CHANNEL MEDIATING SUBSTRATES

Abstract: The present invention relates to channel mediating substrates and particularly to a method for screening novel drug molecules and to a pharmaceutical composition for treatment of disease mediated by reactive oxygen species (ROS) or by a lack of response to ROS. The method for screening drug molecules comprises a) selecting a cell or cell culture type known to respond to oxidative stress and/or ROS by opening an NSNAD channel, b) adding a cell-death monitoring composition to said culture, and c) adding thereto a channel protective structure(s) for the protection thereof from the effects of ROS and cell depolarisation; thereby to establish the channel antagonist status of the channel protective structure.

Description: CHANNEL MEDIATING SUBSTRATES The present invention relates to channel mediating substrates and particularly to a method for screening novel drug molecules and to a pharmaceutical composition for treatment of disease mediated by reactive oxygen species (ROS) or by a lack of response to ROS. There are many diseases in which free radical production has been implicated in the causation of tissue damage, particularly degenerative diseases. There is therefore an ongoing need to provide means for the prevention of cellular necrosis and calcium overload in the putative treatment of the said diseases. Such disease included non-exhaustively:- Insulin dependant diabetes ellitus. Non-insulin dependant diabetes mellitus. Ischaemic (hypoxic) /reperfusion injury of myocardium and kidney (+other tissues) . Acute hypertension/ischaemia of CNS (mainly to prevent cerebral vascular damage) and traumatic injury to brain i.e. Stroke prevention. Parkinson's Disease. Alzheimer's Disease. Batten's Disease. Amyotrophic Lateral Sclerosis. Wilson's Disease. Huntington's Disease. Multiple Sclerosis. Guam neurodegeneration. Progressive supranuclear palsy. Tardive dyskinesia. AIDS through the effects of the HIV type 1 protein induced cytotoxicity. Auto-immune disease eg. inflammatory joint disease (rheumatoid arthritis) . Treatment of paraquat poisoning. Adult respiratory distress syndrome (ARDS) . Diseases due to excessive p02 levels - retinopathy of premature babies. Retrolental fibroplasia. Hyperbaric 0 2 toxicity. There are also some major diseases which may be ROS insensitive and these include tumour cells. It has therefore been suggested that free radical production may be carcinogenic (cf. DNA damage and hence mutagenesis) . There is therefore an ongoing problem to provide means for deliberately killing target cells. Since tumour cells are less susceptible to ROS it follows that it is likely that the nicotina ide adenine dinucleotide-activated non-selective cation (NS HAD ) channel is not available for activation and this is why there is reduced susceptibility to ROS. Alternatively the NS NAD channel is not expressed in these cells and so the natural means to kill the cells has been functionally compromised. It is thus possible using this technology to provide a novel means to induce cell death in specified cells by increasing NS NAD channel activity/sensitivity to ROS and/or to increase the channel availability in such cells by up- regulation of expression. We have now shown inter alia that by using a system such as alloxan/cysteine as a reactive oxygen species (ROS) generator and an insulin-secreting cell line (a cell type known to be particularly susceptible to alloxan/cysteine and ROS) that free radical induction, for example in the presence of metal ions particularly iron, or the reduction of the protective mechanisms present within mammalian cells, for example by inhibiting or reducing levels of GSH, act to increase the susceptibility of the cells to oxidative stress and induce the much earlier activation of a non-selective cation channel resulting in irreversible cell depolarization, consequent calcium overload, and ultimate cell death. This phenomenon is more generally applicable than to the particular system employed in this specific example since it is involved in the responses of various cell types to oxidative stress. The role of ROS in oxidative stress and cellular degeneration is well known. Thus the mechanism we have specifically described for the above pancreatic β-cell is of wide general significance and we believe plays a major role in the general cellular response to oxidative stress (ie. cell suicide processes) . According to a first feature of the present invention therefore there is provided a method for screening drug molecules for efficacy which comprises; a) selecting a cell or cell culture type known to respond to oxidative stress and/or ROS by opening of NS NAD channels, b) adding a cell-death monitoring composition to said culture, and c) adding thereto a channel protective chemical structure(s) for the protection thereof from the effects of ROS and cell depolarization, thereby to establish the channel antagonist status of the channel protective chemical structure. The cell-death monitoring