Background of the Invention

The toxicity of oxygen and more specifically its partial reduction products known as reactive oxygen species (ROS) is commonly designated as oxidative stress. It arises from an imbalance of cellular pro-oxidant and antioxidant processes. Oxidative stress has been implicated in a variety of pathological and chronic degenerative processes including the development of cancer, atherosclerosis, inflammation, ageing, neurodegenerative disorders, cataracts, corneal and retinal degeneration, drug action and toxicity, reperfusion injury after tissue ischemia, and defence against infection. See, for instance, Shoham, A., et al, Free Radical Biology & Medicine (2008), 45, pages 1047-1055 and Gao, X. et al, Proc. Natl. Acad. Sci. USA. (2001), 98(26), pages 15221 -15226, the contents of both of which are incorporated herein by reference. The contents of the publications listed at page 15226 of Gao et al., 2001, supra, are also incorporated herein by reference.

The contents of all the references in the following discussion of the background and the description of the present invention are incorporated herein by reference.

Mammalian cells contribute to their own oxidative stress by generating ROS as part of normal aerobic metabolism, and have developed elaborate and overlapping mechanisms for combating these hazards (Halliwell, B. & Gutteridge, J. M. C. (1999) Free Radicals in Biology and Medicine, Oxford University Press, New York, pp. 1-36). Nevertheless, protective mechanisms are not completely effective, especially during increased oxidative stress.

Sigma receptors are the subject of an evolving research area that could play a key role in therapeutic development for many disease states ^1"4 . Manipulation of sigma receptors can be achieved in a number of ways to yield either cytoprotective or cytotoxic actions. Two sigma receptors are known; sigma 1 receptor has been cloned, but to date sigma 2 receptor has not. A predicted structure for the sigma 1 receptor is illustrated by Ishikawa, M. et al, Journal of Receptor, Ligand and Channel Research, (2010), 3, pages 25-36. Application of sigma receptor antagonists has been reported to increase the rate of cell death and thus is proposed as a putative cancer therapy ⁴ . In contrast, stimulation of sigma 1 receptor has been reported to be cytoprotective in that, for example, excitotoxicity of retinal ganglion cells can be reduced by application of sigma 1 receptor agonists or over- expression of the receptor ^5,6 . It has also been reported that sigma 1 receptor agonists can provide potential benefits against retinal neurodegeneration ⁷ . Sigma receptors are reported to be present on the ER membrane and have regulatory actions on calcium signalling ⁸ . In addition, sigma receptors can positively or negatively regulate apoptosis through activity of the caspase cascade ² .

The ocular lens is a phenomenal biological model; it is isolated from other tissues and has no innervations or vascular system, receiving all required nutrients from the aqueous and vitreous humours that bathe it ¹¹ . Cells within the central anterior epithelium have been present since lens vesicle formation during embryogenesis. Moreover, both cell death and division is negligible in this population. Consequently, for extremely long timeframes these cells are exposed to continuous stress resulting from UV exposure and generation of metabolic products, both of which give rise to oxidative stress, which may ultimately contribute to cataract formation ^I2 ' ¹³ . Cataract renders millions in the world blind and is notably a disease that largely afflicts the elderly. In addition to the reduction in the quality of an individual's life, management of cataract is a significant strain on healthcare budgets ¹⁴ . At present, the only means to treat cataract is by surgical intervention ¹⁵ . It is predicted that the number of annual cataract operations will increase to 20-25 million by 2020 ¹⁶ due to an ever-increasing aged population. Identifying mechanisms to offset cataract formation is therefore of great importance.

The present invention is based on our surprising findings that an ROS-induced endoplasmic reticulum (ER) stress response is an important contributor to oxidative degeneration of the mammalian ocular lens and other degenerative and ageing conditions, and that sigma 1 receptor agonists can suppress, but not ablate, this ROS-induced ER stress response in such a way that the beneficial UPR (unfolded protein response) continues in the affected tissues and organs and induction of apoptosis or cell death is avoided.

This novel understanding of the mechanism which allows the UPR to continue allows the agents for use in the present invention to provide long tenn controlled therapies for a range of degenerative conditions, for example corneal, retinal, lens or skin degeneration, in which the UPR is harnessed and used in the therapies.

