Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/007—Pulmonary tract; Aromatherapy

Definitions

• the present invention relates to a method of treating neonatal asphyxia.
• Neonatal (or perinatal) asphyxia also known as hypoxia-ischemia (HI)
• HI hypoxia-ischemia
• Neonatal asphyxia remains a major cause of chronic neurological morbidity and acute mortality in the newborn (Balduini et al, 2000; Vannucci et al, 1997) and commonly leads to hypoxic-ischemic encephalopathy.
• Neonatal asphyxia meets the criteria for an orphan drug indication since it affects less then 5 patients in 10,000 inhabitants, and is a life- threatening, serious debilitating disease without an established therapy.
• the present invention seeks to provide a method of treating neonatal asphyxia.
• a first aspect of the invention relates to the use of xenon in the preparation of a medicament for the treatment of neonatal asphyxia, wherein said medicament is for use in combination with hypothermia.
• a second aspect of the invention relates to a method of treating neonatal asphyxia in a mammal in need thereof, said method comprising:
• a third aspect of the invention relates to a method of treating neonatal asphyxia in a mammal in need thereof, said method comprising administering a therapeutically effective amount of xenon to the mammal in combination with hypothermia.
• a fourth aspect of the invention relates to the use of xenon in the preparation of a medicament for the treatment of neonatal asphyxia, wherein said treatment comprises administering to a subject simultaneously, sequentially or separately xenon in combination with hypothermia.
• a fifth aspect of the invention relates to the use of xenon, in combination with hypothermia, for the treatment of neonatal asphyxia.
• EAAs glutamate and aspartate are the chief mediators of excitatory synaptic transmission in the mature central nervous system (CNS) (Dingledine and McBain, 1999). They also play a pivotal role in the ontogeny of the immature CNS, where they are involved in a number of physiological processes such as synaptogenesis, neuronal survival, synaptic plasticity, and dendritic and axonal structure. However, excessive activation of these amino acid receptors during development can produce neuronal injury and death. This is termed 'excitotoxicity'.
• Glutamate is the most abundant of the EAAs (Dingledine and McBain, 1999). It is stored in synaptic vesicles and causes calcium-dependent membrane depolarisation of postsynaptic membranes when it is released from the presynaptic terminals. Glutamate exerts its excitatory effect at a variety of receptor subtypes that can be divided into N- methyl-D-aspartate (NMDA) and non-NMDA types, but in the developing CNS it is the NMDA receptor subtype that has been found to play the primary role in brain injury associated with HI (Ikonomidou et al, 1989; Komuro, 1993; MacDonald et al, 1986).
• NMDA N- methyl-D-aspartate
• the NMDA receptor is a major subclass of glutamate receptor and glutamate is believed to be the most important excitatory neurotransmitter in the mammalian central nervous system. Importantly, activation of the NMDA receptor has been shown to be the central event which leads to excitotoxicity and neuronal death in many disease states, as well as a result of hypoxia and ischaemia following head trauma, stroke and following cardiac arrest.
• the NMDA receptor is an ionotropic receptor found ubiquitously throughout the CNS, located on the surface of both postsynaptic and extrasynaptic membranes (Riccio and Ginty, 2002; Sattler et al, 2000). It is coupled to a cationic channel that is permeable to both Na + and Ca 2+ ions and under normal physiological conditions, is blocked by Mg 2+ at a negative resting membrane potential. It becomes unblocked on depolarisation of 005/034966 4 the cell membrane, thus allowing an influx of Ca 2+ through the channel and enabling the receptor to exert its intracellular effects (Hardingham and Bading, 2003).
• NMDA receptors are vital for normal brain function and their importance in normal physiology is demonstrated by their central role in memory and learning (Bliss and Collingridge, 1993). Conversely, pathological activation of NMDA receptors by excess glutamate is the primary cause of neuronal cell death following an ischaemic insult to the brain, due to the disruption of intracellular Ca 2+ regulation. This emphasizes the central role played by NMDA receptors in HI.
• glutamate transporters located in neuronal cells, but expressed preferentially in glial cells.
• glutamate transporters located in neuronal cells, but expressed preferentially in glial cells.
• glutamate uptake carrier There are several different kinds of glutamate uptake carrier, but essentially, they all function in the same way, transporting two Na + cations and one glutamate anion into the cell, while transporting one K + cation and one OH " anion out of the cell and into the extracellular space (Dingledine and McBain, 1999).