composition may be selected from a dye exclusion composition, a fluorescent dye system, a membrane potential sensing fluorescent probe or an intracellular calcium indicator. The dye exclusion composition may for example be trypan blue whereas the fluorescent dye may be utilised in a computer based icrofluorometric technique (eg. FDA/PI) . The membrane potential sensory fluorescent probe may for example be a Bio- oxonal probe such as B438DIBAC4, which monitors the abrupt decline in membrane potential upon NS NAD activation. The intracellular calcium indicator may for example be Fura-2AM The invention may also provide a pharmaceutical composition for the control of channel mediated substrates. In one aspect the NS^ channel may be a novel molecular target for the generation of new drug molecules which will close the channel and thus prevent the depolarisation and consequent calcium overload, either by acting at the cell's membrane directly on the pore-forming sub-units (the channel) or indirectly through associated accessory proteins or second messenger systems. In an alternative embodiment the pharmaceutical composition comprises a purine nucleotide, a cyclic nucleotide or an analogue thereof. These molecular structures enhance NS NAD channel activity through a nucleotide positive regulatory site. The nucleotide positive regulatory site would therefore be a useful target site for drug manipulation. A molecular structure mimicking the actions of the nucleotides may enhance channel opening, even at concentrations of β-NAD + and calcium that do not per se open the channel and so increase the likelihood of cell destruction. Conversely an agent that acted to antagonize the nucleotide interaction or acted as a negative regulator at this site could protect against oxidative stress. An alternative pharmaceutical composition comprises a molecular structure adapted to act at the GSH binding site to agonize (close) or to antagonize (open) the channel. The molecular structure may be GSH or an biologically acceptable analogue thereof. GSH preferably is in an intracellular concentration of 3-lOmM. Application of GSH to the intracellular aspect of the membrane results in a concentration-dependent inhibition of the NS NAD channel. This inhibition is not due to any action through redox coupling to the channel, it is most likely via GSH acting as a ligand to gate the channel into the closed configuration. In an alternative embodiment the pharmaceutical composition comprises a molecular structure adapted to act at the NAD + binding site to agonize (open) or to antagonize (close) the channel. Thus we now believe that this channel is regulated by a combination of intracellular factors, requiring β-NAD + and an increase in the concentration of intracellular calcium for activation (purine nucleotides may increase activity further) , but that under normal levels of GSH channel activity is suppressed. Activation of this channel by oxidative stress may therefore first require levels of GSH to fall below some critical threshold concentration. This is exactly what occurs when cells undergo oxidative stress, i.e there is a pronounced GSH depletion. Further the technology may also be utilised in the opposite sense ie. to deliberately engineer the killing/destruction of targeted cells. Since it is known that the opening of the channel causes a massive influx of cations (sodium and calcium for example) which results in cell destruction, the compositions of the present invention may manipulate the cells either to cause an over expression of the channel or to override the cell controlling mechanisms which keep this channel closed, for example by occluding the inhibition by GSH, thereby engineering a channel opening which further results in cell destruction. According therefore to a further aspect of the present invention there is provided a pharmaceutical composition for the treatment of a disease mediated by ROS or by a lack of response to ROS, which composition comprises; a molecular structure designed to act directly on pore forming sub-units or indirectly on accessory proteins and/or on secondary messenger systems thereby to selectively close or open the channel to prevent or promote depolarisation of the cell. In a particularly preferred form of the invention the molecular structure comprises an NS NAD pore-sequence and optionally a sequence derived for an accessory protein. Further in selected instances the composition may further comprise a free radical (ROS) scavenger. Cloning of a pore sequence and knowledge of the nature of accessory proteins is of assistance in this endeavour but is not essential to the success of the pharmaceutical approach. It is however essential to the genetic approach. Cloning of the channel proteins does make it easier to determine the distribution of this protein in tissues and the changes of expression associated with disease. Additionally it aids in the search for the likely presence of the other members of the general family of free radical sensitive