sub.2O, other gaseous
compounds and/or inhalable medicaments.
[0082] In another preferred embodiment, the xenon is mixed with
another inert gas, such as argon or krypton.
[0083] In another preferred embodiment, the xenon is mixed with
oxygen, or an oxygen-containing gas.
[0084] In one highly preferred embodiment, the medicament is a
binary gaseous mixture which comprises from about 10 to about 80%
xenon by volume, more preferably from about 20 to about 80% xenon
by volume, with the remainder comprising oxygen. In another
preferred embodiment, the medicament comprises from about 30 to
about 75% xenon by volume, with the remainder comprising
oxygen.
[0085] In another highly preferred embodiment, the medicament is a
ternary gaseous mixture which comprises from about 10 to about 80%
xenon by volume, more preferably from about 20 to about 80% xenon
by volume, with the remainder comprising oxygen and nitrogen. In
another preferred embodiment, the medicament comprises from about
30 to about 75% xenon by volume, with the remainder comprising
oxygen and nitrogen.
[0086] In another preferred embodiment, the medicament comprises
about 5 to about 90% by volume of xenon, more preferably, about 10
to about 80% by volume of xenon, more preferably still, about 10 to
about 50% by volume of xenon, more preferably still, about 10 to
about 30% by volume of xenon.
[0087] In another preferred embodiment, the medicament is in the
form of a liquid or solution. In one particularly preferred
embodiment, the medicament is in the form of a lipid emulsion.
[0088] Preferably, the liquid is administered in the form of a
solution or an emulsion prepared from sterile or sterilisable
solutions, which may be injected intravenously, intraarterially,
intrathecally, subcutaneously, intradermally, intraperitoneally or
intramuscularly.
[0089] In one particularly preferred embodiment, the xenon is
administered in the form of a lipid emulsion. The intravenous
formulation typically contains a lipid emulsion (such as the
commercially available Intralipid.RTM.10, Intralipid.RTM.20,
Intrafat.RTM., Lipofundin.RTM.S or Liposyn.RTM. emulsions, or one
specially formulated to maxinise solubility) which sufficiently
increases the solubility of the xenon to achieve the desired
clinical effect. Further information on lipid emulsions of this
sort may be found in G. Kleinberger and H. Pamperl,
Infusionstherapie, 108-117 (1983) 3.
[0090] The lipid phase of the present invention which dissolves or
disperses the gas is typically formed from saturated and
unsaturated long and medium chain fatty acid esters containing 8 to
30 carbon atoms. These lipids form liposomes in aqueous solution.
Examples include fish oil, and plant oils such as soya bean oil,
thistle oil or cottonseed oil. The lipid emulsions of the invention
are typically oil-in-water emulsions wherein the proportion of fat
in the emulsion is conventionally 5 to 30% by weight, and
preferably 10 to 20% by weight. Oil-in-water emulsions of this sort
are often prepared in the presence of an emulsifying agent such as
a soya phosphatide. The lipids which form the liposomes of the
present invention may be natural or synthetic and include
cholesterol, glycolipids, sphingomyelin, glucolipids,
glycosphingolipids, phosphatidylcholine, phosphatidylethanolamine,
phosphatidyl-serine, phosphatidyglycerol, phosphatidylinositol.
[0091] The lipid emulsions of the present invention may also
comprise additional components. These may include antioxidants,
additives which make the osmolarity of the aqueous phase
surrounding the lipid phase isotonic with the blood, or polymers
which modify the surface of the liposomes.
[0092] It has been established that appreciable amounts of xenon
maybe added to a lipid emulsion. Even by the simplest means, at
20.degree. C. and normal pressure, xenon can be dissolved or
dispersed in concentrations of 0.2 to 10 ml or more per ml of
emulsion. The concentration of dissolved gas is dependent on a
number of factors, including temperature, pressure and the
concentration of lipid.
[0093] The lipid emulsions of the present invention may be loaded
with gaseous xenon. In general, a device is filled with the
emulsion and anesthetics as gases or vapours passed through
sintered glass bubblers immersed in the emulsion. The emulsion is
allowed to equilibrate with the anesthetic gas or vapour at a
chosen partial pressure. When stored in gas tight containers, these
lipid emulsions show sufficient stability for the anesthetic not to
be released as a gas over conventional storage periods.
[0094] The lipid emulsions of the present invention may be loaded
so that the xenon is at the saturation level. Alternatively, the
xenon may be present in lower concentrations, provided, for
example, that the administration of the emulsion produces the
desired pharmaceutical activity.
[0095] The concentration of xenon employed in the invention may be
the minimum concentration required to achieve the desired clinical
effect. It is usual for a physician to determine the actual dosage
that will be most suitable for an individual patient, and this dose
will vary with the age, weight and response of the particular
patient. There can, of course, be individual instance where higher
or lower dosage ranges are merited, and such are within the scope
of this invention.
[0096] Preferably, the medicament is in a form suitable for
intravenous, neuraxial or transdermal delivery.
[0097] A further aspect of the invention relates to a method of
preventing and/or alleviating anesthetic-induced neurological
deficits in a subject, preferably a neonatal subject, said method
comprising administering a therapeutically effective amount of
xenon to said subject.
[0098] A further aspect of the invention relates to a method of
treating and/or alleviating and/or preventing anesthetic-induced
neurodegeneration in a subject, preferably a neonatal subject, said
method comprising administering a therapeutically effective amount
of xenon to said subject.
[0099] A further aspect of the invention relates to a method of
treating and/or alleviating and/or preventing anesthetic-induced
neuronal apoptosis in a subject, preferably a neonatal subject,
said method comprising administering a therapeutically effective
amount of xenon to said subject.
[0100] A further aspect of the invention relates to a method of
treating and/or alleviating and/or preventing anesthetic-induced
neuronal injury in a subject, preferably a neonatal subject, said
method comprising administering a therapeutically effective amount
of xenon to said subject.
[0101] A method of preventing and/or alleviating isoflurane-induced
neuronal injury in a subject, preferably a neonatal subject, said
method comprising administering to said subject xenon and
isoflurane.
[0102] A method of preventing and/or alleviating
sevoflurane-induced neuronal injury in a subject, preferably a
neonatal subject, said method comprising administering to said
subject xenon and sevoflurane.
[0103] A method of preventing and/or alleviating desflurane-induced
neuronal injury in a subject, preferably a neonatal subject, said
method comprising administering to said subject xenon and
desflurane.
[0104] A method of providing anesthesia and/or analgesia in a
subject, preferably a neonatal subject, said method comprising
administering xenon in combination with isoflurane, wherein the
amount of xenon is sufficient to alleviate and/or prevent
isoflurane-induced neuronal injury.
[0105] A method of providing anesthesia and/or analgesia in a
subject, preferably a neonatal subject, said method comprising
administering xenon in combination with sevoflurane, wherein the
amount of xenon is sufficient to alleviate and/or prevent
sevoflurane-induced neuronal injury.
[0106] A method of providing anesthesia and/or analgesia in a
subject, preferably a neonatal subject, said method comprising
administering xenon in combination with desflurane, wherein the
amount of xenon is sufficient to alleviate and/or prevent
desflurane-induced neuronal injury.
[0107] Preferred embodiments for all of the above methods are
identical to those given above for the corresponding use
aspects.
[0108] Yet another aspect of the invention relates to an anesthetic
formulation for preventing and/or alleviating one or more
anesthetic-induced neurological deficits in a subject, preferably a
neonatal subject, said formulation comprising xenon and a
pharmaceutically acceptable diluent, excipient and/or carrier.
[0109] Yet another aspect of the invention relate to an anesthetic
formulation for treating and/or alleviating and/or preventing
anesthetic-induced neurodegeneration in a subject, preferably a
neonatal subject, said formulation comprising xenon and a
pharmaceutically acceptable diluent, excipient and/or carrier.
[0110] A further aspect of the invention relates to an anesthetic
formulation for treating and/or alleviating and/or preventing
anesthetic-induced neuronal apoptosis in a subject, preferably a
neonatal subject, said formulation comprising xenon and a
pharmaceutically acceptable diluent, excipient and/or carrier.
[0111] A further aspect of the invention relates to an anesthetic
formulation for treating and/or alleviating and/or preventing
anesthetic-induced neuronal necrosis in a subject, preferably a
neonatal subject, said formulation comprising xenon and a
pharmaceutically acceptable diluent, excipient and/or carrier.