• These ionic pumps act against an electrochemical gradient and thus rely on energy in the form of ATP in order to function correctly.
• Excitotoxicity occurs when the excess extracellular glutamate continuously activates postsynaptic receptors (particularly NMDA receptors) and the resulting calcium influx sets up an osmotic gradient down which water moves causing the cells to swell. Calcium-dependent enzyme systems are also activated within the cell and these two processes result in acute neuronal cell death (Choi and Rothman, 1990).
• Apoptosis and necrosis are usually distinguished by their distinctly different morphological appearances. Apoptosis requires ATP and new protein synthesis, and is identified by cell shrinkage, chromatin-clumping with margination and formation of membrane- enclosed apoptotic bodies, whereas necrosis is recognised by nuclear shrinkage with karyorrhectic and pyknotic nuclear changes (Hill et al, 1995).
• apoptosis is a regular event by which unwanted or damaged neurons 'commit suicide' (Ikonomidou et al, 2001).
• the initial excitotoxic cell death is mediated by excessive activation of NMDA receptors, resulting in the uncontrolled release of glutamate, which damages the surrounding neurons.
• the natural response to damage during synaptogenesis is for the neurons to initiate programmed cell death (Olney, 2003), and this is thought to be a mechanism that is activated to protect the neighbouring tissue (Leist and Nicotera, 1998).
• NMDA receptor plays a major role in the synaptic plasticity which underlies many higher cognitive functions, such as memory and learning, as well as in certain nociceptive pathways and in the perception of pain (Collingridge et al, The NMDA Receptor, Oxford University Press, 1994).
• certain properties of NMDA receptors suggest that they may be involved in the information-processing in the brain which underlies consciousness itself.
• NMDA receptor antagonists are therapeutically valuable for a number of reasons.
• NMDA receptor antagonists confer profound analgesia, a highly desirable component of general anaesthesia and sedation.
• NMDA receptor antagonists are neuroprotective under many clinically relevant circumstances (including ischemia, brain trauma, neuropathic pain states, and certain types of convulsions).
• NMDA receptor antagonists confer a valuable degree of amnesia.
• NMDA antagonists have been investigated as possible neuroprotective agents.
• Many NMDA antagonists, such as MK-801 and ketamine have been shown to be neuroprotective in both in vitro and in vivo models (Albers et al, 1989; Arias et al, 1999; Choi et al, 1988; Kudo et al, 2001).
• NMDA receptor antagonists have also been shown to have psychotomimetic side effects in humans (Krystal et al, 1994) and to cause damage to the posterior cingulate (PC) and retrosplenial cortices (RS) (Olney et al, 1991).
• NMDA receptor antagonists lead to the production of involuntary movements, stimulation of the sympathetic nervous system, induction of neurotoxicity at high doses (which is pertinent since NMDA receptor antagonists have low potencies as general anaesthetics), depression of the myocardium, and proconvulsions in some epileptogenic paradigms e.g., "kindling" (Wlaz P et al, Eur. J. Neurosci. 1994; 6: 1710-1719). There have also been considerable difficulties in developing new NMDA receptor antagonists that are able to cross the blood-brain barrier.
• Xenon is an apolar, inert gas that is a potent NMDA antagonist (Franks et al, 1998). Like other NMDA antagonists, it has also been shown to be neuroprotective against many forms of neuronal injury, both in vitro (Petzelt et al, 2003) and in vivo (Homi et al, 2003; Wilhelm et al, 2002). However, unlike many of the other NMDA receptor antagonists, xenon is not neurotoxic (Ma et al, 2002). A further advantage of using xenon as an NMDA antagonist is that the molecule is an inert, volatile gas that can be rapidly eliminated via respiration.
• Xenon has many other favourable properties. Since its first use in surgery (Cullen SC et al, Science 1951 ; 113:580-582), a number of research groups have shown it has an excellent pharmacological profile, including the absence of metabolic by-products, profound analgesia, rapid onset and recovery, and minimal effects on the cardiovascular system (Lachmann B et al, Lancet 1990; 335:1413-1415; Kennedy RR et al, Anaesth. Intens. Care 1992; 20:66-70; Luttropp HH et al, Acta Anaesthesiol. Scand.