ion channels. The most efficient way to discover novel drug compounds (lead compounds) is through high-throughput screening methods. A simple method is to use cultured cells (eg. our cell line CRI- Gl) which are known to respond to oxidative stress and ROS production by opening the NS^ channels. This can be achieved by the cell death monitoring system set out above. The problem with the dye exclusion method set out above is that it does not tell us anything about the mechanism causing death, only that the cells are dead or dying. An alternative fluorescent dye system is the fluorescein diacetate-propidium iodine (FDA/PI) vital strain technique which again is a computer based microfluorometric technique. This measures the induced change in the plasma membrane permeability and tells if the cell membrane integrity has been terminated, but again does not indicate the cause of the collapse of the membrane potential. Alternatively membrane potential sensing fluorescent probes can give real time signals when the cell membrane potential collapses. By use of these systems, particularly the fluorescent dye systems in combination with other chemical techniques (for example, calcium imaging using Fura-2AM to detect calcium overload) , it is possible to quickly screen the many thousands of possible chemical structures for the protection of cells against the effects of ROS and cell depolarisation, (alternatively against cell hyperproliferation) and therefore establish the most likely structures acting as NS NAD channel antagonists (or agonists) . Once a short list of candidates has been achieved by this screening technique, these can be tested more extensively for their direct or indirect effects on NS NAD channels by conventional electrophysiological techniques. Tissue damage is generally believed to be through the production of ROS, in particular free radicals, such as the hydroxyl free radical which is very reactive and damaging to cells. Therefore as a therapeutic strategy it is desirable to consider a combination therapy which is best suited to maintain the cells in a viable state. Thus having discovered an NS NAD channel antagonist given above, it is desirable to add a free radical scavenger. The reason for this is that the presence of free radicals in cells undoubtedly has many deleterious effects on other cell processes. Therefore to allow the cells to have a best chance for a full functional recovery it is best not only to prevent the death signal (i.e. the opening of the NS NAD channel) but also to limit other damage caused by free radicals such as calcium overload lipid peroxidation, DNA damage etc. Thus the cells would be less functionally compromised during the recovery period from oxidative stress. Switching the NS NAD channel off and maintaining it in the closed state, for example by mimicking the action of GSH, will prevent the activation of this particular cellular destruction pathway during oxidative stress. It has also been suggested that free radical production may be carcinogenic. Accordingly cancer cells may not be as susceptible to ROS as healthy cells. A possible explanation is that NS NAD channels are not available for activation or have reduced sensitivity to ROS or are simply not expressed in tumour cells so that the natural means to kill a cell is functionally compromised. In accordance with the present invention means may be provided to induce death in specific cancer cells by increasing NS NAD channel sensitivity/activity to ROS and/or to increase the channel availability by up regulating expression. In a first method where a channel is present but largely insensitive to ROS a selective activator drug molecule is useful. The selective activator drug molecule may for example mimic β-NAD + which directly activates the channel or interact at the nucleotide regulatory site or the GSH inhibitory site to enhance the action of β-NAD + in the presence of calcium. The same may be found by the methods described above for screening of compounds using a high-throughput system to test for activity. It is then possible to make an inactive form of the drug which will enter cells and which is light activated at the correct wavelength. By using fibre-optics directed into target cells, activation of the molecule is achieved thus opening channels and causing self-destruction. Alternatively a drug vector can be targeted to a component of the channel or/and accessory proteins which act to sensitize the system to ROS (i.e. the channel is switched on at much reduced levels of free radicals/ROS production in the cells) . Once the system has been sensitised the cells can be targeted directly for promotion of oxidative stress by direct irradiation for example, or by addition of antibodies specific to cells so that the natural killer systems of the body will attack the cancer cells, and by the production of free radicals to induce cell destruction. The invention will now be described by way of illustration only with reference to the accompanying drawing