[0112] In a preferred embodiment, the anesthetic formulation of the
invention further comprises an anesthetic agent.
[0113] More preferably, the anesthetic agent is a GABAergic
agent.
[0114] Even more preferably, the anesthetic agent is isoflurane,
sevoflurane or desflurane.
[0115] Another aspect of the invention relates to an anesthetic
formulation comprising 60% xenon, 0.75% isoflurane, 25% oxygen and
with the balance as nitrogen.
Xenon
[0116] As an anesthetic gas, xenon exhibits many desirable
qualities including cardiostability (Stowe et al., 2000), a low
blood-gas coefficient (Nakata et al., 1997) (the explanation for
xenon's fast induction and emergence times), and a potent analgesic
effect (Ma et al., 2004). Given the inevitable restricted
application of this extremely rare and costly gas, xenon may find a
niche as a prophylactic intra-operative neuroprotective anesthetic
(Mayumi Homi et al., 2003).
[0117] The neuroprotective effects of xenon have been observed in
in vivo models of acute neuronal injury involving administration of
excitotoxins to rats (Ma et al 2002), cardiopulmonary bypass in
rats (Mia et al 2003b), middle cerebral artery occlusion in mice
(Homi et al, 2003), cardiac arrest in pigs (Schmidt et al 2005),
and hypoxia-ischaemia in neonatal rats (Ma et al, 2005). Xenon was
a more efficacious neuroprotective agent than either gavestinel (Ma
et al, 2005) or dizolcipine (Ma et al 2003a), two other NMDA
antagonists that have been clinically tested.
[0118] In vitro work has shown that xenon can protect against both
glutamate and oxygen glucose deprivation induced excitotoxicity
(Wilhelm et al., 2002; Ma et al., 2003a). At anesthetic
concentrations it vivo (75%), xenon has been shown to
dose-dependently protect against excitotoxic insults with the same
neuroprotective efficacy as MK3801 (Ma et al., 2002). Additionally,
the same experiments showed that even at this relatively high dose
of xenon, there is no evidence of any neurotoxicity in the
posterior cingulate or retrosplenial corticies. More recent studies
have shown that xenon based anesthesia provides a functional
neurological improvement in rats subjected to cardio-pulmonary
bypass (Ma et al., 2003b).
[0119] Electrophysiology experiments have characterised xenon as a
potent post-synaptic (De Sousa et al., 2000) non-competitive
inhibitor of NMDA receptors with little or no GABA mediated effects
(Franks et al., 1998). Although this may be the mechanism behind
the anesthetic effect, it is almost certain that xenon has other
sites of action that are yet to be elucidated. This theory is
supported by xenon's ability to act in opposition to other NMDA
receptor antagonists, by attenuating their neurotoxic effects
(Nagata et al., 2001).
[0120] To date there is relatively little data on the effects of
xenon on the neonate. In terms of safety, studies have shown that
xenon, in contrast to N.sub.2O, does not interfere with PKC control
of the extending axon in vitro (Fukura et al., 2000) or exhibit
teratogenic properties in vivo (Lane et al., 1980). Concerning
efficacy, xenon has been shown to be an effective analgesic agent
in neonatal rats (Ma et al., 2004).
[0121] The In vivo Rat Model of Anesthesia: Protocol
[0122] Preliminary experiments suggested that 75% xenon+0.75%
isoflurane was too high a dose for neonatal rat pups--inducing
apnoea in 100% of test subjects within 10 min. This is supported by
existing data that xenon is a more potent anesthetic and analgesic
than nitrous oxide (Sanders et al., 2003). 60% xenon+0.75%
isoflurane was substituted as a concentration more likely to be of
equivalent MAC with 75% nitrous+0.75% isoflurane.
[0123] Sprague-Dawley are an inbred strain which display certain
phenotypic differences to rats used in earlier studies.
Specifically, when attempting to replicate previously used
high-dose regimens (Jevtovic-Todorovic et al., 2003), e.g. 75%
N.sub.2O+1% isoflurane+6 mg/kg midazolam, the rats exhibited a high
degree of susceptibility--mortality rates would have been 100% in
the absence of intervention to end anesthetic exposure. Thus, gas
concentrations for each group had to be adapted to induce a state
of anesthesia without causing apnoea.
Characterisation of Neurodegeneration
[0124] The Cupric-Silver technique (DeOlmos Silver Staining) has
repeatedly been shown to be excellent for highlighting the density
and distribution of neurodegeneration (Beltramino et al., 1993;
Jevtovic-Todorovic et al., 2003). The process highlights
argyrophilia (a generalised CNS response to injury (O'Callaghan and
Jensen, 1992)) to reveal cumulative neurodegeneration, so issues
surrounding the small timeframe of marker expression, as in other
techniques identifying gene products and enzyme activation, do not
apply (DeOlmos and Ingram, 1971).
[0125] Caspase-3 immunohistochemistry appeared to be acting as a
suitable marker of neuronal apoptosis. As a cytoplasmic enzyme,
activated caspase-3 stained cells were stained in their entirety,
hence making quantification relatively straightforward.
[0126] At the end of the apoptotic signalling cascade, caspase-9
activates caspase-3 (a cysteine protease), and thus caspase-3 is a
marker of those cells that are downstream of the apoptotic
commitment point. While broadly paralleling silver staining,
caspase-3 immunohistochemistry is superior for both quantification
purposes, and characterisation of physiological cell death (Olney
et al., 2002b).
[0127] C-Fos is one of the immediate early genes that has a role in
linking cytoplasmic events to nuclear gene transcription Walton et
al., 1998). As a regulator of gene expression, c-Fos indicates a
state of neuronal activation, a result of several possible
different external stimuli, including apoptotic cell death
(Dragunow and Preston, 1995) and pain (reviewed in Duckhyun and
Barr, 1995). C-Fos has previously been shown to be a sensitive
marker of the neurotoxicity of NA receptor antagonists in adult
rats (Ma et al., 2002) and is valid for assessment of NMDA receptor
activation (Hasegawa et al., 1998). The c-Fos immunohistochemistry
protocol (Mia et al., 2002), formed the entire basis of
quantification in the spinal cord formalin tests, staining
activated nuclei black.
Anesthetic-Induced Apoptosis
[0128] It can be deduced from existing data that the developing
human brain, both in utero and the first years of life, undergoes a
highly dynamic transformation from a fetal phenotype to one that
resembles the adult phenotype. The hallmarks of this process are an
extremely rapid turnover of synapses (as high as 20% per day (Okabe
et al., 1999)) and a high level of background apoptosis, as
neurones that fail to reach their synaptic targets are eliminated,
presumably to preserve energy-efficiency (Hua and Smith, 2004).
This study confirms that exposure to certain anesthetic agents
during this critical stage of neurodevelopment (synaptogenesis)
causes apoptosis in the developing brain. Experiments have
demonstrated that exposure to isoflurane, a commonly used GABAergic
inhalational agent, induces a 4-fold increase in the level of
apoptosis in the cortex. Also, nitrous oxide (whilst not
manifesting any neurodegenerative properties as an individual
agent) exhibits its neurodegenerative potential by significantly
enhancing isoflurane induced apoptosis to twelve times that seen in
controls. Similar results were obtained from the hippocampus, where
isoflurane and the isoflurane-nitrous oxide mixture increased the
level of apoptosis (4-fold and 7-fold respectively).
[0129] The hippocampus, a specialised fold of cortical tissue
forming part of the limbic system, has an important function in
memory formation (Aggleton and Brown, 1999).
[0130] Neurones in the hippocampus have the ability to exhibit the
phenomenon know as `long term potentiation` (LTP), whereby synaptic
efficacy is progressively strengthened by specific patterns of
neural activity. This process is thought to be the basis of memory
at the cellular level. Classically, hippocampal processing takes
place in both the hippocampus and the parahippocampal gyrus
(subiculum), before being projected to the fornix. Given the extent
of neuronal injury in the hippocampus and subiculum, it is not
surprising that rats exposed to high levels of anesthetics as
neonates show signs of learning deficits as adults
(Jevtovic-Todorovic et al., 2003) backed up by the finding of LTP
suppression in the same study.
[0131] Given the clear implications for paediatric anesthesia, much
work is underway to characterise the mechanism behind this process.
