AU2006236018B2 - Methods for preventing pressure induced apoptotic neural cell death - Google Patents

Description

Our Ref:20128405 P/00/0 I I Regulation 3:2 AUSTRALIA Patents Act 1990 ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): Minas Theodore Coroneo 2 St Pauls Street Randwick New South Wales 2031 Australia Address for Service: DAVIES COLLISON CAVE Patent & Trade Mark Attorneys 255 Elizabeth Street Sydney, New South Wales, Australia, 2000 Invention Title: Methods for preventing pressure induced apoptotic neural cell death The following statement is a full description of this invention, including the best method of performing it known to me: 5951 METHODS FOR PREVENTING PRESSURE INDUCED APOPTOTIC NEURAL CELL DEATH Field of the Invention 5 This invention is concerned with methods and compositions for protecting neural tissue from cell death, more particularly, apoptotic cell death associated with pressure. In a further aspect the invention is concerned with methods and compositions for the treatment of glaucoma, elevated brain pressure, and peripheral nerve damage associated with elevated pressure. 10 Background of the Invention Neuronal tissue or nerve cell death is a major medical problem in human society. Neuronal cell death in the eye may lead to blindness. Glaucoma, is a principal cause of neural cell apoptotic death in the eye and a principal cause of adult blindness. It is the 15 third major cause of visual loss in the elderly, affecting approximately 3% of the population over 50. Neuronal cell death is associated with a range of other medical conditions. These include hydrocephalus, and other brain/skull diseases or injuries. Brain neurone cell death may 20 result in mental impairment, loss of motor functions and the like. Peripheral nerve damage from traumatic injury or surgical complications, for example, in the spine, feet and hands may cause apoptotic cell death. In the spinal column where spinal bones may press upon a nerve trunk causing nerve cell death. Bone and connective 25 tissue pressures in peripheral tissue such as the wrists may cause apoptotic neural cell death and consequent lack of feeling and/or motor movement. Morphologically apoptosis is characterised by progressive condensation of the cytoplasm and nucleus, followed by fragmentation and phagocytosis by other cells (Majino and Joris 30 (1995) Am Pathol 146: 3-15).

-2 Although there are some known inhibitors of apoptosis, there are no effective therapeutic agents for the treatment of apoptotic neuronal cell death. This reflects the lack of understanding of the precise mechanisms involved. 5 In relation to glaucoma, there are now a number of agents which reduce eye pressure, with mixed success. The mechanism of action of such agents is controversial and unclear. Glaucoma remains one of the major causes of blindness in human society (accounting for approximately 15% of cases of blindness). 10 Similarly, apoptotic neural cell death associated with wide range of conditions, such as hydrocephalus, spinal compression, and peripheral neuronal cell pressure mentioned above, remain significant problems, with no effective (non-surgical) therapeutic agents being available. 15 Summary of the Invention In a first aspect of this invention, there is provided a method for protecting neural tissue from pressure induced apoptotic cell death which comprises contacting the cells with at last one compound which blocks the effect of pressure on the cells. 20 In another aspect there is provided a method of protecting neural tissue from pressure induced apoptotic cell death which comprises administering to a subject in need of such treatment at least one compound which blocks the effects of pressure on neuronal cells. In another aspect there is provided a method of protecting neural tissue from pressure 25 induced apoptotic cell death which comprises administering to a subject in need of such treatment at least one compound which blocks stretch-activated channels (either directly or indirectly) or other pressure sensitive cellular mechanisms in neuronal cells. In another aspect of the invention there is provided a method for the treatment of glaucoma 30 which comprises administering to a subject in need of such treatment an effect amount of a composition which blocks the effect of pressure on neural cells in the eye.