Brief Description of the Invention

In a first aspect, the present invention provides the use, in the treatment or prevention of ocular lens degeneration, corneal degeneration or skin degeneration, or any ROS-induced degeneration of other tissues or organs characterised by the ER stress response, or in the preparation of a composition (e.g. a medicament) for use in the treatment or prevention of any one or more of these degenerations, of an active therapeutic agent comprising one or more sigma 1 receptor agonist. In a second aspect, the present invention provides an active agent comprising one or more sigma 1 receptor agonist, for use in the treatment or prevention of ocular lens degeneration, corneal degeneration, skin degeneration or any ROS-induced degeneration of other tissues or organs characterised by the ER stress response. In a third aspect, the present invention provides a composition, for example a pharmaceutical composition (medicament), comprising an effective amount of an active agent comprising one or more sigma 1 receptor agonist, and one or more physiologically compatible carrier, diluent or excipient, for use in the treatment or prevention of ocular lens degeneration, corneal degeneration, skin degeneration or any ROS-induced degeneration of other tissues or organs characterised by the ER stress response.

The conditions of ocular lens degeneration, corneal degeneration and skin degeneration to be treated or prevented according to the present invention include particularly, but not exclusively, ROS-induced degeneration of, respectively, the ocular lens, cornea and skin, characterised by the ER stress response.

The ROS-induced degeneration of other tissues or organs characterised by the ER stress response to be treated or prevented according to the present invention include particularly, but not exclusively, ROS-induced retinal degeneration characterised by the ER stress response. The ROS-induced degeneration of other tissues or organs characterised by the ER stress response may alternatively be ROS-induced degeneration of tissues or organs other than the retina.

A particular ocular lens degenerative disorder to be treated or prevented is cataract. Other degenerative conditions to be treated or prevented include corneal degeneration and skin degeneration, including wrinkling and ageing. The present invention will also find use in the protection of transplant organs such as skin and corneas prior to transplantation or grafting, and such uses and compositions are embraced within the aspects defined above. The compositions of the present invention for use with the eye include, for example, artificial vitreous and aqueous humour for use in eye operations.

In the various aspects of the present invention, in one embodiment the active agent may, if desired, further comprise at least one other component active agent of the same and/or a different type for treating or preventing ocular lens degeneration, corneal degeneration, skin degeneration or any ROS-induced degeneration of other tissues or organs characterised by the ER stress response. The composition may, if desired, contain one or more additional ingredients, which may include effective amounts of one or more components having similar or other physiological activity, suitable amounts of one or more physiologically inert ingredients, or any mixture or combination thereof.

Alternatively, such additional ingredients may be incorporated into other compositions (medicaments), optionally including one or more physiologically compatible carrier, diluent or excipient, for administration separately from the composition according to the present invention, which administration may be prior to, simultaneously with or after the administration of the composition according to the present invention.

The composition according to the present invention may, if desired, consist essentially of the active ingredient and, if present, the one or more physiologically compatible carrier, diluent or excipient. In this embodiment the active agent may suitably consist essentially of the one or more sigma 1 receptor agonist.

The composition according to the present invention may, if desired, consist of the active ingredient and, if present, the one or more physiologically compatible earner, diluent or excipient. In this embodiment the active agent may suitably consist of the one or more sigma 1 receptor agonist.

The expression "sigma 1 receptor agonist" used herein includes all agents which have the physiological effect of activating or stimulating the effects of the sigma 1 receptor in a subject, and therefore include a direct-acting activator or stimulator of the sigma 1 receptor in the subject and an indirect-acting activator or stimulator of the sigma 1 receptor in the subject. The expression "indirect-acting" used herein means that the action is on another substance or group of substances which causes activation or stimulation of the sigma 1 receptor. An indirect sigma 1 receptor agonist includes an agent such as a direct or indirect inhibitor of a sigma 1 receptor antagonist (inhibitor), such that would cause a resultant activation or stimulation of the sigma 1 receptor, for example by endogenous sigma 1 receptor agonists. The expressions "sigma 1 receptor agonism", "sigma 1 receptor activation" or "sigma 1 receptor activation" and the like shall be understood accordingly.

Agonists can be full, partial or super agonists in comparison with endogenous agonists of the receptors. Antagonists to be inhibited can be full or partial inverse agonists or silent antagonists in comparison with endogenous agonists of the receptors, or can block agonism at the receptors indirectly. The present invention is used particularly on mammalian, preferably human, patients and on tissues and organs, including artificial or humanised tissues and organs. The expressions "ocular lens degeneration", "corneal degeneration", "skin degeneration" and "any ROS-induced degeneration of other tissues or organs characterised by the ER stress response" thus apply both to the patient's native organs or tissues as well as transplanted or grafted organs and tissues and ex vivo tissues and organs to be transplanted or grafted.