Activation of both the GABA receptor and the NMDA receptor are
known to influence survival signalling for neurones (Brunet et al.,
2001; Bittigau et al., 2002). With this in mind, the
ethanol-intoxicated mouse has formed the basis of an animal model
for the study of this process. Although caspase-3 is an excellent
marker of apoptotic cells, its position as the end-effector of a
highly divergent death signalling cascade offers little insight
into the mechanism of apoptosis. Caspase-3 activation is a common
step to both the extrinsic `death receptor` mediated and intrinsic
`mitochondrial` pathways of apoptosis (Green, 2000).
[0132] Young et al. hoped to narrow down the search to a single
pathway with a series of elegant experiments. A combination of dual
immunohistochemistry-immunofluorescence, Western blot analysis, and
knock-out mice was used to highlight pathway-specific components,
among them Bax and cytochrome c (intrinsic), and caspase-8
(extrinsic) (Young et al., 2003). It was found that whilst wild
type mice treated with ethanol exhibited the characteristic pattern
of ethanol-induced apoptosis, homozygous Bax-knockout mice given
the same treatment showed virtually no sign of apoptosis at all;
indeed, the level of apoptosis was lower than that seen in the
physiological cell death of controls0. Additionally, they
established that caspase-8 activation does not take place. This
clearly implicates the intrinsic apoptosis pathway in
anesthetic-induced apoptosis. This pathway, centred around the
mitochondria, is controlled by an assortment of pro- and
anti-apoptotic mediators in the cytosol of neurones. In the context
of developing neurones Bcl-X.sub.L (a member of the Bcl-2 family)
is principally anti-apoptotic, whereas Bax is pro-apoptotic (Yuan
and Yanker, 2000). Young et al. hypothesised that ethanol, a dual
NMDA receptor antagonist and GABAergic agent, has the ability to
dislodge Bax from the mitochondrial membrane, where is usually
stored in an inactive state. Once in the cytosol, Bax (if unchecked
by Bcl-X.sub.L) becomes part of an active complex, which is in turn
capable of retying to, and disrupting, the mitochondrial membrane
(Korsmeyer et al., 2002). The subsequent translocation of
mitochondrial contents (specifically cytochrome c--ordinarily part
of cellular energy production) into the cytosol is thought to
produce an extremely powerful pro-apoptotic signal. Cytosolic
cytochrome c forms a complex with Apaf-1 and capsase-8, which then
activates caspase-3, resulting in the initiation of further
cascades, ultimately causing the characteristic cleavage of both
cytoskeletal proteins and DNA (Dikranian et al., 2001). Of course,
from this analysis it is not possible to identify the exact point
at which anesthetics interact with this pathway. Also, individual
classes of agents are capable of inducing apoptosis (e.g.
isoflurane alone (Jevtovic-Todorovic et al., 2003) and ketamine
alone (Ikonomidou et al., 1999)), so use of a dual GABAergic agent
and NMDA receptor antagonist does not distinguish potential
differences between the two receptor interactions, although the
ensuing intracellular cascades may converge downstream (Brunet et
al., 2001; Bittigau et al., 2002). It is entirely possible that
isoflurane and or nitrous oxide can dysregulate the intracellular
Bax Bcl-2 ratio, perhaps by disrupting intracellular calcium
trafficking.
Use of Xenon During Synaptogenesis: Xenon as an Individual
Agent
[0133] The blood brain barrier effectively blocks the translocation
of many water-soluble substances from the blood to the CNS. It
achieves this via a network of tight-junctions, overlapping
astrocyte cover, and the relative absence of transport mechanisms.
However, none of these measures are an effective obstacle to xenon,
a small and apolar atom, which can rapidly attain anesthetic
concentrations in the brain (Sanders et al., 2003). Once at the
synapse, xenon is thought to produce its anesthetic effect through
non-competitive blockade of the NMDA receptor, albeit by a
mechanism that does not produce a typical open-channel block.
[0134] The results of the present study show that inducing a state
of anesthesia with 75% xenon does not cause apoptotic
neurodegeneration in the neonatal brain. Studies have conclusively
proved that blockade of the NMDA receptor is a key element of this
process possibly via deprivation of electrical or trophic
stimulation), with detrimental effects produced with use of MK801,
ketamine, phencyclidine (PCP), and
carboxypiperazin-4-yl-propyl-1-phosphonic acid (CCP) (Ikornomidou
et al., 1999). It is therefore peculiar that xenon, a potent NMDA
receptor antagonist (with 75% xenon equivalent to MK801 in some
contexts (Ma et al., 2002)), does not induce similar apoptotic
neurodegeneration. In light of the volume of biologically plausible
evidence pertaining to lack of trophic stimulation causing
apoptosis during NMDA receptor blockade (reviewed in Haberny et
al., 2002), it is tempting to suggest that xenon has a novel
anti-apoptotic property, mediated by an as-yet undefined target
(which could be membranous, cytoplasmic, mitochondrial or nuclear
given xenon's unusual pharmacodynamic and pharmacokinetic
properties). Whilst at least in theory xenon's unusual block of
NMDA receptors could be responsible (e.g. via an NMDA receptor
subunit that has a different distribution or level of expression in
the neonate such as NR2B or NR2D, or even a preferential effect at
extra-synaptic NMDA receptors (Hardingham et al., 2002)), xenon's
capacity to diametrically oppose NMDA receptor antagonist mediated
neurotoxicity suggests that there are other systems involved
(Nagata et al., 2001). It is therefore possible that as an NMDA
receptor antagonist, xenon could be inducing a degree of
pro-apoptotic signalling via an intracellular signalling cascade,
whilst the theoretical anti-apoptotic action simultaneously
disrupts the very same cascade at a downstream position (and thus
also blocking isoflurane-induced signalling).
Use of Xenon During Synaptogenesis: Xenon in Combination with
Isoflurane
[0135] In this study we demonstrated that concomitant
administration of 60% xenon can inhibit 0.75% isoflurane-induced
apoptosis 4-fold, to a level not statistically different from
controls exposed to air. This leads to the hypothesis that xenon
has an anti-apoptotic effect within the framework of
pharmacologically-induced apoptosis.
[0136] One well defined unique feature of Xenon is its lack of
effect at GABA receptors; it is this attribute which may underpin
some of xenon's atypical effects on the CNS. Consequently it is
reasonably safe to rule out any direct antagonism of isoflurane's
action at the GABA receptor. The only remaining possibility is
downstream disruption of the proposed isoflurane or nitrous
oxide-induced apoptosis pathway, either by xenon directly, or by an
indirect route possibly involving the modulation of other pathways
e.g. dopaminergic (Ma et al., 2002).
[0137] Comparison between the data for xenon alone and xenon in
combination with isoflurane suggests that xenon can lessen the
degree of neuronal injury induced by isoflurane, whilst having
minimal impact on the process of physiological cell death (seen in
controls). This implies that xenon can disrupt pathological
pro-apoptotic signalling. If xenon were to enhance the intrinsic
`survival` pathway (e.g. by upregulation of BCl-X.sub.L), then it
would be reasonable to expect a reduction in the level of PCD on
exposure to 75% xenon--which is clearly not the case. However, this
is highly conjectural given the lack of understanding into the
mechanisms involved; it is not currently known whether the
apoptotic pathways hijacked by conventional anesthetics are
identical to those controlling PCD. Answers may be found in an
in-depth analysis of the different pathways involved in xenon's
mechanism of action when compared to nitrous oxide (a gaseous NMDA
receptor antagonist which is currently the closest comparable
agent).
Clinical Implications
[0138] Given the price and MAC values of xenon, it is an economic
necessity (even with the most advanced reclaim-recycling systems)
as well as a clinical necessity for xenon anesthesia to be
maintained with another agent. The present work with combinations
of agents suggests that use of isoflurane, whilst inducing neuronal
apoptosis as an individual agent, is suitable for this purpose in
neonates.
[0139] Experiments have exposed xenon as potential treatment for
anesthetic-induced apoptosis. Thus, the use of xenon in paediatric
anesthesia (at economically feasible doses) could dramatically
increase the safety of current general anesthetic protocols.
[0140] In summary, these data add credence to the safe and
efficacious use of Xenon in the neonate; xenon is currently the
only known anesthetic shown not to induce neonatal neuronal
apoptosis at clinically applicable doses. This opens the
possibility of xenon-based anesthesia finding a cost-effective
niche within paediatrics as a safe, potently analgesic, and
potentially neuroprotective anesthetic agent.