-3 In another aspect of the invention there is provided a method for the treatment of glaucoma which comprises administering to a subject in need of such treatment a compound which blocks stretch-activated channels (either directly or indirectly) or other pressure sensitive 5 cellular mechanisms in eye neuronal cells. In a another aspect of the invention there is provided a method for the treatment of the effects of elevated brain pressure which comprises administering to a subject in need of such treatment a compound which blocks stretch-activated channels (either directly or 10 indirectly) or other pressure sensitive cellular mechanisms in brain neuronal cells. In a another aspect of the invention there is provided a method for the treatment of peripheral nerve damage which comprises administering to a subject in need of such treatment a compound which blocks stretch-activated channels (either directly or 15 indirectly) or other pressure sensitive cellular mechanisms in said peripheral nerve cells. In a further aspect of the invention there is provided a composition for protecting neural tissue from pressure induced apoptotic cell death which comprises an effective amount of at least one compound which blocks stretch-activated channels (either directly or 20 indirectly) or other pressure sensitive cellular mechanisms in neuronal cells, optionally in association with one or more pharmaceutically acceptable carriers or excipients. In a further aspect of the invention there is provided a composition for the treatment of glaucoma which comprises at least one compound which blocks stretch-activated channels 25 (either directly or indirectly) or other pressure sensitive cellular mechanisms in eye neuronal cells, optionally in association with one or more pharmaceutically acceptable carriers or excipients. In a further aspect of the invention there is provided a composition for the treatment of 30 elevated brain pressure which comprises at least one compound which blocks stretch activated channels (either directly or indirectly) or other pressure sensitive cellular -4 mechanisms in neuronal cells, optionally in association with one or more pharmaceutically acceptable carriers or excipients. In a further aspect of the invention there is provided a composition for the treatment of 5 peripheral nerve damage which comprises at least one compound which blocks stretch activated channels (either directly or indirectly) or other pressure sensitive cellular mechanisms in neuronal cells, optionally in association with one or more pharmaceutically acceptable carriers or excipients. 10 Detailed Description of the Invention This invention provides methods and compositions for protecting neural tissue from pressure induced apoptotic cell death. The invention is based on the finding that elevated pressure on neuronal cells induces apoptotic cell death. The invention is also based on the unexpected finding that compounds which block stretch-activated channels (either directly 15 or indirectly) or other pressure sensitive cellular mechanisms in neuronal cells protect the neuronal cells against pressure induced apoptotic cell death. The effects of pressure on neuronal cells may be blocked though the use of compounds which block stretch-activated channels (either directly or indirectly) or other pressure 20 sensitive cellular mechanisms in neuronal cells, such as those associated with the cell membrane and/or those associated with intracellular membranes, such as those of mitochondria and T tubules. In another aspect the invention is concerned with the method of protecting neural tissue 25 from pressure induced apoptotic cell death which comprises administering to a subject in need of such treatment at least one compound which blocks stretch-activated channels (either directly or indirectly) or other pressure sensitive cellular mechanisms in said neuronal cells. 30 Stretch-activated channels have been described by various authors, and may be regarded as being associated with mechanoelectric transduction (see Zeng et al (2000) Heart and -5 Circulatory Physiology 278 (2): H548). Stretch-activated channels (SACs) are found in a variety of cells including cardiomyocytes (see Hu and Sachs (1996) J Membr Biol 154: 205-216). Examples of stretch activated channels include stretch activated channels from of a family of two-pore domain K' channels (K 2 p) of which 8 have been cloned in rodents 5 and humans. There are 4 classes: " TWIK- 1 & TWIK-2 (Tandem of P domains in Weak Inward rectifier K+ channels) are weak inward rectifiers; * TREK-1 (TWIK-Related K* channel ) & TRAAK (TWIK-related Arachidonic Acid (AA)-stimulated K+ channel ) are polyunsaturated fatty acids (FA) and stretch-activated 10 K* channels (see Meadows et al, Brain Research 2001; 892, 94-101); * TASK-1 and TASK-2 (TWIK-related Acid-Sensitive K+ channels) are acid-sensitive K* channels; " KCNK6 and KCNK7 are silent subunits that probably need a partner to become active. (see Maingret F, Patel AJ, Lesage F, Lazdunski M, Honore E. Lysophospholipids open the 15 two-pore domain mechano-gated K(+) channels TREK-1 and TRAAK. J Biol Chem. 2000;275:10128-33). TRAAK appears to be restricted to the central nervous system, spinal cord and retina. TREK-1 is ubiquitous with strong expression in the central nervous system. Both TREK-1 20 and TRAAK are outward rectifier K* channels opened by membrane stretch, cell swelling, and/or shear stress (all pressure effects). At atmospheric pressure, basal activity is negligible and channels are opened by convex curvature of the plasma membrane. Mechano-gating does not require the integrity of the cytoskeleton and the activating force is apparently directly coming from the cell membrane .bilayer. Cytoskeleton disruption 25 potentiates the opening by membrane stretch, suggesting that these channels are tonically repressed by the cytoskeleton. Blocking of stretch-activated channels may be measured according to conventional physiological techniques, such as by voltage clamp recordings from isolated cells subject -6 to membrane stretching, for example resulting from increased pressure or induced physical stretching, such as subjecting isolated cells to controlled strain such as longitudinal stretch. Under these conditions, stretch-activated channels may be measured by elicited electrical current. The elicited current may represent inward cationic currents such as described by 5 Zeng et al (2000) Heart and Circulatory Physiology 278 (2): H548. Agents which block stretch-activated channels bind to or are associated with the channels so as to reduce or abolish the elicited currents. Preferably stretch induced currents are reduced by the compounds, for example, by between 10% and 100%, such as 10% and 90%, 10% and 80%, 10% and 70%, 10% and 60%, 10% and 50%, 10% and 40%, 10% and 30%, 10% and 10 20%. In another aspect this invention is concerned with the method of protecting neural tissue from pressure induced apoptotic cell death which comprises administering to a subject in need of such treatment at least one compound which blocks stretch-activated channels 15 (either directly or indirectly) or other pressure sensitive cellular mechanisms in neuronal cells. In another aspect of the invention there is provided a method for the treatment of glaucoma which comprises administering to a subject in need of such treatment at least one 20 compound which blocks stretch-activated channels (either directly or indirectly) or other pressure sensitive cellular mechanisms in neuronal cells. In another aspect of the invention there is provided a method for the treatment of elevated brain pressure which comprises administering to a subject in need of such treatment at 25 least one compound which blocks stretch-activated channels (either directly or indirectly) or other pressure sensitive cellular mechanisms in brain neuronal cells. In a another aspect of the invention there is provided a method for the treatment of peripheral nerve damage which comprises administering to a subject in need of such 30 treatment at least one compound which blocks stretch-activated channels (either directly or indirectly) or other pressure sensitive cellular mechanisms in peripheral nerve cells.