Detailed Description of the Invention Sigma 1 Receptor Agonist

The sigma 1 receptor agonist may be a specific to the sigma 1 receptor or non-specific to sigma 1 , potentially having agonistic or antagonistic effects at other receptors, potentially including the sigma 2 receptor.

The sigma 1 receptor agonist may exhibit selective agonism at the sigma 1 receptor, whereby the agonistic or antagonistic character of the agent can be selected, for example according to the effector pathways or the tissue type (functional selectivity). Examples of sigma 1 receptor agonists include, for example, (+)-benzomorphans such as (+)-pentazocine or (+)-N-allylnormetazocine ((+)-SKF- 10047); PRE-084; igmesme; SA- 4503 (cutamesine); 4-phenyl-l -(4-phenylbutyl)piperidine (4-PPBP); dehydroepiandrosterone (DHEA) (e.g. as sulphate) or pregnenolone (e.g. as sulphate) or other endogenous steroids acting as sigmal receptor agonists; haloperidol; dimemorfan; donepezil; tetrahydro-N,N-dimethyl-5,5-diphenyl-3-furan-methanamine (e.g. as hydrochloride) (ANAVEX1 -41) and fluvoxamine.

The formulae for most of these compounds, and others, are given in Ishikawa, M. et al, Journal of Receptor, Ligand and Channel Research, (2010), 3, pages 25-36. Compositions and Administration Routes

The active agent(s) and composition(s) of the present invention may be administered by any suitable method. The compositions may typically be formulated in conventional manner, as is well known in the art.

Suitable administration routes for treatment of the cornea or skin include topical administration, transdermal administration (e.g. by transdermal infusion or by active transdermal delivery, e.g. by means of a propellant such as a pressurised gas) and local injection or infusion. Oral administration, for example in pill, tablet, capsule or liquid form or in foodstuffs or drinks, or nasal spray or drops, or a parenteral route such as systemic injection or infusion, are alternative administration routes, but are generally less preferred. Where the organ to be treated is the ocular lens, the active agent may suitably be introduced into the aqueous and/or vitreous humour of the eye, or a substitute aqueous or vitreous humour which replaces the natural material, for example in a surgical procedure. Where the organ to be treated is the cornea, the active agent may suitably be introduced into the aqueous humour of the eye, or a substitute aqueous humour which replaces the natural material, for example in a surgical procedure. Topical compositions for administration to the eye (for example, for treating the cornea) include eye drops and creams. Topical compositions for administration to the skin include sprays, lotions, creams, pastes, patches (e.g. transdermal patches).

Where appropriate, compositions may be formulated as slow release and/or slowly degradable compositions in conventional manner.

These composition forms will usually include one or more pharmaceutically acceptable ingredients which may be selected, for example, from adjuvants, carriers, binders, lubricants, diluents, sodium chloride, stabilising agents, buffering agents, emulsifying agents, wetting agents, viscosity-regulating agents, surfactants, preservatives and colorants. As will be understood by those skilled in the art, the most appropriate method of administering the active ingredients is dependent on a number of factors. The compositions for use in the present invention may, if desired, be presented as a dry product for reconstitution with water or other suitable vehicle before use.

For control of release of the active agent(s) according to the present invention a number of effective methods are available. See, for example, Wagh V.D., Inamdar B., Samanta M.K., Polymers used in ocular dosage form and drug delivery systems. Asian J Pharm 2, 2008, 12-17 and the literature references cited therein, the contents of which are incorporated herein by reference. The use of polymers (e.g. cellulose derivatives such as hydroxypropylmethylcellulose (HPMC) and hydroxypropylcellulose (HPC), poly(acrylic acid) (PAA), polyacrylates, cyclodextrins and natural gums, polyorthoesters (POEs) and mucoadhesive polymers, semisolids such as gels, films and other inserts, resins such as ion exhange resins, iontophoretic delivery, and colloidal particles such as microspheres and nanoparticles, may be particularly mentioned. Further discussion of each of these approaches is given in the Wagh paper cited above and the literature references therein, incorporated herein by reference.

The compositions of the present invention may, for example, comprise artificial vitreous or aqueous humour for use in eye surgery. This composition may, for example, be prepared by supplementing a humour substitute with one or more sigma 1 receptor agonist.