Xenon/Sevoflurane Combinations
[0141] A further aspect of the invention relates to a combination
comprising xenon and sevoflurane. Preferably, the combination is a
synergistic combination.
[0142] Studies by the applicant have shown that surprisingly,
combinations of xenon and sevoflurane at concentrations at which
they are completely ineffective as individual agents, provide
striking neuroprotection when combined and administered to a
subject prior to hypoxic injury, i.e. xenon and sevoflurane in
combination exhibit a surprising and unexpected synergistic
protection against subsequent hypoxic injury.
[0143] Without wishing to be bound by theory, it is believed that
the protective effect is anti-necrotic, rather than anti-apoptotic,
i.e. the protective effect arises from the prevention of cell death
by necrosis. Cell death can occur by apoptosis or necrosis. In the
former, a stimulus initiates a cascade of events which ultimately
leads to cell death; apoptosis is often referred to as "programmed
cell death" and is a part of normal physiological development. In
contrast, necrosis involves a stimulus which directly induces the
death of the cell and is always a pathologic process.
[0144] Studies by the applicant have demonstrated that doses of
xenon and sevoflurane that are ineffective when administered as
individual agents work synergistically in combination, resulting in
a greater reduction in LDH release than corresponding
concentrations of the gases used alone. Experiments have shown that
neither sevoflurane at 0.67%, nor xenon at 12.5%, produces a
significant reduction in LDH release; thus using xenon or
sevoflurane as individual preconditioning agents offers no
significant protection from ischaemic damage. However when the two
gases are used in combination as preconditioning agents, LDH
release is significantly reduced. Further details of these
experiments may be found in the accompanying examples.
[0145] In one preferred embodiment, the xenon is administered in a
sub-therapeutically effective amount. In this context, the term
"sub-therapeutically effective amount" means an amount which is
lower than that typically required to produce anesthesia.
Generally, an atmosphere of about 70% xenon is sufficient to induce
or maintain anesthesia. Accordingly, a sub-therapeutic amount of
xenon corresponds to less than about 70% xenon.
[0146] Likewise, in one preferred embodiment, the sevoflurane is
administered in a sub-therapeutically effective amount. In this
context, the term "sub-therapeutically effective amount" means an
amount which is lower than that typically required to produce
anesthesia. Generally, an atmosphere of about 2.5% sevoflurane is
sufficient to maintain anesthesia. Accordingly, a sub-therapeutic
amount of sevoflurane corresponds to less than about 2.5%
sevoflurane.
[0147] Another aspect of the invention relates to a pharmaceutical
composition comprising xenon and sevoflurane and a pharmaceutically
acceptable diluent, excipient or carrier. Preferably, the
pharmaceutical composition is an anesthetic formulation.
[0148] In one preferred embodiment, the formulation comprises from
about 10 to about 30% xenon and from about 1 to about 5%
sevoflurane (v/v), with the balance comprising oxygen or nitrogen,
or a mixture thereof. More preferably, the formulation comprises
from about 10 to about 20% xenon and from about 2 to about 4%
sevoflurane, with the balance comprising oxygen or nitrogen, or a
mixture thereof.
[0149] In one highly preferred embodiment of the invention, the
formulation comprises about 12.5% xenon, about 0.67% sevoflurane,
about 25% oxygen and the balance nitrogen.
[0150] A further aspect of the invention relates to an anesthetic
formulation for preventing and/or alleviating one or more
sevoflurane-induced neurological deficits in a subject, said
formulation comprising xenon and a pharmaceutically acceptable
diluent, excipient and/or carrier.
[0151] Another aspect of the invention relates to an anesthetic
formulation for treating and/or alleviating and/or preventing
sevoflurane-induced neurodegeneration in a subject, said
formulation comprising xenon and a pharmaceutically acceptable
diluent, excipient and/or carrier.
[0152] Yet another aspect of the invention relates to an anesthetic
formulation for treating and/or alleviating and/or preventing
sevoflurane-induced neuronal apoptosis in a subject, said
formulation comprising xenon and a pharmaceutically acceptable
diluent, excipient and/or carrier.
[0153] One aspect of the invention relates to the use of xenon and
sevoflurane in the preparation of a medicament for providing
neuroprotection and/or anesthesia and/or analgesia.
[0154] Another aspect of the invention relates to the use of xenon
in the preparation of a medicament for providing neuroprotection
and/or anesthesia and/or analgesia, wherein said medicament is for
use in combination with sevoflurane.
[0155] Another aspect of the invention relates to the use of
sevoflurane in the preparation of a medicament for providing
neuroprotection and/or anesthesia and/or analgesia, wherein said
medicament is for use in combination with xenon.
[0156] A further aspect of the invention relates to the use of (i)
xenon, and (ii) sevoflurane, in the preparation of a medicament for
alleviating and/or preventing sevoflurane-induced neuronal injury
in a subject.
[0157] Another aspect of the invention relates to the use of xenon
in the preparation of a medicament for preventing and/or
alleviating one or more sevoflurane-induced neurological deficits
in a subject. Preferably, the neurological deficit is associated
with neuronal necrosis.
[0158] Another aspect of the invention relates to the use of xenon
in the preparation of a medicament for treating and/or alleviating
and/or preventing sevoflurane-induced neurodegeneration in a
subject.
[0159] Another aspect of the invention relates to the use of xenon
in the preparation of a medicament for treating and/or alleviating
and/or preventing neuronal necrosis in a subject.
[0160] Another aspect of the invention relates to the use of xenon
in the preparation of a medicament for treating and/or alleviating
and/or preventing sevoflurane-induced neuronal apoptosis in a
subject.
[0161] Another aspect of the invention relates to the use of xenon
in the preparation of a medicament for preventing and/or
alleviating sevoflurane-induced neuronal injury in a subject.
[0162] Another aspect of the invention relates to the use of xenon
and sevoflurane in the preparation of a medicament for providing
anesthesia in a subject, wherein the amount of xenon is sufficient
to alleviate or prevent sevoflurane-induced neuronal injury.
[0163] Yet another aspect of the invention relates to the use of
xenon in the preparation of a medicament for treating and/or
alleviating and/or preventing neuronal necrosis, or a condition
associated with neuronal necrosis.
[0164] Conditions associated with neuronal necrosis include, for
example, ischaemic infarction and traumatic infarction.
[0165] A further aspect of the invention relates to a method of
providing neuroprotection and/or anesthesia and/or analgesia in a
subject, said method comprising administering to said subject a
therapeutically effective amount of a combination of xenon and
sevoflurane.
[0166] Preferably, the xenon and sevoflurane are administered prior
to hypoxic-ischaemic injury, more preferably, at least 1 hour, more
preferably at least 2 hours prior to hypoxic-ischaemic injury. In
one particularly preferred embodiment, the xenon and sevoflurane
are administered from about 2 to about 24 hours prior to
hypoxic-ischaemic injury.
[0167] Preferably, the subject is a mammal, more preferably, a
human.
[0168] For all aspects of the invention, preferably the subject is
a neonatal subject.
[0169] In one preferred embodiment, the xenon and sevoflurane are
administered to the neonatal subject by administering to the mother
prior to and/or during labour, or prior to and/or during a cesarean
section.
[0170] Another aspect of the invention relates to a method of
preventing and/or alleviating sevoflurane-induced neurological
deficits in a subject, said method comprising administering a
therapeutically effective amount of xenon to said subject.
[0171] Another aspect of the invention relates to a method of
treating and/or alleviating and/or preventing sevoflurane-induced
neurodegeneration in a subject, said method comprising
administering a therapeutically effective amount of xenon to said
subject.
[0172] Another aspect of the invention relates to a method of
treating and/or alleviating and/or preventing sevoflurane-induced
neuronal apoptosis in a subject, said method comprising
administering a therapeutically effective amount of xenon to said
subject.
[0173] Another aspect of the invention relates to a method of
treating and/or alleviating and/or preventing sevoflurane-induced
neuronal necrosis in a subject, said method comprising
administering a therapeutically effective amount of xenon to said
subject.
[0174] Another aspect of the invention relate to a method of
treating and/or alleviating and/or preventing sevoflurane-induced
neuronal injury in a subject, said method comprising administering
a therapeutically effective amount of xenon to said subject.