-7 In a further aspect of the invention there is provided a composition for protecting neural tissue from pressure induced apoptotic cell death which comprises at least one compound which blocks stretch-activated channels in neuronal cells, optionally in association with 5 one or more pharmaceutically acceptable carriers or excipients. In a further aspect of the invention there is provided a composition for the treatment of glaucoma which comprises at least one compound which blocks stretch-activated channels (either directly or indirectly) or other pressure sensitive cellular mechanisms in neuronal 10 cells, optionally in association with one or more pharmaceutically acceptable carriers or excipients. In a further aspect of the invention there is provided a composition for the treatment of elevated brain pressure which comprises at least one compound which blocks stretch 15 activated channels (either directly or indirectly) or other pressure sensitive cellular mechanisms in neuronal cells, optionally in association with one or more pharmaceutically acceptable carriers or excipients. In a further aspect of the invention there is provided a composition for the treatment of 20 peripheral nerve damage which comprises a protectant which blocks stretch-activated channels (either directly or indirectly) or other pressure sensitive cellular mechanisms in neuronal cells, optionally in association with one or more pharmaceutically acceptable carriers or excipients. 25 Compounds which block the apoptotic effect of pressure on neuronal cells, and in turn which may block stretch-activated channels (see Hamill 0, McBride DW, Pharmacol Rev 1996; 48: 231-252) include: gandoliniuAm (Gd 3 ), a lanthanide; agonists of Gd 3 on neural cells (identifiable by, for example, competitive binding analysis); pyrazine-carboxamides such as amiloride and its analogues (as described by Kleyman and Cragoe (1998) JMembr 30 Biol 105:1-21 and Kleyman and Cragoe (1990) Methods Enzymol 191:739-754); aminoglycoside antibiotics (such as verdamycin, gentamycin, sisomycin, streptomycin, -8 dihydrostreptomycin, netilmycin, amikacin, ribostamycin, dibekacin, and kanamycin); quinidine, sipatrigine, and other blockers including: Na channel blockers, Ca channel blockers, K channel blockers; Ca ions; protons; aluminium ions; tubocurarine; halothane and other inhalational anaesthetics; quinine; integrin-blocking peptides and 5 antibodies, cisplatin; tarantula spider venom; colchicine and vinblastine. At least one active compound is used in the compositions and methods of this invention. For example, two or more compounds may be used in combination. Such combinations may involve synergistic interactions. The effects may occur directly on the stretch-activated channels (SAC) or indirectly via actions. on the cytoskelton, extracellular matrix or on 10 mechanosensitive enzymes (phospholipase A2 and phospholipase C). Inhibitors of these mechanisms are also within the scope of this invention. The invention is not limited to the aforementioned compounds and includes any compound which blocks the apoptotic effects of pressure on neuronal cells. Suitable compounds can 15 be readily identified by testing apoptotic protecting activity under pressure, whether, for example, under atmospheric or hydrostatic pressure or such as by physical stretching of cells. Where neuronal cells are subjected to elevated pressure, such as 100 mm Hg for two hours or more, pressure induced apoptotic cell death occurs, as can be determined by apoptosis assays (see Agar A et al J Neurosci Res 2000; 60: 495-503). Compounds 20 which inhibit such cell death may be used in this invention. Similarly, compounds which block stretch-activated channels in neuronal cells can be readily-identified by conventional physiological techniques, such as patch/voltage clamp recordings from isolated neuronal cells subject to elevated pressure as described above 25 (Zeng et al (2000) Heat and Circulatory Physiology 278(2):H548). Compounds which block elicited currents may be used in this invention. Compositions according to the invention may be formulated with standard buffers, excipients, carriers, diluents and the like. Examples of carriers include: water, 30 physiologically saline, isotonic solutions containing dextrose, glycerol or other agents conferring isotonicity, lower alcohols, vegetable oils, polyethylene glycol, glycerol -9 triacetate and other fatty acid glycerides. Examples of other carriers which may be used include cream forming agents, gel forming agents, and the like, compounding and tabletting agents. Excipients include buffers, stabilisers, emulsion forming agents, colouring compounds, salts, amino acids, antibiotics and other anti-bacterial compounds 5 chelating agents and the like. More than one excipient and carrier may be used. The amount of compounds used to protect neural tissue from pressure induced apoptotic cell death will depend upon various factors including the neural tissue to be treated, such as that in the eye, in the brain, or in peripheral tissue such as in the hand, leg, foot, 10 fingers, oral cavity, nose or ear, the manner