In one embodiment of the present invention a number of ingredients are administered via separate pharmaceutical preparations. In this embodiment the different pharmaceutical preparations of active ingredients may be administered simultaneously, sequentially or separately.

Therefore, in one aspect, the present invention provides a kit comprising a preparation of a first active ingredient which is one or more sigma 1 receptor agonist, and a preparation of a second active ingredient, and optionally instructions for the simultaneous, sequential or separate administration of the preparations to a patient in need thereof.

The second active ingredient may be selected from a wide range of physiologically active agents, according to the likely or expected symptoms that may need to be relieved in the subject being treated. Such agents may include, for example, an anti-inflammatory agent; a lipid lowering agent such as a statin or a fibrate; a modulator of blood cell morphology such as pentoxyfylline; a thrombolytic or an anticoagulant such as a platelet aggregation inhibitor; a CNS agent such as an antidepressant (such as sertraline); an agent for the treatment of acute or chronic pain, such as a centrally or peripherally-acting analgesic (for example an opioid or derivative thereof), carbamazepine, phenytoin, sodium valproate, amitryptiline or other anti-depressant agent(s), paracetamol, or a non-steroidal antiinflammatory agent); a parenterally or topically-applied (including inhaled) local anaesthetic agent such as lignocaine or a derivative thereof; or any mixture or combination thereof.

The dosage of active agent(s) to be applied is readily obtained by tests and establishing the dosage is within the skill of the reader, based on the experimental work described below, hi general, a dosage of active agent(s) such that the concentration contacting the organ or tissue to be treated is in the range of about 0.1 to about 100 μΜ, for example about 1 to about 50 μΜ, for example about 1 to about 25 μΜ, for example about 10 μ , gives satisfactory results. However, the dosage may be varied within and outside these ranges, to suit the nature of the condition to be treated or prevented, the circumstances of the patient or organ or tissue to be treated, and the nature of the active agent(s). Thus, when an organ or tissue is treated ex vivo, for example when an organ or tissue is bathed, treated or stored with or in a composition comprising the active agent(s) according to the present invention prior to transplant or graft surgery, the concentration of the active agent(s) in the bathing, treating or storage composition may be higher. Brief Description of the Drawings

In the accompanying drawings:

Figures 1 and 2 show the effects of hydrogen peroxide on FHL-124 cell survival. The basic experiment was the same for the two figures, with different statistical analysis for Figure 1 compared with Figure 2 (details of the statistical analyses are given in the figure legends); Figures 3 and 4 show the effects of hydrogen peroxide on caspase expression in FHL- 124 cells. The basic experiment was the same for the two figures, with different statistical analysis for Figure 3 compared with Figure 4 (details of the statistical analyses are given in the figure legends);

Figures 5 and 6 show the effects of hydrogen peroxide on ER stress proteins in FHL-124 cells. The basic experiment was the same for the two figures, with different statistical analysis for Figure 5 compared with Figure 6 (details of the statistical analyses are given in the figure legends);

Figures 7 and 8 show the effects of hydrogen peroxide on sigma 1 receptor expression in FH-124 cells. The basic experiment was the same for the two figures, with different statistical analysis for Figure 7 compared with Figure 8 (details of the statistical analyses are given in the figure legends);

Figures 9 and 10 show the effects of the sigma 1 receptor agonist (+)-pentazocine against hydrogen peroxide induced loss of cell viability. The basic experiment was the same for the two figures, with different statistical analysis for Figure 9 compared with Figure 10 (details of the statistical analyses are given in the figure legends);

Figures 11 and 12 show (+)-pentazocine suppression of hydrogen peroxide induced Bip (binding immunoglobulin protein) and phosphor-EIF2a expression in FHL-124 cells. The basic experiment was the same for the two figures, with different statistical analysis for Figure 11 compared with Figure 12 (details of the statistical analyses are given in the figure legends);

Figures 13 and 14 show (+)-pentazocine inhibition of hydrogen peroxide induced cleavage of pro-caspase 3 and 12. The basic experiment was the same for the two figures, with different statistical analysis for Figure 13 compared with Figure 14 (details of the statistical analyses are given in the figure legends);

Figures 15 and 16 show the nil effect of (+)-pentazocine on hydrogen peroxide induced disruption of calcium signalling. The basic experiment was the same for the two figures, with different statistical analysis for Figure 15 compared with Figure 16 (details of the statistical analyses are given in the figure legends);

Figure 17 shows the results of the described experiment on whole human lens organ cultures;

Figures 18 and 19 show lactate dehydrogenase levels in the culture medium of whole human lens organs. The basic experiment was the same for the two figures, with different statistical analysis for Figure 18 compared with Figure 19 (details of the statistical analyses are given in the figure legends);

Figures 20 and 21 shows TUNEL assay detection of apoptosis following hydrogen peroxide exposure. The basic experiment was the same for the two figures, with different statistical analysis for Figure 20 compared with Figure 21 (details of the statistical analyses are given in the figure legends).