[0175] Yet another aspect of the invention relates to a method of
preventing and/or alleviating sevoflurane-induced neuronal injury
in a subject, said method comprising administering to said subject
xenon and sevoflurane.
[0176] Another aspect of the invention relates to a method of
providing anesthesia and/or analgesia in a subject, said method
comprising administering xenon in combination with sevoflurane,
wherein the amount of xenon is sufficient to alleviate and/or
prevent sevoflurane-induced neuronal injury.
[0177] Another aspect of the invention relates to a method of
treating and/or alleviating and/or preventing neuronal necrosis, or
a condition associated with neuronal necrosis, in a subject, said
method comprising administering a therapeutically effective amount
of xenon to said subject.
[0178] Another aspect of the invention relates to a method of
treating and/or alleviating and/or preventing neuronal necrosis, or
a condition associated with neuronal necrosis, in a subject, said
method comprising administering a therapeutically effective amount
of xenon to said subject.
[0179] Yet another aspect of the invention relates to the use of
xenon and isoflurane in the preparation of a medicament for use as
a preconditioning agent for protecting against hypoxic injury.
[0180] As used throughout, the term "preconditioning agent" refers
to a medicament that is capable of alleviating and/or preventing
neuronal damage that may arise from a subsequent hypoxic injury.
Typically, preconditioning agents may be administered prior to
potentially injurious events such as invasive surgery,
cardiopulmonary bypass (CPB), organ transplant, labour, prior to
uterine implantation of fertilized embryo (as part of in vitro
fertilization), neuromuscular surgical procedures, brain tumour
resection and the like. Preconditioning agents may also be
administered after one or more injurious events where the subject
may be at risk of subsequent further injurious events, for example,
stroke patients.
[0181] Preferably, when used as a preconditioning agent, the xenon
is administered prior to hypoxic-ischaemic injury, more preferably,
at least 1 hour, more preferably at least 2 hours prior to
hypoxic-ischaemic injury. In one particularly preferred embodiment,
the xenon is administered from about 2 to about 24 hours prior to
hypoxic-ischaemic injury.
[0182] Yet another aspect of the invention relates to the use of
xenon in the preparation of a medicament for use as a
preconditioning agent for protecting against hypoxic injury,
wherein said medicament is for use in combination with
sevoflurane.
[0183] Yet another aspect of the invention relates to the use of
sevoflurane in the preparation of a medicament for use as a
preconditioning agent for protecting against hypoxic injury,
wherein said medicament is for use in combination with xenon.
[0184] A further aspect of the invention relates to a method of
protecting a subject from hypoxic injury, said method comprising
administering to said subject a therapeutically effective amount of
a combination of xenon and sevoflurane.
[0185] The present invention is further described by way of
non-limiting example and with reference to the following figures
wherein:
[0186] FIG. 1 shows rats anesthetised for a period of 6 hrs
(neurodegeneration experiments) or 105 min (formalin test). Once
the brains were removed, sections were cut to include the region of
interest: a coronal section -3.6 mm from the bregma
(neurodegeneration experiments) or a transverse section of the
lumbar enlargement of the spinal cord (formalin test). FIG. 1A:
Set-up for closed-circuit xenon anesthesia. FIG. 1B: Diagram
depicting a sagittal view through the neonatal rat brain, and the
transverse slice used for counting. FIG. 1C: Diagram of a
transverse section through the lumbar enlargement of the spinal
cord of the neonatal rat--dotted lines represent boundaries of
counting regions, taken from a previously used protocol (Duckhyun
and Barr, 1995).
[0187] FIG. 2 shows silver stained sections. DeOlmos silver
staining was employed to determine potential areas of interest for
immunohistochemistry. Rats were anesthetised with various gas
combinations, had their brains removed, and sections cut for
DeOlmos silver staining. Areas of non-specific neurodegeneration
are stained black (.times.4 magnification). FIG. 2A:
Photomicrograph of the cortex of a control animal, showing low
silver uptake. FIG. 2B: Photomicrograph of the cortex of a rat
exposed to 75% nitrous oxide+0.75% isoflurane, showing silver
uptake in specific cortical layers. FIG. 2C: Photomicrograph of the
hippocampus of a control animal, showing low silver uptake. FIG.
2D: Photomicrograph of the hippocampus of a rat exposed to 75%
nitrous oxide+0.75% isoflurane, showing extensive silver
uptake.
[0188] FIG. 3 shows cortical and hippocampal apoptotic
neurodegeneration induced by exposure to anesthetics in neonatal
rats: mean data. Apoptotic neurodegeneration induced in the cortex
and hippocampus by mock anesthesia or exposure to anesthetics (75%
nitrous oxide, 75% xenon, 0.75% isoflurane, 75% nitrous oxide+0.75%
isoflurane or 75% xenon+0.75% isoflurane) as measured with
caspase-3 immunostaining in the cortex of 7 day old neonatal rats.
FIG. 3A: Mean data from cortex (mean.+-.SD, n=3) from all treatment
groups. **p<0.01 vs air; ***p<0.001 vs air. FIG. 3B: Mean
data from hippocampus (mean.+-.SD, n=3) from all treatment groups.
**p<0.01 vs air.
[0189] FIG. 4 shows cortical apoptotic neurodegeneration in
neonatal rats exposed to individual anesthetic agents.
Photomicrographs (.times.4 magnification) showing caspase-3
immunostaining of the cortex, highlighting cells destined for
apoptosis (black staining). Photomicrographs (.times.4
magnification) correspond to gas exposure: air (A), 75% nitrous
oxide (B), 75% xenon (C), or 0.75% isoflurane (D) for 6 hrs.
[0190] FIG. 5 shows cortical apoptotic neurodegeneration in
neonatal rats C-posed to combinations of anesthetic agents.
Photomicrographs (.times.4 magnification) comparing caspase-3
staining in the cortex of neonatal rats exposed to either 75%
nitrous oxide+0.75% isoflurane (A), or 60% xenon+0.75% isoflurane
(B) for 6 hrs. Despite the fact that both nitrous oxide and xenon
are characterised as NMDA receptor antagonists, they exhibit
diametrically opposite properties when modulating
isoflurane-induced apoptosis (enhancing and attenuating
respectively). High power light microscopy (.times.20
magnification) confirmed that entire neurones where being stained,
in keeping with caspase-3 being a cytoplasmic enzyme (C).
[0191] FIG. 6 shows hippocampal apoptotic neurodegeneration induced
by exposure to anesthetics in neonatal rats. Following a 6 hr gas
exposure, caspase-3 immunostaining of the hippocampus was performed
to highlight cells destined for apoptosis (black staining).
Photomicrographs (at .times.4 magnification) correspond to gas
exposure: air (A), 75% nitrous oxide (B), 75% xenon (C), 0.75%
isoflurane (D), 75% nitrous oxide+0.75% isoflurane (E), and 60%
xenon+0.75% isoflurane (F).
[0192] FIG. 7 shows the results from formalin testing. The
analgesic potential of (75% nitrous oxide+0.75% isoflurane) was
compared to (60% xenon+0.75% isoflurane) using a formalin test to
quantify the nociceptive response to a formalin injection to the
left-hind paw via c-Fos expression in the spinal cord. FIG. 7A:
Mean data (mean.+-.SD, n=3) from all treatment groups. ***
p<0.001 vs formalin injected controls; .sup.+p<0.05 vs
N.sub.2O+Iso. FIG. 7B: Photomicrograph of spinal cord slice for 75%
nitrous oxide+0.75% isoflurane. FIG. 7C: Photomicrograph of spinal
cord slice for 60% xenon+0.75% isoflurane.
[0193] FIG. 8 shows the flow diagram of LDH assay protocol.
[0194] FIG. 9 shows the flow diagram of the protocol to assess the
necrotic, viable and apoptotic cell populations after
preconditioning.
[0195] FIG. 10 shows the graph of LDH release against xenon
concentration. The cells were preconditioned for 2 hours followed
by OGD) (oxygen glucose deprivation).
[0196] FIG. 11 shows the graph of LDH release against sevoflurane
concentrations. The cells were preconditioned for 2 hours followed
by OGD.
[0197] FIG. 12 shows the graph of LDH release against Xenon
preconditioning, sevoflurane preconditioning an combination of
preconditioning. The cells were preconditioned for 2 hours followed
by OGD.