of delivery, the severity of the condition being treated, and the judgement of the prescribing physician. By way of example, compounds of the invention may be delivered as a solution for installation, such as an eye drop, ear drop, nose drop; an injectible sterile subcutaneous or intravenous solution; in the form of a tablet, capsule, suppository, dragee; or in the form of a transdermal 15 composition; all of which are well known in the pharmaceutical field and described for example in Remington's Pharmaceutical Sciences Mack Publishing Company, Philadelphia. Generally, the concentration of active agents, which may be regarded as therapeutically effective, will be in the order of 0.001 m to 500mM, such as from 0.1 m to 100 m, 50 m to 100 m, 100 m to 500 m, 500 m to ImM, or 1mM to 500mM. 20 In relation to the treatment of glaucoma compositions of the invention may be administered to the eye, such as by way of eye drop or intraocular injection or as a systemic medication such as a tablet etc. 25 The present invention in one of its aspects represents a significant advance in relation to the treatment of glaucoma. Theories of glaucoma pathogenesis to date are controversial and unclear. Whilst elevated pressure in the eye is a characteristic of glaucoma, the ways in which retinal ganglion cell death is mediated, and may be prevented, are unknown. The inventor's work indicates that pressure alone may be the stimulus for 30 apoptosis in neuronal cells, both in culture and in vivo. Blocking the apoptotic effect of -10 pressure on neuronal cells, such as by inhibiting stretch-activated channels, provides therapeutic outcomes. Compositions for the treatment of glaucoma may be administered to a subject one or 5 more times per day, on or alternative days as a single administration or on a weekly basis. It is preferred that the compositions are administered to the eye for the treatment of glaucoma on a daily basis, generally from 1 to 3 times per day, such as at 5 to 8 hour intervals. 10 In the treatment of elevated brain pressure, such as that due to hydrocephalus, compounds of the invention may be formulated by conventional means known in the art so as to cross the blood-brain barrier. Such compositions may be administered parenterally or non-parenterally as described above, such as by way of oral administration in the form of a capsule, table or the like, rectal or vaginal administration, 15 intravenous administration or intramuscular administration. In the treatment of pressure induced apoptotic neuronal cell death in peripheral nerves, administration of the compounds of the invention will depend upon the site of the neurons/condition being treated. By way of example, increased neuronal cell pressure in 20 the spine may be treated by way of transdermally active compositions, intramuscular injection, intralumbar injection, intravenous administration, oral administration, rectal administration or inhalation administration. Example 1 25 Pressure-induced Primary Retinal Ganglion Cell (RGC) apoptosis is believed by the applicant to be mediated by stretch activated channels. Recently a stretch activated receptor has been identified in animal and human RGCs and their amino acid sequence determined. TRAAK is a mechanogated K* channel, opened by membrane stretch and activated by arachidonic acid. We have confirmed the presence of TRAAK in the RGC-5 - 11 line and shown arachidonic acid induction of apoptosis. A second receptor of relevance is TREK-1 (see below). The RGC-5 cell line is a vector transformed neuronal line derived from primary rat RGC 5 cultures. Developed by Prof N. Agarwal at the University of North Texas, Fort Worth, it has been characterized by morphology, cell markers and PCR analysis. The pressure chamber in-vitro system used in this experiment is as previously described by Agar A, Yip SS, Hill MA, Coroneo MT. "Pressure related apoptosis in neuronal cell lines" 10 JNeurosci Res. 2000;60:495-503. This system determines neuronal apoptosis in response to elevations in ambient hydrostatic pressure. Experiments to date have exposed various neurones including RGC-5 neurones to pressure conditions analogous to normal intra ocular pressure (15mm Hg), chronic glaucoma (30mm Hg) and acute glaucoma (100mm Hg). Compared to non-pressurized controls, increased proportions of apoptotic RGC-5 15 neurones have been found at all these pressure levels. Further, this effect increases with increasing pressure. These stretch activated channels are from of a family of two-pore domain K' channels (K2p) of which 8 have been cloned in rodents and humans. There are 4 classes: 20 * TWIK-1 & TWIK-2 (Tandem of P domains in Weak Inward rectifier K* channels ) are weak inward rectifiers; 0 TREK-1 (TWIK-Related K* channel ) & TRAAK (TWIK-related Arachidonic Acid (AA)-stimulated K* channel ) are polyunsaturated fatty acids (FA) and stretch-activated K+ channels (see Meadows et al, Brain Research 2001; 892, 94-101); 25 e TASK-1 and TASK-2 (TWIK-related Acid-Sensitive K* channels) are acid-sensitive K+ channels; * KCNK6 and KCNK7 are silent subunits that probably need a partner to become active.