Test Data and Detailed Description of the Drawings

The following non-limiting test data are provided for further illustration and explanation of the present invention and the accompanying drawings. The experimental system we have employed serves as an effective model to test the relationship between sigma receptors, ER stress and oxidative stress in ocular and non-ocular cells and tissues, including the cornea, retina and skin, that are subject to oxidative damage ⁹ ' ^I0 . The following abbreviations are used herein: H202 = hydrogen peroxide; SEM = standard error of the mean; Sig-IR = sigma 1 receptor; PTZ = pentazocine; PBS = phosphate buffered saline; LDH = lactate dehydrogenase; TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labelling; FITC = fluorescein isothiocyanate; DAPI = 4',6-diamino-2-phenylindole; BSA = bovine serum albumin. Other abbreviations are standard or are explained in the text. Materials and Methods FHL124 cell culture

The human lens epithelial cell line (FHL-124) was a kind gift from Dr. J. . Reddan (University of Michigan, USA). FHL-124 is a non-virally transformed cell line generated from human capsule-epithelial explants ²⁰ showing a 99.5% homology (in transcript profile) with the native lens epithelium ²¹ . FHL-124 were routinely cultured at 35 °C in a humidified atmosphere of 95% air and 5% C0 ₂ , in Eagle's Minimal Essential Medium (EMEM) (Gibco, Paisley, UK) supplemented with 5% v/v foetal bovine serum (Gibco) and 0.005% w/v gentamicin. FHL124 cells were seeded on tissue culture dish (30k/400 μΐ for western blot experiments) and 96-well plates (1 Ok/well for MTS assay).

Cell survival assay

®

FHL-124 cell number was measured using the CellTiter 96 AQueous Non-Radioactive

(a)

Cell Proliferation Assay (Promega). This is a colorimetric method for determining the number of viable cells in proliferation, cytotoxicity or cliemosensitivity assays based on the reduction of MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-( 4- sulfophenyl)-2H-tetrazolium, inner salt]. This was used in a 96 well plate format. The plate was read at 490 nm using a Wallac "Victor2" 1420 multilabel counter using Perkin Elmer Workout (VI .5) software.

Western Blot Analyses

The cells were lysed on ice in buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 % Triton X-100, 1 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride, ImM phenylmethylsulfonylfluoride and 10 μ^πιΐ aprotinin) (Wang et al, 2005). Lysates were pre-cleared by centrifuging at 13000 rpm at 4°C for 10 minutes, and the protein content assayed by BIO-RAD protein assay, so that equal amounts of protein per sample were loaded onto 10% SDS-PAGE gels for electrophoresis and transferred onto a nitrocellulose membrane with BIO-RAD Trans- Blot semi-dry Transfer Cell. Proteins were detected using the ECL plus blotting analysis system (Amersham biotech) with anti-Bip/GRP78 and anti-pro-caspase-12 (BD Biosciences); anti-P-actin and anti-pro-caspase-3 (Cell Signalling Technology); anti-EIF- 2α (BIOSOURCE); anti-phospho-EIF-2a (Upstate biotechnology); anti-IRE-1 , anti- Sigma-1 receptor and anti-ATF-6 (Abeam, UK).

Whole Human lens culture

The whole human lens culture was employed as described previously ²² . Donor eyes were obtained from the East Anglian Eye Bank. The research followed the tenets of the Declaration of Helsinki regarding the use of human material. After removal of the cornea for transplantation, the eyes were placed in sterile containers and covered with Eagle's minimum essential medium (EMEM) containing 200 U/ml penicillin and 200 μg/ l streptomycin. They were stored at 4°C before dissection. Lenses were placed in culture within 24 hours post-mortem.