[0198] FIG. 13 shows combination preconditioning, using FACS
analysis of necrotic, viable and apoptotic cell populations.
EXAMPLES
Materials and Methods
[0199] This study conforms to the UIK Animals (Scientific
Procedures) Act of 1986 and the study protocol has Home Office
approval.
Example 1
Exposure to Anesthetic Gases
Animals
[0200] 7 day old Sprague-Dawley rat pups were placed in individual
wells of a custom-built anesthetic chamber, and randomised to
groups A-F to receive one of 6 gas combinations for 6 hours.
Previous work has established that NMDA receptor antagonists have
their maximal neurodegenerative affect 7 days after birth
(Ikonomidou et al., 1999).
Gas Delivery
[0201] Group B received 75% nitrous oxide and 25% oxygen as
delivered by a calibrated anesthetic machine, whereas group C
received 75% xenon along with 25% oxygen through a customised
anesthetic machine modified for xenon delivery (Ohmeda, modified by
Air Products, Surrey, UK). Group D were exposed to 25% oxygen along
with 0.75%; isoflurane. The remaining 2 groups were exposed to
combinations of gases--namely 25% oxygen+75% A nitrous oxide+0.75%
isoflurane (group E) and 25% oxygen+60% xenon+15% nitrogen+0.75%
isoflurane (group F). The high cost of xenon precludes its use in
an open-circuit, consequently group C and group F received gases
using a closed-circuit system (FIG. 1A), whereas gases for all
other groups were delivered in a high-flow open-circuit.
Monitoring
[0202] All rats were kept normothermic throughout using a water
bath combined with a thermostat. Gas concentrations were monitored
with a S/5 spirometry module (Datex-Ohmeda, Bradford, UK), and the
rats themselves were regularly checked for signs of respiratory
distress. Given the inert chemical characteristics of gaseous
xenon, a special 439XE monitor (Air Products, Surrey, UK), was used
to verify the delivery of anesthetic concentrations of xenon, based
on radiofrequency analysis.
Tissue Perfusion, Harvesting, and Fixation
[0203] Rats destined for immunohistochemistry were sacrificed with
100 mg kg.sup.-1 sodium pentobarbital IP immediately
post-anesthesia, whereas those rats for DeOlmos silver staining
were allowed to recover for 18 hrs before undergoing the same
procedure. A thoracotomy was performed, and the aorta cannulated
via a needle inserted into the apex of the heart. The pup was then
perfused with 10 ml of 1% heparin solution, with the excess
solution leaving through an incision in the right atrium. To fix
the tissues, 20 ml of 4% paraformaldehyde in 0.1M phosphate buffer
was injected by the same transcardial route. The whole brain was
then removed and allowed to fix in paraformaldehyde perfusate and
refrigerated at 4.degree. C. 24 hours later, the brains were
transferred to a solution of 30% sucrose with phosphate buffer and
1% sodium azide, and were kept refrigerated until the brains sank
(approximately 48 hours).
Sectioning
[0204] Once processed, a block was cut to safely include the area
of interest--a coronal section -3.6 mm from the bregma (FIG. 1B).
The blocks were then embedded and frozen into O.C.T. solution. The
block was then cut coronally into approximately 120 slices, each 30
.mu.m thick, with a cryostat (Bright Instrument Company Ltd.,
Huntingdon, UK). The cut sections were transferred to a 6 well
plate containing phosphate buffered saline (PBS).
Staining Protocols
DeOlmos Silver Staining
[0205] DeOlmos silver staining was carried out according to an
established protocol (DeOlmos and Ingram, 1971). The floating
sections were mounted onto adhesive polysine slides, washed in
distilled water, and then incubated in a copper-silver mixture
(1000 ml 2.5% silver nitrate, 15 ml 0.5% cupric nitrate, 40 ml of
pyridine and 30 ml of 95% ethyl alcohol). After 4 days the sections
were removed, treated with 100% acetone for 5 min, and then
transferred to freshly prepared ammoniacal silver nitrate stock
(300 ml of distilled water, 200 ml of 0.36% NaOH, 90 ml
concentrated ammonium hydroxide and 10 ml of 20% silver nitrate)
for 15 min. Immediately following the ammoniacal silver nitrate,
the slides were placed in a reducer solution made of 24 ml 10%
non-neutralised formalin, 14 ml of 1% citric acid, 200 ml of 100%
ethanol and 1762 ml of distilled water for 2 min. To complete
processing, the sections had their background stained yellow with
0.5% potassium ferricyanide, were bleached for 1 min in 1% sodium
thiosulphate and then washed in distilled water. They were then
gently dehydrated in 70%, 90% and 100% ethanol. The ethanol was
then cleared with two 5 min exposures to 100% xylene. While still
wet with xylene, the slides had 2 drops of styrolite coverslip
media (BDH, Poole, UK) added, and were then coverslipped. Having
tapped out the air bubbles, the slides were allowed to dry
overnight before light microscopy.
Caspase-3 Immunohistochemistry
[0206] A random well from each cut block, each containing around 20
representative slices of the total block, was transferred to a
marked silk-bottomed well using a 3 ml Pasteur pipette. The
sections were then washed in 5 ml of PBS for 5 min on a shaker set
at 75 rpm. This washing procedure was repeated twice more,
replacing the PBS each time. To quench the sections, they were
incubated at room temperature on a shaker for 30 min in a solution
comprising 35 ml methanol, 15 ml of PBS and 500 .mu.l of stock 30%
H.sub.2O.sub.2. The quenching solution was then removed, and the
sections washed three times in PBS. Sections were blocked for 60
min at room temperature with 50 ml of PBST (PB containing 0.5%
Triton-X (Promega Corporation, Madison, Wis.)), and 1500 .mu.l of
normal goat serum (NGS) (Vector Laboratories Inc., Burlingame,
Calif.). For incubation with the primary antibody, the sections
were kept overnight at 4.degree. C. on a shaker set at 50 rpm in a
solution made up of 16 .mu.l (1:1500) rabbit anti-cleaved caspase-3
antibody (New England Biolabs, Hertfordshire, UK), 50 ml of PBST
and 500 .mu.l of NGS. The next day the sections were washed 3 times
in PBST and then incubated with the secondary antibody for 60 min
in a solution made up with 50 ml of PBST, 750 .mu.l of NGS and 250
.mu.l of goat anti-rabbit IgG antibody (Chemicon International,
Temecula, Calif.). Following a further 3 washes in PBST, the
sections were incubated in freshly prepared ABC solution from a
Vectastain ABC kit (Vector Laboratories Inc., Burlingame, Calif.)
for 60 min. The ABC solution was then washed off with 3 changes of
PBS, whilst fresh 3,3'-diamino-benzidine (DAB) solution was
prepared, which included distilled water, buffer, DAB stock,
H.sub.2O.sub.2 and nickel solution from a peroxidase substrate kit
(Vector Laboratories Inc. Burlingame, Calif.). The slices were
immersed in DAB solution for 4 min at room temperature, immediately
washed 3 times with PBS to end staining, and then washed 3 times
with distilled water.
[0207] To mount the sections onto microscope slides, the well
contents were floated into distilled water and the individual
sections transferred to superfrost slides using a fine paintbrush.
Once mounted, slides were allowed to dry overnight. To complete
processing of the slides, the samples slides were then dehydrated,
cleared and coverslipped as for the DeOlmos silver staining.
C-Fos Immunohistochemistry
[0208] The c-Fos immunohistochemistry was performed in parallel
with the caspase-3 immunohistochemistry with only three changes to
the protocol. Whereas NGS was used in the caspase-3 protocol,
normal donkey serum (NDS) (Chemicon International, Temecula,
Calif.) was used for the c-Fos wells. The primary antibody used was
20 .mu.l (1:2500) of goat anti-c-Fos antibody (Santa Cruz
Biotechnology, Santa Cruz, Calif.), and the secondry antibody was
250 .mu.l of donkey anti-goat antibody (Chemicon International,
Temecula, Calif.). All other stages of c-Fos immunostaining were
identical to caspase-3 immunohistochemistry protocol.
Quantification
[0209] The number of degenerating or activated neurones was
determined by counting the number of DAB stained (black) cells in a
coronal section of one hemisphere around -3.6 mm from the bregma
visualised on a BX-60 light microscope (Olympus, Southall, UK) and
example photomicrographs were taken with a Axiocam digital camera
(Zeiss, Gottingen, Germany). Data was collected for both the cortex
and the hippocampus across 3 slices, after which the mean number of
degenerating neurones was calculated. Those sections stained with
the silver staining method were photographed down the microscope
without any formal counting.
