Patent Application: US-65306500-A

Abstract:
a method is provided for therapeutic use of a class of compounds that are effective in protecting nerve cells from deterioration and cell death arising from degenerative disease , trauma or aging and may be used to achieve a similar effect in male and female subjects with minimal adverse side effects . the method comprises administering a therapeutically effective dose of a natural or synthetic bioflavonoid that acts as an mapk cascade antagonist . examples of bioflavonoids that may be used in the present method are apigenin and 2 -- oxanaphthalen - 4 - one .

Description:
“ neuroprotection ” is defined here and in the claims as the inhibition of progressive deterioration of neurons that leads to nerve cell death . “ neurodegenerative disorder ” is defined here and in the claims as a disorder in which progressive loss of neurons occurs either in the peripheral nervous system or in the central nervous system . examples of neurodegenerative disorders include : chronic neurodegenerative diseases such as alzheimer &# 39 ; s disease , parkinson &# 39 ; s disease , huntington &# 39 ; s chorea , diabetic peripheral neuropathy , multiple sclerosis , amyotrophic lateral sclerosis ; aging ; and acute neurodegenerative disorders including : stroke , traumatic brain injury , schizophrenia , peripheral nerve damage , hypoglycemia , spinal cord injury , epilepsy , and anoxia and hypoxia . these examples are not meant to be comprehensive or limiting in any way but serve merely as an illustration of the term “ neurodegenerative disorder .” the present invention is directed to a novel method that inhibits nerve cell death caused by activation of intracellular enzymes that can cause nerve cell death . for example , the present method inhibits nerve cell death caused by the intracellular enzymes known as the mapk cascade . the method is used for treating and preventing nerve cell death caused by acute or chronic neurodegenerative disorders . the method comprises supplying at least one bioflavonoid that acts as an inhibitor of intracellular enzymes , such as mapk , that , when activated , cause nerve cell damage and death , and administering a therapeutically effective dose of the bioflavonoid to an animal or human . the class of neuroprotective compounds that may be used in the present method include ( a ) compounds that can be extracted from plants , for example apigenin , or ( b ) synthetic compounds of a similar composition , for example 2 -( 2 ′- amino - 3 ′ methoxyphenyl )- oxanaphthalen - 4 - one . the bioflavonoid may be administered orally , intramuscularly , transdermally , buccally , intravenously or subcutaneously . also , the bioflavonoid may be administered by dose or by controlled release vehicles . pharmaceutically acceptable salts of the compounds may also be used . in one embodiment of the invention , the bioflavonoid 2 -( 2 ′- amino - 3 ′ methoxyphenyl )- oxanaphthalen - 4 - one ( referred to herein as “ pd098059 ”) is administered as an intracellular enzyme inhibitor . the pd098059 is administered to achieve a blood plasma concentration of pd098059 of up to 100 μm , with a plasma concentration preferably between about 30 μm and about 90 μm . as described in example 1 below , this level of pd098059 has been shown to be effective in providing neuroprotection to nerve cells in the presence of intracellular enzymes that can cause neuron cell death . in another embodiment of the invention , the bioflavonoid apigenin is administered as the enzyme inhibitor . the apigenin is administered to achieve a blood plasma concentration of up to 100 μm , with a plasma concentration preferably between about 25 μm and 100 μm . as described in example 1 below , this level of apigenin has been shown to be effective in providing neuroprotection to nerve cells in the presence of intracellular enzymes . as will be apparent to one skilled in the art from the disclosure herein , other bioflavonoids , or pharmaceutically acceptable salts of the bioflavonoids , may be used in the present method to achieve neuroprotection . specific examples of compounds that may be used in the method of the present invention are apigetrin , apiin , pelargidenon , versulin , cossmetiin , apioside , cosmosiin , cosmosiine cosmosioside , baicalin , cirsimaritin , 6 - hydroxyluteolin , luteolin , plantaginin , rhoifolin , sorbavin , afrelin , hyperin , isoquercitrin , isorhamnetin , kaempferitrin , kaempferol - 7 - glucoside , oxyayanin - a , quercetin , quercitrin , rhamnetin and rutin . furthermore , any of these compounds may be supplied in the form of a prodrug that may be metabolized to form an active mapk cascade antagonist . administration of any of the compounds of the invention may include the use of a single compound or a mixture of neuroprotective compounds . the neuroprotective compounds used in the present invention can be administered in pharmaceutical compositions . the recommended route of administration of the neuroprotective compound includes oral , intramuscular , transdermal , buccal , intravenous and subcutaneous . methods of administering the compound of the invention may be by dose or by controlled release vehicles . the pharmaceutical compositions may be in the form of tablets , dragees , capsules , pills , solutions , suspensions , emulsions or ophthalmic preparations or any other appropriate form for delivery of pharmaceutical compositions . the pharmaceutical compositions in solid form may contain non - aqueous diluents , including for example fillers and extenders , binding agents , moisturizing agents , disintegrating agents , surface active agents , adsorptive carriers , lubricants or any other appropriate diluent known to one skilled in the art . pharmaceutical compositions in liquid form may contain liquid diluents such as , for example , water , ethyl alcohol , propylene glycol or any other appropriate diluent known to one skilled in the art . for parenteral administrations , solutions and suspensions should be sterile and , if appropriate , blood - isotonic . as used herein , the term “ therapeutically effective dose ” means a dose of a mapk cascade inhibitor which will treat or protect a subject from acute or chronic neurodegenerative disorders such as for example alzheimer &# 39 ; s disease . therapeutically effective doses of the mapk cascade inhibitor can be determined according to standard medical principles under the direction of a physician or veterinarian . as described in detail in examples 1 - 3 below , assays have been conducted demonstrating the neuroprotective effects that are achieved by the method of the present invention . the efficacy of the method of the present invention has been demonstrated in experiments conducted on cultured hippocampal tissue explants prepared according to the method of stopini et al . ( stoppini l ., buchs p . a . and muller d . a simple method for organotypic cultures of nervous tissue . j . neurosci . meth ., 37 , 173 , 1991 ). seven day old wistar rat pups were anaesthetized with fluothane , decapitated and transverse hippocampal slices ( 400 microns ) were cultured for 12 days on 6 - well tissue culture plates containing 1 ml of culture medium . the medium composition was 50 % minimal essential medium ( mem ), 25 % horse serum , 25 % earl &# 39 ; s balanced salt solution with d - glucose and hepes , penicillin g and streptomycin sulfate , 5000 units / ml and 5 μg / ml respectively and the ph , 7 . 15 . slices were cultured at 36 . 5 ° c ., 100 % humidity , 95 % air and 5 % co2 atmosphere , and were fed twice weekly by 50 % medium exchange . all drugs were dissolved in water or dimethylsulfoxide ( dmso , final concentration was 0 . 1 %) to make stock solutions . controls for dmso effects were performed where appropriate . to apply a drug , the stock solution was dissolved in 1 ml of fresh culture medium to make a desired concentration . then , hippocampal slices were transferred for a specified time interval to the fresh medium containing the drug . to terminate the drug action , slices were again transferred to a fresh medium containing 2 mg / ml propidium iodide ( pi ). the medium temperature was kept at 36 ° c . during the transfers . since the change of medium induced nerve cell death , control experiments were performed by transferring hippocampal slices an equal number of times into the fresh medium with the intervals between transfers equal to the time interval that slices were exposed to the drug . in a separate series of experiments the medium replacement was performed twice with a 30 minute interval in order to induce cell death . cell death was measured as the pi fluorescence intensity by using mrc - 600 laser scanning confocal imaging system . pi was added to the medium 30 minutes prior to taking control images ( immediately before the drug treatment ). images were taken immediately before and 24 hours after the drug treatment or , where cell death was induced by the fresh medium , 24 and 72 hours after the last medium exchange . each experiment was performed at least in triplicate , on 5 - 6 slices each time and data expressed as an average of all experiments . cell death index was derived from pi fluorescence intensity normalized to the intensity values obtained after the 48 hour exposure of slices to an ambient temperature of 4 ° c . image analysis was performed off - line using scionimage ( scion corporation ) image analysis software . differences were considered significant where p & lt ; 0 . 