• xenon is a small, uncharged atom, it can easily pass through the blood-brain barrier thus producing a rapid onset of action (Nakata et al, 2001). It also has a very low blood: gas partition coefficient lending to fast emergence from xenon anaesthesia (Goto et al, 1997). As well as these advantages, xenon is non-explosive, non-toxic and unreactive (Shichino et al, 2002), and this makes xenon an ideal candidate for use as a neuroprotectant in the neonate.
• neuroprotectant means an agent that is capable of providing neuroprotection, i.e., protecting a neural entity, such as a neuron from ongoing injury from,for example, an ischaemic injury or traumatic injury.
• hypothermia exerts its neuroprotective effect
• many theories have been postulated.
• the mechanisms by which hypothermia is protective are temperature and time-dependent, and may act at more than one point along the cascade of events that leads to HI injury (Yager and Asselin, 1996). This is supported by the fact that a moderate temperature of
• hypothermia Many other mechanisms of protection by hypothermia have been suggested, including the reduction of reactive oxygen species (Taylor et al, 2002), a reduction in tissue acidosis (Chopp et al, 1989) and the attenuation of post-HI neuronal apoptosis (Xu et al, 2002).
• a first aspect of the present invention relates to the use of xenon in the preparation of a medicament for the treatment of neonatal asphyxia in a neonatal subject, wherein said medicament is for use in combination with hypothermia.
• hypothermia refers to subjecting a particular subject (in this case, a neonatal subject) to hypothermic conditions, for example, by lowering the body temperature, preferably by 3-5°C, through passive or active techniques.
• subjecting to hypothermic conditions leads to a decrease in metabolism of body tissues of the subject, thereby decreasing the need for oxygen.
• hypothermia in the treatment of neonatal asphyxia has been well documented in the art (see for example, Volpe, 2001 ; Gunn et al, 2000).
• hypothermia could be use in combination with the administration of xenon.
• xenon has neuroprotective properties.
• WO 01/08692 the contents of which are incorporated herein by reference, relates to the use of xenon as a neuroprotectant and/or as an inhibitor of synaptic plasticity.
• xenon would be effective as a neuroprotectant in the context of the presently claimed invention.
• the xenon is admixed with a pharmaceutically acceptable diluent, excipient or carrier.
• 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).
• suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like.
• suitable diluents include ethanol, glycerol and water.
• 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).
• binders include starch, gelatin, natural sugars such as 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.
• suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
• Preservatives, stabilizers and dyes may be provided in the pharmaceutical composition.
• preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
• Antioxidants and suspending agents may be also used.
• the present invention is also applicable to the treatment of animals.
• the invention further relates to the use of xenon in combination with a veterinarily acceptable diluent, excipient or carrier.
• the xenon is typically administered in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.
• the xenon may also be administered in combination with another pharmaceutically active agent.
• the agent may be any suitable pharmaceutically active agent including anaesthetic or sedative agents which promote GABAergic activity. Examples of such GABAergic agents include isoflurane, propofoi and benzodiazapines.
• the xenon is administered in combination with a volatile anesthetic agent, preferably isoflurane, sevoflurane or desflurane.
• 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.
• 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.
• the xenon may be administered by any suitable delivery mechanism, or two or more suitable delivery mechanisms.
• the xenon is administered by perfusion.
• 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.
• 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.
• 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 surgery.
• the medicament is in gaseous form.
• the xenon is administered by inhalation. More preferably, the xenon is administered by inhalation of a 70-30% v/v xenon/oxygen mixture.
• the xenon is administered in the form of a 20-70% v/v xenon air mixture.
• the medicament is in the form of a liquid or solution.
• 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.
• 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®10, Intralipid®20, Intrafat®, Lipofundin®S or Liposyn® emulsions, or one specially formulated to maximise 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.
• 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.
• 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.
• xenon can be dissolved or dispersed in concentrations of 0.2 to 10 ml or more per ml of emulsion.
• concentration of dissolved gas is dependent on a number of factors, including temperature, pressure and the concentration of lipid.
• the lipid emulsions of the present invention may be loaded with gaseous xenon.
• a device is filled with the emulsion and anaesthetics as gases or vapours passed through sintered glass bubblers immersed in the emulsion.
• the emulsion is allowed to equilibrate with the anaesthetic gas or vapour at a chosen partial pressure.
• these lipid emulsions show sufficient stability for the anaesthetic not to be released as a gas over conventional storage periods.
• the lipid emulsions of the present invention may be loaded so that the xenon is at the saturation level.