- 12 (see Maingret F, Patel AJ, Lesage F, Lazdunski M, Honore E. Lysophospholipids open the two-pore domain mechano-gated K(+) channels TREK-i and TRAAK. J Biol Chem. 2000;275:10128-33). 5 TRAAK appears to be restricted to the central nervous system, spinal cord and retina. TREK-i is ubiquitous with strong expression in the central nervous system. Both TREK-i and TRAAK are outward rectifier K+ channels opened by membrane stretch, cell swelling, and/or shear stress (all pressure effects). At atmospheric pressure, basal activity is negligible and channels are opened by convex curvature of the plasma membrane. 10 Mechano-gating does not require the integrity of the cytoskeleton and the activating force is apparently directly coming from the cell membrane bilayer. Cytoskeleton disruption potentiates the opening by membrane stretch, suggesting that these channels are tonically repressed by the cytoskeleton. 15 Immunolocalisation by specific antibodies has shown that these 2 channels have different subcellular locations - whereas TRAAK is mainly present in soma and to a lesser degree in axons and dendrites, TREK-1 is concentrated in dendrites. Thus both channels are relevant in relation to retinal ganglion cell pressure responses in glaucoma. We have confirmed the presence of TRAAK in both human retina and our retinal cell line RGC-5. 20 The pressure to half-maximum activation derived from the pressure chamber in this example is 36mm Hg for TREK-1 and 46mm Hg for TRAAK. This is of special significance since intraocular pressure of 30 mm Hg is clinically recognized to be the pressure above which retinal ganglion cell damage is highly likely -treatment to lower 25 pressure is usually commenced when eye pressure is at 30 mm Hg or higher. It has also recently been shown that patients with carpal tunnel syndrome had a mean carpal canal pressure of 32 mm Hg (normal 9.6 mm Hg) (see Szabo R, Chidgey LK. J Hand Surg (Am). 1989;14:624). Thus it appears that human neural tissue is sensitive to pressures of approximately 30mm Hg.