wherein Figure 1 shows in graphic form a whole-cell current-clamp recording from CRI/Gl cells. At α ImM alloxan + ImM cysteine was applied and at β ImM H 2 0 2 was applied; Figure 2 shows in graphical form single channel recordings from CRI/Gl cells. At δ ImM alloxan + ImM cysteine was applied and at γ 4.4mM H 2 0 2 was applied; Figure 3 shows in graphic form single channel recordings from CRI/Gl cells. In Figure 3A (o) denotes the channel activated by ImM alloxan + ImM cysteine, (□) denotes the channel activated by 4.4mM H 2 0 2 and (•) denotes the channel activated by β-NAD + in inside-out patches from CRI-G1. Figure 3B shows the representative traces of single-channel currents recorded from CRI-G1 cells following depolarization by ImM alloxan + ImM cysteine (e) and 4.4mM H 2 0 2 (ζ) and the single-channel currents reorded from an excised inside-out patch following the addition of β-NAD + (θ) ; Figure 4 shows whole-cell current-clamp recordings from CRI/Gl cells further comprising 50mM nicotinamide(η) , lOOOUml "1 catalase(κ) or ImM dimethylthiourea (DMTU) (ξ) ; whereas Figure 5 shows whole-cell current clamp recordings from CRI/Gl cells undergoing oxidative stress further comprising 10 mM H 2 0 2 (σ and u) . In the above Figures 1-5 the downward arrows indicate initiation of the drug challenge to cells and these agents are therefore present throughout the rest of the recording. Example l - Figure l shows "A" a representative record of the time course of membrane potential change following formation of the whole cell configuration in the absence of drugs. The electrode contained (in mM) 140 KC1, 0.6 MgCl 2 , 4.51 CaCl 2 , 5.0 EGTA, 10 HEPES, pH 7.2 (free Ca 2+ of 1 μM) and the bath solution consisted of normal saline (in mM) ; 135 NaCl, 5 KC1, 1 MgCl 2 , 1 CaCl 2 , 10 HEPES, pH7.4. The rapid hyperpolarisation of the cell due to activation of K ATP channels as ATP is washed out from the cell can be seen. The membrane potential was well sustained over a 45 minute period prior to an appearance of a slow depolarisation to -40mV, thus allowing the examination of the actions of the test compounds B and C. Representative recordings of CRI-G1 membrane potential changes followings application of 1 mM alloxan + 1 mM cysteine (B) or l mM H 2 0 2 (C) in the bathing solution are shown. Note the sudden appearance of complete and irreversible depolarisation for both treatments. Thus specifically the whole-cell current-clamp recordings from an insulinoma cell-line CRI/Gl show that following formation of the whole-cell configuration the initial mean resting membrane potential was -42.0 ± 1.3 V (n=10) . Dialysis of the cell with ATP free solution resulted in cell hyperpolarisation with a concomitant decrease in input resistance which stabilised after 5 or 10 minutes at a membrane potential of -70 ± 1.2 mV (n=10) . This increase in conductance is due to the run up of ATP-sensitive K + (K ATP ) channels as the cytoplasmic ATP is washed out from the cells. The cells remain at this hyperpolarised membrane potential for approximately 45 minutes before the K ATP channel activity decreases again due to run-down and the cells slowly depolarise towards a new stable membrane potential value of approximately -40 V. The application of alloxan (1 mM) a diabetogenic agent in combination with the reducing agent cysteine (1 mM) , agents which have been shown to cause destruction of pancreatic β- cells and insulinoma cells i vitro (and in vivo) caused the appearance of a slow irreversible depolarisation to -2.0 ± 0.8 mV (n=10) . This was actually observed in every cell tested. The depolarisation induced by alloxan/cysteine began 10.4 ± 2.1 minutes following application of drugs to the bath and took 8.4 ± 0.9 minutes to complete and was often associated with a decrease in cell input resistance indicating that an increased ionic conductance had occurred. Cells remained completely depolarised over the remaining course of recording (up to 45 minutes) whether the alloxan/cysteine remained in contact with the cells or was washed out following depolarisation. This effect was not seen in the absence of alloxan/cysteine (n=8) when comparable time studies were analysed. Thus complete depolarisation was not observed over identical time periods. Microscopic examination of the cells following alloxan/cysteine treatment showed that cells underwent morphological changes such as membrane shrivelling and blebbing consistent with cell necrosis and ultimately cell death, before finally detaching from the plate some 2-4 hours later. These changes did not occur in control cells which were not treated with alloxan/cysteine and did not occur, or were substantially delayed, in cells treated with 1 mM alloxan/1 mM cysteine in the presence of catalase or 50 mM nicotinamide or DMTU (treatments which remove ROS) (see Figure 4) . Example 2 - In order to establish the nature of ionic conductance underlying this irreversible depolarisation, cell- attached single-channel recordings were performed. Figure 2A