• the xenon may be present in lower concentrations, provided, for example, that the administration of the emulsion produces the desired pharmaceutical activity.
• 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 instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
• the medicament is in a form suitable for intravenous, neuraxial or transdermal delivery.
• the xenon is administered simultaneously, in combination, sequentially or separately with hypothermia.
• xenon is administered concurrently with hypothermia
• in combination is used to mean the xenon is administered, if not simultaneously, then “sequentially” within a timeframe in which the xenon and the hypothermia both exhibit a therapeutic effect, i.e. they are both are available to act therapeutically within the same time-frame.
• administration "sequentially” may permit the xenon to be administered within 5 minutes, 10 minutes or a matter of hours before the hypothermia, provided the circulatory half-life of the xenon is such that it is present in a therapeutically effective amount when the neonatal subject is exposed to hypothermic conditions.
• the neonate is subjected to hypothermia prior to treatment with xenon.
• the xenon is administered sequentially with hypothermia.
• the xenon is administered sequentially before the hypothermia.
• the xenon is administered separately before the hypothermia.
• the xenon is administered sequentially after the hypothermia.
• the xenon is administered separately after the hypothermia.
• the xenon is administered sequentially or simultaneously with hypothermia, more preferably simultaneously.
• the xenon is administered in a therapeutically effective amount. In another preferred embodiment, the xenon is administered in a sub-therapeutically effective amount. In other words, the xenon is administered in an amount that would be insufficient to produce the desired therapeutic effect if administered in the absence of hypothermic conditions.
• the combination of xenon and hypothermia has a synergistic effect, i.e., the combination is synergistic.
• the xenon is administered prior to the hypoxic insult.
• the xenon is administered to the neonate via the mother prior to birth, for example, by administering to the mother prior to or during labour.
• the xenon is administered to the mother for up to about 48 or 24 hours prior to birth, more preferably up to about 12 hours, more preferably up to about 6 hours or 3 hours or 1 hour prior to birth.
• the neonate is then subjected to hypothermic conditions.
• Another aspect of the invention relates to a method of treating neonatal asphyxia in a mammal in need thereof, said method comprising:
• the hypothermia is maintained for a period of at least about 6 hours, more preferably at least about 12 hours, after the hypoxic-ischemic (HI) insult.
• HI hypoxic-ischemic
• hypothermia is maintained for a period of from about 6 to about 24 hours after the hypoxic-ischemic (HI) insult.
• HI hypoxic-ischemic
• the hypothermia is maintained for a period of at least about 6 hours, more preferably at least about 12 hours, after birth. In one preferred embodiment, the hypothermia is maintained for a period of from about
• treatment in accordance with the method of the invention is initiated within about 6 hours of the hypoxic-ischemic (HI) insult, and more preferably within about 2 hours of the hypoxic-ischemic insult.
• HI hypoxic-ischemic
• Hypothermia may be produced passively, by allowing the temperature to drift downwards and not purposefully sustain body temperature. Being poikilothermic, neonates rapidly assume the temperature of their surroundings. Alternatively the patient may be actively rendered hypothermic by deliberately reducing their ambient temperature.
• a second aspect of the invention relates to a method of treating neonatal asphyxia in a mammal in need thereof, said method comprising:
• the mammal is a newborn subject in the first four weeks after birth. More preferably, the mammal is in the first two weeks, more preferably still, the first week after birth.
• the mammal is a human.
• the mammal is subjected to conditions of mild hypothermia.
• mild hypothermia typically refers to a decrease in the core temperature from 37°C to about 33°C
• the temperature of the mammal is maintained at a temperature of from about 31 °C to about 36°C. More preferably, the temperature of the mammal is maintained at a temperature of from about 32 ° C to about 36 °C, more preferably from about 32 °C to about 35 °C, more preferably still from about 33 °C to about 35 ° C.
• Preferred embodiments for the second aspect of the invention are the same as those described above in respect of the first aspect.
• Another aspect of the invention relates to a method of treating neonatal asphyxia in a mammal in need thereof, said method comprising administering a therapeutically effective amount of xenon to the mammal in combination with hypothermia.
• Yet another aspect of the invention relates to the use of xenon in the preparation of a medicament for the treatment of neonatal asphyxia, wherein said treatment comprises administering to a subject simultaneously, sequentially or separately xenon in combination with hypothermia.