Lenses were dissected by anterior approach and incubated for 30 minutes in bicarbonate- C0 ₂ -buffered EMEM (pH 7.4), containing 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 g/ml amphotericin, and 50 μg/ml gentamicin. Thereafter, the lenses were maintained in EMEM with 50 μg/ml gentamicin at 35°C.

After a pre-culture period of 24 to 72 hours to ensure no damage had arisen from the isolation procedure, lenses were allowed to expose to (+)-pentazocine (10 μΜ) prior to adding 300 μΜ H ₂ 0 ₂ .During the experimental period, lens images were taken at the starting point (T=0) and 24 hour time point using a charge-coupled device (CCD) camera (UVP, Cambridge, UK) with Synoptics software (Synoptics, Cambridge, UK). By the end of 24 hours culture in presence of experimental conditions, the medium was collected for LDH assay and the whole epithelium was dissected and fixed in 4% paraformaldehyde for TUNEL assay.

Cell death assay (LDH assay)

A non-Radioactive Cytotoxicity Assay (Cyto Tox 96R, Promega) was used to measure the release of LDH from the cultured Human lenses. The procedure followed the manufacturer's protocol. The plate was read at 490nm with Wallac "Victor2" 1420 multi- label counter using Perkin Elmer Workout (VI .5) software. TUNEL assay

Human lens epithelia were dissected from the culture lenses at end-point then fixed in 4% parafonnaldehyde at room temperature for 30 min. The preparations were then washed three times with PBS prior to cell permeabilisation with 0T % Triton X-100, 0T % sodium citrate on ice (4°C) for 2 min. Cells were then incubated with 500 μΐ Enzyme solution containing FITC conjugated terminal deoxynucleotidyl transferase from calf thymus [EC 2.7.7.31] (diluted 1 : 10 in label solution) at 37°C in the dark for 60 min. Negative controls were incubated in label solution alone. The preparations were then rinsed three times with PBS. To visualize chromatin, DAPI diluted 1 : 100 inl%BSA/PBS was applied for 10 min at room temperature. Finally, after 2x5 min washing with PBS, the stained preparations were mounted in Hydromount mounting medium on a clean glass slide. Images were obtained using a CCD Upright Zeiss fluorescent microscope and Zeiss Axio vision 4.5 software. All images were captured using the same time of exposure. Images were processed to optimise to the peak fluorescence. The positive cell number and total cell number on five random regions of each slide were counted.

Statistical analyses

A i-test analysis was performed using excel software to determine any statistical difference between groups (Excel; Microsoft, Redmond, WA). A 95% confidence interval was used to assess significance.

Tests and Results

Exposure of the human lens cell line FHL 124 to increasing doses of H ₂ 0 ₂ led to reduced cell viability (Figures 1 and 2). Moreover, reduction in pro-forms of caspase 12 and 3 were observed in response to H ₂ 0 ₂ (Figures 3 and 4), suggesting the mode of cell death was apoptosis.

In order to determine a putative involvement of endoplasmic reticulum (ER) stress in H ₂ 0 ₂ mediated events, the expression pattern of ER stress response proteins was determined using western blot methods. The ER is the site of protein synthesis, where folding and trafficking are initiated, and also the mediator of internal and external stresses

17 ER stress can arise because unfolded or mis-folded proteins are produced within the ER. Normal levels of unfolded proteins can be counteracted by a number of ER chaperone proteins, but if the level continues to increase, for example through prolonged oxidative or osmotic stress, then it is detected by the molecule Bip which in turn activates one or more of 3 stress pathway initiators (PERK, IREl and ATF6) ¹⁷ .

ER stress can also be seen as a depletion of ER calcium with a concomitant rise in cytosolic calcium. Once a prolonged stress is sensed then a range of external pathways are initiated ¹ . For example, when EIF2cc is phosphorylated, then translation of certain proteins is inhibited. This apparently is a protective pathway, which presumably results from an inhibition of the synthesis of unfolded proteins. In response to 30uM H202, level of Bip, ATF6, IREl and pEIF2a were significantly increased within 4 hours of exposure (Figures 5 and 6).

Moreover, it was also observed that expression of sigma 1 receptor was markedly increased in response to H ₂ 0 ₂ induced oxidative stress (Figures 7 and 8). As sigma 1 receptor is reported to suppress oxidative stress ⁷ and is located on the ER membrane in close proximity to IP3R ⁸ , we tested the hypothesis that activation of sigma 1R could yield cytoprotection against oxidative stress through modulation of the ER stress response.