illustrates a typical response to the addition of 1 mM alloxan/1 mM cysteine to the bathing solution. In keeping with the whole-cell membrane potential data, there was a delay of approximately 4.3 ± 0.8 minutes (n=8) following addition of alloxan/cysteine before the appearance of an inward current. The appearance of this current was sudden and often many channels appeared to be activated over a very short time period (10-30 seconds) as shown in Fig. 2A. This current reached a peak and was observed to decline again until the patch was effectively silent once more. However this was simply due to the complete collapse of the cell resting membrane potential (as shown in Fig. 1) and the consequent decrease in driving force for current flow. This was shown by application of voltage to the recording pipette whereupon discrete channel events were observed again. Channel activity could be observed in the cell-attached mode with a voltage applied to the pipette (Figure 2B) for as long as recordings were made thereafter (approximately 20 minutes) . Application therefore of alloxan/cysteine induced the appearance of this inward current in 15 out of 20 patches examined. Alloxan is believed to cause β-cell death through the production of hydrogen peroxide and subsequent formation of free oxygen radicals, in particular the hydroxyl free radical. Application of H 2 0 2 directly to insulinoma cells reproduced the actions of alloxan/cysteine exactly i.e. induction of irreversible depolarisation with a concomitant increase in cellular conductance, the identical morphological changes and eventual cell death (as determined by detachment of cells from the plate) . The delay before the onset of irreversible depolarisation by H 2 0 2 was concentration dependant; 1 mM required 15.5 ± 4 minutes (n=4) , 10 mM 8.7 ± 1.8 minutes (n=6) and 44 mM 3.8 ± 8.7 minutes (n=6) . In this time matched control experiment membrane potential was monitored for 60 minutes and no spontaneous depolarisations of this nature were observed. For all three concentrations of H 2 0 2 the depolarization event was significant (P < 0.01). Example 3 - A channel activated by application of H 2 0 2 was identical to that opened by the presence of alloxan/cysteine as can be observed on the cell-attached recordings following depolarization of the cells (compare Figures 2C and 2B/3B) . Note the appearance of single channel currents (Figures 2C/3B) characterized by long stable open times and identical amplitude when compared with the channel activated by alloxan/cysteine (Figure 2A, B / 3B) . The channel activated by alloxan/cysteine or H 2 0 2 applications has a number of unique properties. Channel activity is characterised by very long, stable open events having a duration of seconds with relatively few closures during an opening burst. Construction of current-voltage relations, following the collapse of the cell membrane potential, indicate that the channel is characterised by a linear relation with a reversal potential 0 mV under both conditions for activation (Figures 2B and 3B) . The single channel conductance as measured following depolarisation in cell- attached recordings was 68.8 ± 1.1 pS (n=3) and 71.0 ± 3.4 pS (n=4) for alloxan/cysteine and H 2 0 2 activation respectively. Excision of the patch from the cell following activation of this channel resulted in the complete and immediate cessation of channel activity. Direct application of ImM alloxan/1 mM cysteine (n=5) or up to 44 mM H 2 0 2 (n=10) to the cytoplasmic aspect of inside-out membrane patches did not cause the restoration of channel activity. However the application of β-NAD + (0.2-0.5 mM) to the bathing solution resulted in the appearance of a channel with properties indistinguishable from those of the channel described in cell-attached recordings. The β-NAD + -activated channel was also characterised by long open durations (seconds) and a linear current-voltage relation with a slope conductance of 71.3 ± 1.8 pS and a reversal potential of OmV (Figures 2B, C and Figures 3B,C). The channel described in the cell-attached and inside-out recording configurations is a non-selective cation channel for the following reasons. In cell-attached recordings the electrode contained normal saline (NaCl) and the predominant cation intracellularly is potassium. In inside-out patches the membrane was exposed to normal saline on both sides. Further experiments with cation and anion substitutions using inside-out patches clearly indicate that the β-NAD + channel is permeable to monovalent cations such as Na+, K+ and Cs+ and has an appreciable permeability to Ca 2+ but is impermeable to Cl (Herson et al., 1997b). Therefore it is likely that the channel activated by alloxan and H 2 0 2 in intact cells is the same as that opened by β-NAD + in inside-out patches (Reale et al., 1995; Herson and Ashford, 1997) . Indeed the frequency with which this channel is detected in inside-out patches is 75%, a figure virtually identical to that observed for the incidence of the