- 13 Both TREK-1 and TRAAK are resistant to TEA and 4-AP and slightly sensitive to Ba 2 . At high concentrations, TREK-1 is blocked by quinidine and activated by inhalational anaesthetics, halothane, chloroform, diethylether and isoflurane. Both channels are 5 activated by riluzole a putative neuroprotectant. TREK-1 but not TRAAK is inhibited by activators of Protein Kinase C and A. TREK-1 is opened by intracellular acidification and this shifts the pressure activation relationship so that channels open at atmospheric pressure. Mechanical activation of TREK-1 and TRAAK is mimicked by polyunsaturated fatty acids such as arachadonic acid (AA), oleate, linoleate, eicosapentaenoate, 10 docosahexaenoate and the anionic amphipath trinitrophenol. To investigate the potential for pharmacomodulation of such receptors we have conducted experiments testing a blocker of such channels in this bioassay. Gadolinium (Gd 3 ) a blocker of stretch-activated channels has been used experimentally to block the adverse 15 effects of airway pressure-induced permeability in isolated rat lungs (see Parker JC, Ivey CL, Tucker JA. "Gadolinium prevents high airway pressure-induced permeability increases in isolated rat lungs" JAppl Physiol. 1998;84:1113-8). We surprisingly report here its use in inhibiting pressure induced apoptosis in retinal ganglian cells, and neural cells. 20 To date we have also shown that arachidonic acid (1,10, 50,100 microM/L, n=4 at each concentration) induces apoptosis in these and other cell lines and that this effect is also blocked by Gadolinium. 25 We have used Gadolinium (20 microM/L, n=8 ) to inhibit pressure induced apoptosis in the RGC-5 cells as well as in other neural cell lines. These data taken together indicate that the effector mechanism for retinal ganglion and neural cell death involves stretch activated channels, principally TRAAK and TREK-1 and -14 that blocking these channels may inhibit cell death in clinical conditions such as glaucoma and pressure induced neural damage. Example 2 - glaucoma model in the rat 5 There are a number of experimental animal models for human glaucoma including a recently established rat model in which chronic ocular hypertension is induced (WoldeMussie E, Ruiz G, Wijono M, Wheeler LA. Neuroprotective effect of Brimonidine in chronic ocular hypertensive rats. IOVS 2000; 41:S830). In this model intraocular pressures are elevated by laser photocoagulation of episcleral and limbal vessels (retarding 10 the egress of aqueous humour from the eye), the levels of pressure being up to 2 fold in 2 to 3 weeks. This elevated pressure results in retinal ganglion cell death as occurs in glaucoma and 33±2.9% of retinal ganglion cells are lost in this model. This model is used in tests. In a groups of experimental animals intraocular pressure is 15 elevated and the animals split into two treatment groups. One group is treated with systemic gadolinium and the other group with sipatrigine (see below). Reduction in pressure-induced retinal ganglion cell loss compared untreated controls may be observed, supporting the therapeutic treatments of this invention. 20 Example 3 - human studies In humans, acute glaucoma is a condition in which there is a sudden rise of eye pressure, usually brought about by closure of the drainage angle of the eye (iris blocks the angle). Damage to the retinal ganglion cells and iris precedes damage to most other tissues in the eye. Despite treatment to lower eye pressure, significant and often severe retinal ganglion 25 cell damage occurs. Controlled studies are carried out using blockers of stretch-activated channels to reduce the severity of retinal ganglion cell damage in patients with acute glaucoma. All patients who - 15 are subject to conventional treatment to lower intraocular pressure as soon as diagnosis is made. They are then randomized to control and experimental groups. The experimental group is treated with systemic sipatrigine, or local sipatrigine (by eyedrop), a potent inhibitor of TREK-1 and TRAAK channels which has previously been safely used in the 5 treatment of stroke (see Dawson DA, Wadsworth G, Palmer AM. "A comparative assessment of the efficacy and side-effect liability of neuroprotective compounds in experimental stroke" Brain Res. 2001;892:344-50.). Better outcomes in retinal ganglion cell survival (and therefore field of vision) in the sipatrigine treated group may be achieved, thus forming the basis of using this invention in other forms of glaucoma or 10 neural damage induced by pressure. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps 15 but not the exclusion of any other integer or step or group of integers or steps. The reference to any prior art in this specification is not, and should not be taken as an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia. 20