• a further aspect of the invention relates to the use of xenon, in combination with hypothermia, for the treatment of neonatal asphyxia.
• the neonatal rat HI model is very established and has been validated for use in a number of previous studies (Levine, 1960; Rice et al, 1981).
• the age of the rats used in this model has been found to correspond to the brain maturity of the term human neonate (Clancy et al, 2001; Ikonimidou et al, 1989) and thus a reasonably accurate comparison can be made between the two.
• the temperature of the rat pups was monitored using a probe that was inserted into the cortex of one of the pups. The probe took approximately 15 minutes to equilibrate and this was allowed for by delaying the start time of the experiment until the probe began recording the correct temperature.
• the anaesthetic gas xenon has been shown to exhibit neuroprotection in several models of adult neuronal injury.
• the results of this study corroborate previous findings that xenon has significant neuroprotective properties and in addition, suggest that this neuroprotection extends to neonatal models of brain injury induced by hypoxia-ischaemia.
• xenon significantly protects against neonatal HI via an anti-apoptotic mechanism. Both apoptosis and necrosis are important components of neuronal loss after HI injury, but apoptosis appears to be the more important type of cell death in determining neonatal outcome (Taylor et al, 1999).
• Apoptotic death is often preceded by the activation of many genes, (including transcription factors) which may be either pro-apoptotic or anti-apoptotic. As xenon appears to interfere with apoptotic cell death, it is possible that it may exert its effect on one of these genes, or at some point along the apoptotic pathway.
• genes including transcription factors
• xenon appears to interfere with apoptotic cell death, it is possible that it may exert its effect on one of these genes, or at some point along the apoptotic pathway.
• the extrinsic pathway the extrinsic pathway
• the intrinsic pathway the extrinsic pathway
• the extrinsic pathway (also referred to as the "death receptor pathway”) involves the binding of cytokines to death receptors which activate caspase-8 and this in turn, activates the "executor caspsase", caspase-3, which goes on to induce apoptotic cell death (Mehmet, 2000).
• the intrinsic pathway is heavily dependent on mitochondria and involves an increase in mitochondrial membrane permeability caused by the pro-apoptotic protein bax. This leads to the release of cytochrome c, the formation of a complex of cytochrome c, Apaf-1 (apoptosis protease-activating factor- 1) and caspase-9, and the subsequent activation of caspase-3.
• xenon acts on either one of these pathways, but there is evidence to suggest that apoptotic neurodegeneration induced by HI is mediated via the mitochondrial pathway and the initiation of bax-dependent mitochondrial changes (Taylor et al, 1999). Further to this, the NMDA antagonist ketamine has been shown to protect against incomplete cerebral ischaemia and reperfusion by early modulation of the balance between pro- and anti-apoptotic proteins, namely by inhibiting the Hl-induced bax increase (Engelhard et al, 2003). Thus it is possible that xenon could inhibit apoptosis by downregulating bax.
• Bcl-2 is an anti-apoptotic protein that acts to decrease the permeability of the mitochondria and hence inhibit the release of cytochrome c. Its overexpression has been shown to decrease neuronal damage caused by transient global cerebral ischaemia in gerbils (Engelhard et al, 2003). Therefore, the upregulation of bcl-2 is another potential target for xenon. As xenon is apolar and fat soluble, it is able to distribute itself widely throughout the body. It can penetrate membranes and consequently, it may also have an effect in the nucleus by altering gene transcription to upregulate survival pathways, or inhibiting the RNA and protein synthesis of pro- apoptotic molecules. Anti-necrosis by xenon was shown to be statistically significant in the cortex at 48 h, but not in the gyrus ( Figure 16). At all other time groups, xenon was not anti-necrotic.
• xenon exerts an anti-necrotic effect in the cortex at 48 h, it may be that while xenon is unable to prevent necrosis that occurs before its administration (as in the 16 and 24 h groups), it is some how able to combat the secondary necrotic wave that occurs after its administration.
• Initial necrosis occurs as early as 3 h after the HI insult (Northington et al, 2001) and at this point xenon has not yet been administered. It is therefore unlikely to be able to arrest or reverse a process that has already occurred.
• the secondary necrotic wave occurs at a time at which xenon has been present in the brain for 48 h, and this suggests that the presence of xenon at the advent of necrosis may be able to decrease this type of cell death. Further work must to be completed to ascertain the exact mechanism of this interaction.