Data Analysis
[0210] All results are expressed as mean.+-.standard deviation.
Statistical analysis comprised a parametric repeated measures
analysis of variance of means followed by a Newman-Keuls test for
multiple comparisons across groups A-F. A P value of <0.05 was
considered statistically significant.
Formalin Testing
[0211] Formalin testing was carried out according to an established
protocol (Ma et al., 2004) to compare group E with group F. Rats
from one litter were randomised to one of 4 groups to receive
different injections and gases: air+formalin, air+saline, 60%
xenon+0.75% isoflurane+formalin or 75% nitrous oxide+0.75%
isoflurane+formalin. All rats were exposed to the respective gas
mixture for 15 min, and then had the left-hind paw injected with
either formalin (10 .mu.l of 5% formalin) or an equivalent volume
of saline. Following a further 90 min of gas exposure, the
animals/spinal cord samples were sacrificed, perfused and fixed as
in the main study. Out of the whole spinal cord, a block was cut,
comprising the lumbar enlargement. 30 .mu.m transverse sections
were cut on a cryostat, and the sections processed for c-Fos
immunohistochemistry. After staining, 3 sections exhibiting maximal
c-Fos expression were selected and photographed from each group,
and the spinal cord divided into regions as reported previously
(FIG. 1C) (Ducklyuan and Barr, 1995). The mean number of c-Fos
positive cells was then calculated by region for statistical
analysis.
Results
DeOlmos Silver Staining
[0212] As a non-specific marker of regions undergoing
neurodegeneration, the DeOlmos silver staining particularly
highlighted both the hippocampus and specific cortical layers.
These areas showed extensive silver uptake, denoted by black
staining in sections exposed to anesthetics, as opposed to controls
subjected to mock anesthesia where uptake was minimal (FIG. 2).
Caspase-3 Immunohistochemistry
Cortical Activated Caspase-3
[0213] Neuronal cells exhibiting caspase-3 activation were readily
distinguishable from the background as black cell body and axonal
staining. The staining established the level of background level of
cortical capase-3 activation in rats exposed to air as 19.3.+-.6.4
(mean.+-.SD), n=4 (FIG. 3A). As individual agents, neither 75%
N.sub.2O nor 75% xenon induce a significant increase in caspase-3
positive cells (22.5.+-.5.9, n=3 and 19.7.+-.9.6, n=3 respectively;
p>0.05 vs air) whereas administration of 0.75% isoflurane alone
produced a moderate level of activated caspase-3 staining
(76.5.+-.11.4, n=5; p<0.01 vs air) (FIG. 4).
[0214] When combined with 0.75% isoflurane, 75% N.sub.2O
considerably enhances isoflurane-induced apoptosis (232.0.+-.19.9,
n=6; p<0.001 vs air) while 60% xenon reduces the injury
(26.7.+-.3.9, n=4; p>0.05 vs air) (FIG. 5).
Hippocampal Activated Caspase-3
[0215] Neither 75% N.sub.2O nor 75% xenon exhibited a significant
increase in caspase-3 positive cells above baseline (3.7.+-.1.4 and
5.0.+-.3.2 respectively vs 5.2.+-.1.8 in controls; p>0.05) (FIG.
3B). In contrast, 0.75% isoflurane alone significantly increases
the number of degenerating neurones (22.1.+-.9.6; p<0.01 vs
air), as did the combination of 0.75% isoflurane and 75% N.sub.2O
(34.8.+-.20.2; p<0.01 vs air) (FIG. 6). Dual administration of
60% xenon with 0.75% isoflurane reduced the degree of neuronal
injury to 5.8.+-.2.6; p=<0.05 vs air.
Spinal Cord C-Fos Expression (Formalin Test)
[0216] Both gas combinations (75% N.sub.2O+0.75% isoflurane and 60%
xenon+0.75% isoflurane) exhibited an analgesic effect by
suppressing c-Fos expression in all regions of the spinal cord vs
formalin-injected positive controls (p<0.001) (FIG. 7). In
laminae A/B, where c-Fos expression was maximal, the xenon
combination conferred a greater analgesic effect than that induced
by the nitrous combination (15.0.+-.1.7 vs 22.3.+-.4.3
respectively; p<0.05).
Example 2
Methods
Neuronal Glial Co-Culture
[0217] Whole cerebral neocortices (devoid of the hippocampus, basal
ganglia and meninges) were obtained from early post natal (1-2 day
old) pups of BALB/c mice. The pups were anaesthetised with
isoflurane and then decapitated with the heads placed immediately
into 4.degree. C. HSG solution, an isotonic, high sucrose glucose
solution made primarily from Hank's balanced salt solution (HBSS,
GibroBRL) enhanced with NaHCO.sub.3 (0.04 M), sucrose (0.2 M) and
D-Glucose (0.3 M) also containing antibiotic-anti-mycotic solution
(AAS, GibroBRL). Throughout the micro dissection process, brain
tissues were kept in 4.degree. C. HSG solution.
[0218] The brain tissue was then immersed in 0.25% trypsin and a
placed in a shaking air chamber for 50 minutes at 37.degree. C.
filled with 5% CO.sub.2 and 95% room air. DNase was then added to
the mixture and placed back into the shaking air chamber for a
further 15 minutes. The mixture was then centrifuged at 1600 rpm
for 10 minutes at 4.degree. C. and the supernatant was carefully
discarded. The cells were then resuspended and then plated at a
density of 6.25.times.10.sup.4 cells/cm.sup.2 on 24-multiwell
plates (Costar, Cambridge, Mass.) and cultured in a medium
consisting of Eagle's minimum essential medium augmented with 20 mM
glucose, 26 mM NaHCO.sub.3, 10% foetal bovine serum, 10%
heat-inactivated horse serum, AAS (Gibco, Paisley, UK), 2 mM
glutamine (Sigma, Poole, UK) and 10 ng/ml murine epidermal growth
factor (EGF) (GibcoBRL). Glial cells reached confluence about one
week after plating.
[0219] Using a similar procedure cortical neuronal cells were
obtained from fetal BALB/c mice at 14-16 days of gestation and
plated at a density of 1.25.times.10.sup.5 cells per cm.sup.2 on
the confluent monolayer of glial cells derived from the
corresponding genetic strain. Neuronal cells reached confluence 10
days after plating.
Pure Neuronal Culture
[0220] Neuronal cells were harvested from 19 day old embryonic mice
by caesarean section for pregnant BALB/c mice. 6-9 mouse brains
were removed from fetal mice and dissected to isolate whole
cerebral neocortices devoid of the hippocampus, basal ganglia and
meninges. Again throughout the micro dissection process, brain
tissues were kept in 4.degree. C. HSG solution. From here, a
similar plating procedure described above was performed. The cells
were plated at a density of 1.2.times.10.sup.5 cells, per cm.sup.2
on 24-multiwell plates (Cater, Cambridge, Mass.) and the cultures
were maintained at 37.degree. C. in a humidified 5% CO.sub.2
environment. Neurobasal Media supplemented with B27, glutamine and
AAS was used to resuspend the neuronal cells and as culture medium.
For every 10 ml of Neurobasal Media, the following supplements were
added: 200 .mu.l B27, 100 .mu.l antibiotic and 25 .mu.l glutamine.
Medium replacement for these cells was performed on day 2, 5 and 7
with pre-warmed 37.degree. C. culture medium (Neurobasal Media,
B27, Glutamine and AAS). On day 5 after neuronal plating, 100
.mu.l/10 ml cytosine arabinoside (CA hydrochloride, Sigma) was
added to the cell cultures to halt non-neuronal cell division.
Neuronal cell cultures were ready to use on day 7.
Preconditioning
[0221] Cells were preconditioned using a purpose built 1.4 litre
airtight, temperature controlled gas chambers. The chambers had
inlet and outlet valves and an internal electric fan to ensure
effective and continuous delivery of gases. Gas flow rate was 100
ml/min, and so chambers were flushed and allowed to equilibrate for
45 minutes before establishing a closed system. Cells were
preconditioned for 2 hours inside the closed system with the
appropriate gas concentrations using flow meters. Sevoflurane was
delivered using a vaporiser (Datex-Ohmeda).