Application of 10 and 30μΜ pentazocine, a sigma 1 R agonist significantly inhibited the H ₂ 0 ₂ induce loss of cell viability (Figures 9 and 10). In addition, oxidative stress induced reduction of pro-caspase 3 and 12 was inhibited by the presence of pentazocine (Figures 13 and 14). Critically, the induction of ER stress proteins Bip and EIF2a following oxidative insult were suppressed by pentazocine (Figures 11 and 12).

In order to test whether the results observed were a consequence of a reduced ER stress resulting from a direct action of pentazocine on H ₂ 0 ₂ we assessed changes in calcium signalling. Application of 10μΜ ATP, a known calcium mobilising stimulus initiated a rapid mobilisation of intracellular calcium. Application of 50μΜ H ₂ 0 ₂ did not significantly affect baseline intracellular calcium concentrations; however, following this period of exposure, application of 10μΜ ATP resulted in a very weak response relative to pre-treatment response (Figures 15 and 16). The response to ATP following maintenance for 20 minutes in control medium or medium containing 30μΜ pentazocine had no significant effect on baseline intracellular calcium or the ATP induced response (Figures 15 and 16). Application of pentazocine in the presence on H ₂ 0 ₂ had no effect on the suppression of ATP calcium signalling (Figures 15 and 16).

This data indicates that the protective mechanisms of pentazocine are associated with managing the ER stress pathways induced by oxidative stress rather than a direct action on H ₂ 0 ₂ . These data also explain why sigma 1 receptor activation by pentazocine does not yield 100% protection. Oxidative stress is likely to affect many processes, but it would appear that ER stress is a significant contributor to the deleterious effects of oxidative damage.

The data obtained from the cell line strongly suggest that activation of sigma 1R using pentazocine would provide therapeutic protection against oxidative stress. We therefore tested this concept in a whole lens human organ culture system. Exposure of 300 uM H ₂ 0 ₂ to cultures lead to damage of the lens epithelium and opacity relative to untreated controls (Figures 17 to 19). Application of pentazocine to the lens had no effect on cell viability or clarity. Addition of pentazocine in the presence of H ₂ 0 ₂ significantly reduced the level of opacity and loss of cell viability (Figures 17 to 19). At end-point, the TUNEL assay was used to assess rates of apoptosis. Application of H ₂ 0 ₂ resulted in a significant number of apoptotic cells being detected; this number was significantly reduced when pentazocine was applied in the presence of H ₂ 0 ₂ (Figures 20 and 21). Conclusions

Stimulation of the sigma 1 receptor provides significant protection against oxidative damage. This protection is likely to result from a suppression, but not ablation of the ER stress response thus permitting the unfolded protein response to be effective, but not inducing apoptotic signals. The current findings have direct relevance to the prevention of cataract, but moreover the principles presented here can be applied to prevent the effects of oxidative damage throughout the eye. In particular, the use of sigma 1 receptor agonists could serve to protect ocular cells in the cornea and retina through suppression of ER response pathways. Maintenance of viable cell populations in the cornea is essential if the tissue is to be to be used for transplant purposes ¹⁸ .

Addition of sigma 1 receptor agonists to corneal storage media could putatively aid cell survival and overall integrity of the tissue. This in turn would yield higher numbers of corneal tissue of appropriate quality for transplant. Moreover, damage to both the retina and lens can result from vitrectomy i.e. removal of the vitreous humour and replacement with artificial vitreous ¹⁹ . This method of surgery is associated with formation of a nuclear cataract a relatively short period after operation ^!9 . While this form of surgery is essential for the patients, the secondary problems that result need to be managed to improve the quality of care. Supplementing the artificial vitreous with a sigma 1 receptor agonist could consequently provide protection for cells through regulation of ER stress pathways.

Moreover, this principle could be applied to non-ocular tissues such as the skin; reducing oxidative stress resulting from UV exposure in this organ could reduce the onset of damage and potential oncogenesis. This could be treated through inclusion of sigma 1 agonists in topically applied lotions or creams. The application of sigma 1 receptor agonists is therefore varied and could improve overall medical provision, such as an improvement in corneal transplant success, the delay of cataract formation and reducing the impact of vitrectomy on both the lens and retina; reduction of skin damage.

The foregoing describes the present invention broadly and without limitation to particular embodiments. Variations and modifications as will be readily apparent to those skilled in the art are intended to be included in the scope of the application and subsequent patents.

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