• hypothermia is anti-necrotic and in the gyrus, it is anti-apoptotic ( Figure 16).
• the data in this study do not explain this effect, but one possible justification could be that different brain regions express differential vulnerability (Northington et al, 2001).
• the secondary necrotic wave (discussed above) occurs at a time at which hypothermia has already been administered, and this may make it more effective.
• the gyrus there is no delayed necrosis and thus no anti-necrotic effect is observed.
• Anti-apoptosis appears to be the neuroprotective mechanism in this region, and it is possible that the expected anti-apoptotic neuroprotective effect of hypothermia, that is not evident at the earlier time intervals, may be exposed after longer periods.
• the present study has shown using an in vivo rat model to show that xenon is neuroprotective in the neonate, and significantly protects against apoptosis induced by hypoxic-ischaemic injury.
• the data in this study also suggest that when xenon and hypothermia are used in combination in the same model, they interact synergistically to dramatically decrease apoptotic cell death. Accordingly, this combination may represent an effective treatment to protect against the devastating neurological consequences of neonatal asphyxia.
• Figure 1 shows the relationship between damage, as measured by loss of brain weight (ratio of right hemisphere/left) and the duration of the hypoxic period (in minutes) in Sprague-Dawley rats.
• Figure 2 shows brain sections from Sprague-Dawley rats that have suffered 90 minutes of hypoxia-ischemia injury.
• Figure 3 shows the major cellular damage which is evident in Sprague-Dawley rats 24 hours after 90 minutes of hypoxia-ischemia.
• Figure 4 shows the concentration-dependence of xenon neuroprotection (ratio right hemisphere weight/left against xenon concentration).
• Figure 5 shows the effect of 70% xenon on neurological functions assessed remotely after hypoxic-ischemic (HI) insult.
• Figure 6 compares the neuroprotective effect (ratio of right hemisphere/left) observed with with N 2 and xenon respectively, when xenon is administered 2 hours post HI insult.
• Figure 7 shows the effect of mild hypothermia on the neuroprotective effect of xenon (LDH release against xenon concentration, % atm).
• Figure 8 shows a van't Hoff plot of the natural logarithm of LDH release plotted against reciprocal absolute temperature.
• Figure 9 shows a photograph of the purpose-built airtight chambers used for gas delivery. The water bath and closed circuit xenon delivery system are also depicted.
• Figure 11 shows (A) Sagittal section of a rat brain modified from website: faculty.virginia.edu/... /RatBrainLabels.jpg.
• the broken line represents the area of the brain from which coronal sections (B) were taken, approximately -3.6 mm from the bregma.
• (B) shows a cresyl violet-stained coronal section.
• the boxes indicate the areas in which the counting frames were placed and the cells were analysed - the top box indicates the cortex and the bottom one, the gyrus.
• X overlies a hole that was intentionally created with a safety pin in order to demonstrate the non-ligated (contra lateral) hemisphere.
• Figure 12 shows a photomicrograph of the cortex (cresyl-violet stained), taken with a 1 OOx oil emersion lens and an Axiocam digital camera, demonstrating the difference in morphological appearance between an apoptotic, necrotic and viable cell.
• Viable cells stain less intensely than either type of cell death and therefore have a paler cytoplasm, whereas dead cells are more darkly stained.
• Necrotic and apoptotic cells are differentiated on the basis of their different nuclear appearances - necrotic nuclei are large and irregularly shaped, whereas apoptotic nuclei are small, shrunken, and spherically shaped.
• Figure 14 shows a photomicrograph demonstrating the cortex and gyrus in the sham, positive control and 75 % xenon animals at 16 hours.
• the 75 % group is more similar in appearance to the sham group than the positive control group. This confirms the neuroprotective effect of xenon at 16 hours.
• the gyrus of the control group is distorted in shape due to the increased amount of cell death and vacuolation.
• Figure 15 shows that xenon is neuroprotective at 24 h via an anti-apoptotic mechanism.
• Figure 16 shows that xenon is neuroprotective at 48 h via an anti-apoptotic mechanism More specifically, Figure 16 shows graphs for apoptotic and necrotic neuronal death induced by hypoxic-ischaemia, and the effects of 75% xenon and 33°C hypothermia on such cell death at 48 h in (A) the cortex, and (B) the gyrus. Xenon is neuroprotective via an anti-apoptotic mechanism in both the cortex and the gyrus. In addition, xenon has an anti-necrotic effect in the cortex.