Claims (9)

1. A method of protecting neural tissue from pressure induced apoptotic cell death which comprises administering to a subject in need of such treatment at least one 5 compound which blocks stretch-activated channels (either directly or indirectly) or other pressure sensitive cellular mechanisms in neuronal cells.

2. A method for the treatment of glaucoma which comprises administering to a subject in need of such treatment at least one compound which blocks stretch-activated 10 channels (either directly or indirectly) or other pressure sensitive cellular mechanisms in eye neuronal cells.

3. A method for the treatment of the effects of elevated brain pressure which comprises administering to a subject in need of such treatment at least one compound which 15 blocks stretch-activated channels (either directly or indirectly) or other pressure sensitive cellular mechanisms in brain neuronal cells.

4. A method for the treatment of peripheral nerve damage which comprises administering to a subject in need of such treatment at least one compound which 20 blocks stretch-activated channels (either directly or indirectly) or other pressure sensitive cellular mechanisms in neuronal cells.

5. A composition when used for the treatment of glaucoma which comprises at least one compound which blocks stretch-activated channels (either directly or indirectly) 25 or other pressure sensitive cellular mechanisms in neuronal cells, optionally in association with one or more pharmaceutically acceptable carriers or excipients.

6. A composition when used for the treatment of elevated brain pressure which comprises at least one compound which blocks stretch-activated channels (either 30 directly or indirectly) or other pressure sensitive cellular mechanisms in brain P .WPDOCSiCRN\GDR\SpccU028405 claims 2nd SPA do.3/09/2009 - 17 neuronal cells, optionally in association with one or more pharmaceutically acceptable carriers or excipients.

7. A composition when used for the treatment of peripheral nerve damage which 5 comprises at least one compound which blocks stretch-activated channels in peripheral neuronal cells (either directly or indirectly) or other pressure sensitive cellular mechanisms, optionally in association with one or more pharmaceutically acceptable carriers or excipients. 10

8. A method according to any one of claims 1-4 wherein said at least one compound is selected from gandolinium (Gd 3 *), a lanthanide; agonists of Gd 3 on neural cells (identifiable by, for example, competitive binding analysis); pyrazine-carboxamides such as amiloride and its analogues; aminoglycoside antibiotics (such as verdamycin, gentamycin, sisomycin, streptomycin, dihydrostreptomycin, netilmycin, 15 amikacin. ribostamycin, dibekacin, and kanamycin); quinidine, sipatrigine, and other blockers including: Na channel blockers, Ca channel blockers, K channel blockers; Ca ions; protons; aluminium ions; tubocurarine; halothane and other inhalational anaesthetics; quinine; integrin-blocking peptides and antibodies, cisplatin; tarantula spider venom; colchicine and vinblastine. 20

9. A composition according to any one of claims 5-7 wherein said at least one compound is selected from gandolinium (Gd 3 '), a lanthanide; agonists of Gd 3 on neural cells (identifiable by, for example, competitive binding analysis); pyrazine carboxamides such as amiloride and its analogues; aminoglycoside antibiotics (such 25 as verdamycin, gentamycin, sisomycin, streptomycin, dihydrostreptomycin, netilmycin, amikacin, ribostamycin, dibekacin, and kanamycin); quinidine, sipatrigine, and other blockers including: Na channel blockers. Ca channel blockers, K channel blockers; Ca ions; protons; aluminium ions; tubocurarine; halothane and other inhalational anaesthetics; quinine; integrin-blocking peptides and antibodies, 30 cisplatin; tarantula spider venom; colchicine and vinblastine.