alloxan/cysteine and H 2 0 2 activated channels in cell-attached recordings. Example 4 - Intact cells of the CRI-G1 insulin-secreting cell line were loaded with fura-2 AM 30 min prior to imaging and intracellular calcium levels were estimated from the ratio of 510 n emission from excitation by 340 and 380 nm wavelength light. Whole cell experiments were performed with patch pipettes containing (mM) : 140 KC1, 0.6 MgCl 2 10 HEPES (pH 7.2) and 50 μM fura-2 and the bath solution contained (mM) : 135 NaCl, 5 KC1, 1 MgCl 2 , 1 CaCl 2 , 10 HEPES (pH 7.4). Nominally free calcium was obtained by adding 10 mM EGTA to the appropriate solution. In 3 intact cells lOmM H 2 0 2 resulted in a biphasic rise in intracellular calcium, with the first small phase occurring after 5 min with the 340/380 ratio rising from 1.13 ± 0.09 (90 nM) to 3.38 ± 0.35 (736 nM) . The second dramatic rise occurred after 20 min and reached dye saturation (> 10 μM) . In agreement with the intact cell, the addition of 10 mM H 2 0 2 during whole-cell current clamp experiments resulted in a biphasic calcium response in all six cells tested. The first phase occurred within 2-3 min and the 340/380 ratio rose from 0.45 ± 0.02 (46 nM) to 0.87 ± 0.08 (150 nM) . The second phase coincided exactly with the hydrogen peroxide induced depolarization and resulted in a second dramatic increase in intracellular calcium, the ratio rising to 1.94 ± 0.16 (630 nM) . The cation channel involved in this depolarization is calcium permeable and therefore we tested whether removal of extracellular calcium abolishes the second rise in calcium. In intact cells 10 mM H 2 0 2 , in the absence of extracellular calcium, caused only the initial rise which occurred after approximately 5 min and the 340/380 ratio rose from 0.65 ± 0.01 (50 nM) to 0.75 ± 0.02 (110 nM) and this was maintained over the duration of the experiment, n=5 (30 min) . The addition of extracellular calcium resulted in a rapid and dramatic increase in intracellular calcium, reaching dye saturation. The removal of intracellular calcium, by the addition of 10 mM EGTA to the pipette solution abolishes any change in intracellular calcium as well as preventing the H 2 0 2 induced depolarization (n=4) . The results of the above experiment, see Figure 5, indicate that during H 2 0 2 challenge to CRI-G1 insulin-secreting cells, there is a rise in intracellular Ca 2+ that precedes the activation of NS NAD channels. The first increase in intracellular Ca 2+ is small (from <100nM to 700-800 nM) whereas the second increase is much more dramatic (> 10 μM) see Figure 5A. In the absence of extracellular Ca 2+ only the first rise in intracellular Ca 2+ is observed whereas chelation of intracellular Ca 2+ with EGTA abolished all changes in intracellular Ca 2+ concentration associated with the H 2 0 2 challenge as well as preventing the H 2 0 2 induced depolarization see Figure 5B. We interpret these data as follows; subsequent to the H 2 0 2 challenge, there is an initial mobilization of intracellular Ca 2+ , from intracellular stores(see Figure 5A) , and this, in association with a decline in GSH levels, results in NAD+ (and Ca 2+ ) dependent activation of the NS NAΓJ channel which in turn allows substantial entry of extracellular Ca 2+ through the activated channels (and so the second, larger, rise in intracellular Ca 2+ ' see Figure 5A) and further potentiation of the depolarizing current. In addition clearly any other mechanism acting in vivo which contributes to an increased internal Ca 2+ concentration in cells undergoing oxidative stress will enhance the opening NS NAD channels. In particular with respect to CNS diseases purported to involve oxidative stress as a key element for presentation, increased neuronal excitation through the opening of NMDA type receptor-channels by glutamate may enhance neurotoxicity. There is a great deal of evidence for this mechanism as a contributing factor to many CNS degenerative diseases currently in the scientific literature. Example 5 - Whole-cell current-clamp recordings from CRI-G1 cells. The electrode contained (in mM) ; 140 KCL, 0.6 MgCl 2 , 4.51 CaCl 2 , 5.0 EGTA, 10 HEPES, pH 7.2 (free Ca 2+ of lμM) and the bath solution consisted of normal saline. A representative record of the time course of membrane potential change following formation of the whole-cell configuration and the subsequent application of (A) 1 mM alloxan/cysteine + 50 mM nicotinamide and (B) 1 mM alloxan/cysteine + 1000 U ml "1 catalase and 1 mM alloxan/cysteine + 1 mM DMTU. Note that, over the time scale of these experiments (35-40 minutes) , the presence of 50 mM nicotinamide, 1000 U ml "1 catalase or 1 mM DMTU prevented the appearance of the alloxan/cysteine depolarisation (compare with Fig. IB, C) . From the forgoing it will be seen that alloxan induced destruction of pancreatic β-cells is known to be associated with the production of reactive oxygen species and is considered to be a model of how β-cell cytotoxicity occurs in Type 1 diabetes. The