• Figure 17 shows that a combination of xenon and hypothermia interact synergistically to produce anti-apoptotic neuroprotection. More specifically, Figure 17 shows graphs for apoptotic and necrotic neuronal death induced by hypoxic-ischaemia, and the effect of a combination of 20% xenon and 35°C hypothermia on such cell death at 16, 24 and 48 h in (A) the cortex, and (B) the gyrus. No difference was seen when the 20% xenon group and the 35°C hypothermia group were compared to positive controls, thus at these values there is no neuroprotection.
• Neonatal Asphyxia Model Seven day old postnatal Sprague-Dawley rats underwent right common carotid artery ligation under surgical anaesthesia (1%-1.5% isoflurane in pure oxygen). After ligation, the animals were returned to their mothers and placed in a specially designed area with constant of room temperature (23°C) and humidity (48%). One hour after surgery, neonatal rats were placed in specially designed chamber with 8% oxygen combined with 0, 20, 40, 60 or 70% Xenon (with nitrogen making up the balance) for 90 min at 37°C (temperature kept by water bath running outside chambers). At post-experimental day 7, rats (14 day old) were killed and their brains removed.
• Rat pups in some groups were allowed to live up to 30 days of postnatal age, at which time their neuromotor function and co-ordination were assessed with established protocols (Neuromotor testing and Rotarod testing).
• Figure 2 Brain sections from animals that suffered 90 minutes of hypoxia-ischemia injury are shown in Figure 2.
• Figure 2 centre
• the brain slices on the right are from animals that have suffered the same hypoxia-ischemia but have been breathing 70% xenon during the hypoxic period. These brains look close to normal showing the remarkable neuroprotection afforded by xenon.
• Figure 3 The major cellular damage which is evident 24 hours after 90 minutes of hypoxia- ischemia is shown in Figure 3.
• the concentration-dependence of xenon neuroprotection (ratio right hemisphere weight/left against xenon concentration) is shown in Figure 4.
• Figure 4 shows the ratios of ipsilateral/contralateral hemispheric weight of 14 day rat brain after hypoxia/ischemia with or without various concentrations of xenon at 7 days old. Neuroprotection is evident even at sub-anaesthetic concentrations. Control animals were subjected to carotid ligation but no hypoxia was given. Results are mean ⁇ SEM
• the neuroprotective effect (ratio of right hemisphere/left) observed with with N 2 and xenon respectively is shown in Figure 6, when xenon is administered 2 hours post HI insult.
• the data show that xenon is effective in providing neuroprotection even if it is administered 2 hours after the end of the hypoxic period.
• the ratios of ipsilateral/contralateral hemispheric weight of 14 day rat brain after 90 min hypoxic-ischemia insult and then 2 hrs recovery following exposure with 70%N or 70%) Xe + 30% O 2 for 90 min at 7 days old. Results are mean ⁇ SEM (n 6).
• FIG. 7 The effect of mild hypothermia on the neuroprotective effect of xenon (LDH release against xenon concentration, % atm) is shown in Figure 7.
• Modest hypothermia produces a very large and unexpected enhancement in xenon neuroprotection. Cooling by 4 degrees greatly enhances the potency of xenon in blocking LDH release.
• this figure shows the effect of a combination of xenon and hypothermia on oxygen-glucose deprivation (OGD)-induced lactate dehydrogenase (LDH) release.
• OGD oxygen-glucose deprivation
• LDH lactate dehydrogenase
• Figure 7 shows the results of exposing neuronal cultures to 75 minutes OGD in the presence of increasing concentrations of xenon, either at 37°C (red), or at 33°C (blue).
• the ED 50 values for xenon at 37°C vs xenon at 33°C were 35.9 +/- 2.15% and 1 1.5 +/- 2.0%) (means +/- SEM) respectively.
• Neuronal injury is expressed as a percentage of the maximal LDH release after 75 minutes of OGD and 6 hours of recovery in the absence of either xenon or hypothermia. Points represent mean values, with error bars indicating standard errors.
• Example 2 More detailed studies are outlined below in Example 2.
• rat pups were anaesthetised with 2% isoflurane before undergoing permanent unilateral ligation of the right common carotid artery, using a midline neck incision and 5.0 silk suture. Once surgery was complete, pups recovered from the anaesthesia and were then returned to the dam until the time of experimentation.