[0222] Preparation of gas impregnated solutions--Deoxygenated
balanced salt solution (BSS) was made by bubbling 5% CO.sub.2 and
95% N.sub.2 through sintered gas bubblers into the BSS in a
Drechsel bottle in a 37.degree. C. incubator.
Oxygen Glucose Deprivation
[0223] To model ischaemic damage in the brain, neuronal cells were
subjected to oxygen glucose deprivation. Twenty four hours after
preconditioning cells, cultures were washed twice with HEPES buffer
solution (120 mM NaCl, 5.4 mM KCL, 0.8 mM MgCl.sub.2, 15 mM glucose
and 20 mM HEPES, titrated to pH 7.4 using 1M NaOH). They were then
washed once with pre-warmed deoxygenated BSS minus glucose (116 mM
NaCl, 5.4 mM KCL, 0.8 mM MgSO.sub.4, 1 mM NaH.sub.2PO.sub.4, 1.8 mM
CaCl.sub.2, 26 mM NaHCO.sub.3) and then titrated to a pH of 7.4
using 2M HCl. The culture medium was then replaced with 600 .mu.l
of deoxygenated BSS and then immediately placed into a 37.degree.
C. air tight gas exposure chamber and left to equilibrate to an
anaerobic environment consisting of 5% CO.sub.2 and 95% N.sub.2.
The cells were exposed to this anoxic environment for 75 minutes.
Oxygen glucose deprivation was terminated by removing the cultures
from the gas chamber and changing the media; cultures destined for
lactate dehydrogenase (LDH) assay were washed once and replaced
with Eagle's minimal essential medium enhanced with 25 mM glucose
and 38 mM NaHCO.sub.3, whereas pure neuronal cultures for FACS were
washed once and replaced with Neurobasal Media supplemented with
B27, glutamine and AAS.
LDH Measurement
[0224] The amount of neuronal damage was assessed by the amount of
LDH released into the culture medium, using a standardised
colorimetric enzyme kit (Sigma Poole, UK). This technique has been
previously described (Wilhelm et al 2002). LDH assessment was
performed 16 hours after oxygen glucose deprivation (FIG. 8).
FACS Assessment
[0225] Twenty fours hours after oxygen glucose deprivation, cells
were stained for FACS analysis. The culture medium was removed and
washed twice with HEPES buffer solution. 100 .mu.l of 1.times.
binding buffer (BB) solution (50 mM HEPES, 750 mM NaCl, 12.5
mMCaCl.sub.2, 5 mM MgCl.sub.2, 20% BSA) with 0.4 .mu.l/ml Annexin V
(Sigma-Aldrich, Poole, UK) was then added and left to incubate on
ice for 10 minutes. Cells were then washed twice with 1.times. BB,
and then 0.8 .mu.g/ml propidium iodide (Sigma-Aldrich, Poole, UK)
in 1% fetal bovine serum (FBS) in phosphate buffer solution (PBS)
was added and left to incubate on ice for 5 minutes. This was
followed with washing twice with 1% PBS in PBS and then adding 400
.mu.l 0.25% trypsin/EDTA and left to incubate for 5 minutes at
37.degree. C. 800 .mu.l 1% FBS in PBS was then added to stop the
reaction, cells were removed and added to tubes for centrifugation
at 1200 g for 10 minutes. The supernatant was discarded and the
cells were re-suspended with 300 .mu.l 1% FBS in PBS. Steps, if
possible, were performed on ice to reduce the amount of neuronal
death.
[0226] A FACSCalibur (Becton Dickinson, Sunnyvale, Calif.) with a
single argon laser was used for flow cytometric analysis.
Excitation was carried out at 488 nm and the emission filters used
were 515-545 BP (green; FITC) and 600LP (red; PI). At least 10,000
cells per sample were analysed. Data acquisition was performed with
Cell Quest 3.3 (Becton Dickinson) and data analysis was performed
with Cell Quest Pro (Becton Dickinson) (FIG. 9).
Statistical Analysis
[0227] Statistical analysis was performed using Instat. Data was
expressed as mean+/-SEM. Statistical analysis of the data within
and between groups was performed with analysis of variance for
repeated measures followed by the Student-Newman-Keuls test.
Results are considered to be significant if P<0.05.
Results
Xenon Preconditioning
[0228] Preconditioning with xenon for 2 hours produces a
concentration dependent reduction in LDH release following oxygen
glucose deprivation (FIG. 10). LDH release was significantly
reduced by xenon at 50% and at 75%, to 55+/-12% and to 49+/-12% of
control values respectively (p<0.05). Xenon at 12.5% reduced LDH
release to 83+/-7% and xenon at 25% reduced LDH release to 70+/-11%
of controls. Xenon at 12.5% and 25% displayed a trend of decreasing
LDH release with increasing concentrations, however the results
were not significant (p>0.05).
Sevoflurane Preconditioning
[0229] Sevoflurane preconditioning for 2 hours also produces a
concentration dependent reduction in LDH release (FIG. 11).
Concentrations of sevoflurane greater than 1.9% produced a
significant reduction in LDH release. Concentrations of sevoflurane
less than 1.9% did not significantly reduce LDH release and thus
did not offer neuronal cells any protection from oxygen glucose
deprivation p>0.05). Sevoflurane at 2.7% resulted in a
significant decrease of LDH to 64+/-6% of control (p<0.05). LDH
release was maximally reduced at concentrations of 3.3% sevoflurane
to 37+/-5% of controls (p<0.001). Sevoflurane at 0.67% was found
to be ineffective, producing a reduction of LDH release to 97+/-5%
of controls, and sevoflurane at 1.3% also did not produce any
reduction in LDH release (100+/-11% of controls).
Xenon and Sevoflurane Combination Preconditioning
[0230] Ineffective doses of xenon and sevoflurane in combination
worked synergistically together resulting in a greater reduction in
LDH release than corresponding concentrations of the gases used
alone. In earlier experiments (FIG. 12), data showed that
sevoflurane at 0.67% and xenon at 12.5% were found not to produce
significant reduction in LDH release, and hence offered no
significant protection from ischaemic damage (p>0.05). However
when the two gases are used in combination as preconditioners, LDH
release was significantly reduced to 59+/-5% of controls
(p<0.001).
[0231] Assessment of Necrotic, Viable, and Apoptic Cell Populations
with Combination Preconditioning
[0232] To extrapolate the mechanisms behind xenon, sevoflurane, and
combination preconditioning, FACS was used to determine whether the
gases exerts its effects via an anti-apoptotic or anti-necrotic
mechanism. For this technique it is necessary to use pure neuronal
cultures.
[0233] Controls were unstained cells with no injury and no
preconditioning, in order to determine whether viable cells gave
off fluorescence and to define a viable cell region. The
effectiveness of xenon and sevoflurane combination used as
preconditioners to reduce the amount of neuronal injury following
oxygen glucose deprivation is consistent with data from the LDH
assay (FIG. 13). Sham preconditioning (injured cells with no
preconditioning), 12.5% xenon and 0.67% sevoflurane had a
significantly smaller viable cell population compared to controls
(p<0.001). Combination preconditioning had a viable cell
population of 23+/-1%, confirming synergy of the two gases in
reducing the amount of neuronal injury in an oxygen glucose
deprivation model compared to 9% in 12.5% xenon and 0.67%
sevoflurane (P<0.001).
[0234] Control groups had a necrotic population of 17+/-1%, whereas
sham preconditioning, 12.5% xenon preconditioning, 0.67%
sevoflurane preconditioning had necrotic populations of 70+/-2%,
75+/-2%, and 81+/2% respectively. However xenon and sevoflurane in
combination had a higher apoptotic population of 35%+/-3%, compared
to xenon alone and sevoflurane alone, with apoptotic populations of
9+/-1% (p<0.001) and 17+/-1% (p<0.001) respectively.
[0235] A combination of xenon and sevoflurane had a significantly
reduced necrotic cell population of 41+/-2% (<0.001). These data
suggest that xenon and sevoflurane when used in combination as
preconditioners provide substantial neuroprotection through an
anti-necrotic mechanism.
[0236] Various modifications and variations of the described
methods of the invention will be apparent to those skilled in the
art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific preferred embodiments, various modifications of the
described modes for catrying out the invention which are obvious to
those skilled in chemistry or related fields are intended to be
within the scope of the following claims.
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