cellular target for the reactive oxygen species (free radicals) has not been elucidated. However, from the data presented herein we suggest that alloxan, through the formation of H 2 0 2 , causes the opening of a novel non-selective cation channel which results in the sudden, complete and irreversible depolarisation of the cell as a prelude to cell destruction. Opening of this channel is most likely mediated by H 2 0 2 reacting with catalytically active ferrous iron to form hydroxyl radicals. The result of the activation of this channel type is not only total cell depolarisation, with consequent calcium entry via activation of voltage gated calcium channels, but also the uncontrolled and long term entry of sodium and calcium ions through the non-selective channel itself. There are many examples of cell destruction mediated through the uncontrolled entry of calcium (eg. via NMDA or nicotinic receptor activation) or the opening of L-type calcium channels by serum IgM from Type 1 diabetes patients. We now suggest that the free-radical-sensitive channel we have reported here is a novel means by which a cell undergoing oxidative stress dies. In summary the biophysical characteristics of the NS AD channel are that the channel is permeable to the main physiological inorganic cations (K + , Na + , Ca 2+ and Mg 2+ ) . It has extremely slow kinetics such that the open times are measures in seconds and it has a large conductance(70-9OpS) . The channel can only be activated from the intracellular aspect by the pyridine nucleotide β-NAD + (half-maximal activation at approximately lOOμM) , which is present in all mammalian cells in a concentration of 200-300μM. Hence activation of the NS NAD channel occurs within the physiological range of NAD + in cells. The activation of the channel by β-NAD + displays an absolute requirement for the presence of Ca 2+ and Mg 2+ ions in the intracellular environment.

Claims: CLAIMS 1. A method for screening drug molecules for efficacy which comprises : - a) selecting a cell or cell culture type known to respond to oxidative stress and/or ROS by opening an NS NAD channel, b) adding a cell-death monitoring composition to said culture, and c) adding thereto a channel protective structure(s) for the protection thereof from the effects of ROS and cell depolarisation; thereby to establish the channel antagonist status of the channel protective structure. 2. A method according to claim 1 wherein the cell-death monitoring composition is selected from a dye exclusion composition, a fluorescent dye system, a membrane potential sensing fluorescent probe or an intracellular calcium detection system. 3. A method according to claim 2 wherein the dye exclusion composition is selected from trypan blue or wherein the fluorescent dye utilises a computer based micro-fluorometric technique or a membrane potential sensing fluoresence probe or wherein Fura-2AM is utilised in the intracellular calcium detection system. 4. A pharmaceutical composition for the treatment of a disease mediated by ROS or by a lack of response to ROS which composition comprises a molecular structure adapted to act directly on pore-forming sub-units or indirectly on accessory proteins and/or secondary messenger systems thereby to selectively close or open a NS NAD channel and selectively prevent or promote depolarisation of the cell. 5. A pharmaceutical composition according to claim 4 wherein the molecular structure comprises an NS NAD pore sequence and optionally a sequence derived for an accessory protein. 6. A pharmaceutical composition according to either of claims 4 or 5 wherein the molecular structure is adapted to act at the NAD + binding site to agonize (open) or to antagonize (close) the channel. 7. A pharmaceutical composition according to any one of claims 4-6 wherein the molecular structure is adapted to act at the GSH binding site to agonize (close) or to antagonize (open) the channel. 8. A pharmaceutical composition according to any preceding claim where in the molecular structure is adapted to act on a nucleotide positive regulatory site. 9. A pharmaceutical composition according to claim 8 wherein the molecular structure is a purine nucleotide, a cyclic nucleotide or a biologically acceptable analogue thereof. 10. A pharmaceutical composition according to any one of claims 4-9 wherein the molecular structure is GSH or a biologically acceptable analogue thereof. 11. A pharmaceutical composition according to claim 10 wherein the GSH is in an intracellular concentration of 3- lOmM. 12. A pharmaceutical composition according to any preceding claim wherein the composition further comprises a free radical scavenger.

Inventors: ASHFORD MICHAEL LAWRENCE JAMES, HERSON PACO SAMUEL, ASHFORD, MICHAEL, LAWRENCE, JAMES, HERSON, PACO, SAMUEL
Applicants: ASHFORD MICHAEL LAWRENCE JAMES, HERSON PACO SAMUEL, UNIV ABERDEEN, ASHFORD, MICHAEL, LAWRENCE, JAMES, HERSON, PACO, SAMUEL, UNIVERSITY OF ABERDEEN
Application year: 1997
Main classification: G01N33
IPC codes: A61K31, G01N33