• Control groups consisted of (a) non-treated littermates that underwent incision but no ligation (i.e. sham animals), used as negative controls, and (b) littermates exposed to
• mice Following HI and recovery, experimental rats were treated with 90 minutes of one of the five experimental interventions below. Each of the five treatments was carried out on separate groups of rats.
• This temperature was chosen as it represents "mild” hypothermia, and was thus thought to be clinically relevant, providing a good balance between side effects and benefit.
• the pups recovered with their mother until the time of sacrifice.
• the pup with the temperature probe in place was culled immediately after the experiment, and its brain was not used for analysis.
• Paraffin Embedding After post-fixing, brains for histology were dehydrated using a Histokinette 2000 tissue-embedding processor (Leica U.K. Ltd., Milton Keynes, U.K.) and then embedded in paraffin wax blocks. The paraffin-embedded brains were cut in coronal sections at a thickness of 5 ⁇ m using a microtome (Leica, Germany).
• Figure 10.3 illustrates the area of the brain from which the slices were taken. Approximately 20 slices were taken from each brain in the region of the hippocampus, (as this is the area most vulnerable to HI injury) around -3.6mm from the bregma.
• the brains were cryoprotected in 30% sucrose in PBS (this also contained 2mg/ml sodium azide) and stored in the fridge for 48 h, or until they had sunk to the bottom of the tube.
• the brains were then frozen in O.C.T. compound (BDH, Poole, England) at -22°C and coronal sections were cut at 30 ⁇ m on a sliding cryostat (Bright Instrument Company Ltd., Huntingdon, U.K.). Sliced brains were stored in the fridge in wells containing 0.1 M PBS and 1 mg/ml sodium azide, before being stained for immunohistochemistry (see below).
• Paraffin-embedded sections were mounted on slides and stained with cresyl violet for histology as described previously (Wilhelm et al, 2002).
• Cresyl violet is a basic stain that binds to acidic components of neuronal cytoplasm such as RNA-rich ribosomes, and also the nuclei and nucleoli of nerve cells. This technique was used to assess cell viability and whether non-viable cells exhibited apoptosis or necrosis on the basis of validated morphological criteria
• Each experimental group consisted of three time groups (16, 24, 48 h), and each time group contained three animals, (thus there were nine animals in total for each experimental group). Three slides per animal were chosen from the area of the brain that was -3.6mm from the bregma ( Figure 11). Slides were then sorted into time groups and the examiner was blinded to the intervention.
• Two areas from the ipsilateral side of each brain were analysed using a BX60 light microscope (Olympus, Southall, U.K.) - one in the cortex and one in the gyrus of the hippocampus ( Figure 11).
• a 40x objective lens with a grid was used to count the total number of cells that appeared in the grid. Cells were scored as either viable, apoptotic, or necrotic based on their morphological appearance, and the percentage of each cell type was noted down for each of the brain areas.
• An Axiocam digital camera (Zeiss, Gottingen, Germany) was used along with the microscope to take photomicrographs of the brain slices.
• Xenon and Hypothermia as Independent Agents Xenon is neuroprotective in the neonate by an anti-apoptotic mechanism
• hypothermia Treatment with a combination of 20% xenon + 35°C hypothermia demonstrates synergistic neuroprotection via an anti-apoptotic mechanism.
• xenon (20%) or hypothermia (35°C) in combination By using proven ineffective interventions of either xenon (20%) or hypothermia (35°C) in combination, a profound synergistic neuroprotection was demonstrated in both areas of the brain, and across all three of the time groups (16, 24 and 48 h) via an anti-apoptotic mechanism.
• Post-ischaemic application of the combination treatment significantly reduced the degree of apoptotic cell death, and increased the proportion of viable cells (see Figure 17).
• N-methyl-D-aspartate antagonists ready for clinical trial in brain ischemia? Ann Neurol 25:398-403.
• Nicholls D.G., Budd, S.L., 2000. Mithochondria and neuronal survival. Physiol Rev 80:315-360.
• Vannucci, R.C. 1997. Hypoxia-ischemia: Clinical Aspects in: Fanaroff, A. A., Martin, R.J., (Eds), Neonatal-perinatal medicine IV, Mosby- Yearbook Inc, Philadelphia, pp877-891. Vannucci, R.C., Connor, J.R., Mauger, D.T., Palmer, C, Smith, M.B., Towfighi, J.,

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