05 ( unpaired two - tailed t - test ). as illustrated in fig1 exposure of hippocampal slices to n - methyl - d - aspartate ( nmda ) ( 2 . 5 - 500 μm ) for 30 minutes resulted in marked cell death in the ca1 - ca4 region of the hippocampus . the effect was concentration - dependent and could be blocked by the nmda receptor antagonist d - apv but not the na + channel blocker tetrodotoxin or non - nmda receptor antagonists . these data suggest that cell death was mediated by nmda receptors and was not dependent on neurotransmitter release . assuming that exposure of slices to a temperature of 4 ° c . killed 100 % of cells , 50 μm of nmda killed approximately 30 % of all cells and this concentration of nmda ( and the corresponding level of death ) was used as a reference point in all further experiments . in another region of the hippocampus , the dentate gyrus , nmda ( 2 . 5 - 500 μm ) did not induce concentration - dependent increase in cell death , which reflects selective vulnerability of the hippocampal neurons . to show that mapk inhibitors are effective in reducing nerve cell death , hippocampal slices were treated for 2 hours with the mapk cascade inhibitors pd098059 ( 30 - 90 μm ) or apigenin ( 25 - 100 μm ) prior to application of excitotoxin n - methyl - d - aspartate ( nmda , 50 μm ). as shown in fig2 pd098059 significantly reduced nmda toxicity . the reduction of fluorescence intensity as compared to fig1 illustrates the neuroprotective effect of pd098059 . pd098059 was also effective in reducing nmda - induced cell death when applied immediately after nmda , suggesting that this neuroprotective action was unlikely due to nmda receptor / channel blockade but is attributed to mapk inhibition . in contrast , as shown in fig4 wortmannin ( 1 - 5 μm ) an inhibitor of another intracellular kinase , phosphoinositol - 3 kinase ( pi - 3k ), did not reduce nmda toxicity . this demonstrates that the inhibitor must specifically inhibit the mapk cascade to impart the neuroprotective effects of the present invention . as shown in fig3 the pkc inhibitor staurosporine , which indirectly blocks mapk activation was also somewhat effective in protective nerve cells against nmda toxicity . however , staurosporine was clearly not as effective as the direct mapk inhibitor pd098059 . the neuroprotective activity of the mapk cascade inhibitor pd098059 in comparison to the other compounds tested is summarized in the graph in fig5 . as shown in fig5 at 24 hours after nmda exposure , the number of dead cells in hippocampal explants is 45 % lower in the presence of 0 . 2 μm of staurosporine . the number of dead cells in explants treated with pd098059 was 95 % lower than in explants treated with nmda alone , and 33 % lower than in explants treated with staurosporine . wortmannin did not exhibit any significant reduction in nerve cell death was without any effect . in another example of the method of the present invention , cell death was induced by diluting the culture medium with freshly prepared culture medium . there was a significant increase in cell death in slices subjected to transfers to a fresh medium at 24 and 72 hours after the first transfer , in comparison to cultures that received no transfers . the increase occurred in both ca1 - ca4 and dentate gyrus areas and was not blocked by nmda receptor antagonist ap5 ( 20 μm ) suggesting that this was not an nmda - dependent process . as shown in fig6 addition of pd098059 ( 30 - 90 μm ) for 2 hours prior to the exposure of cultures to the fresh medium significantly reduced cell death at 24 hours and at 72 hours . wortmannin ( 1 μm ), which inhibits growth factor receptor signaling , significantly enhanced cell death at 24 and 72 hours suggesting that growth factor withdrawal contributed to cell death due to the culture medium exchange . in a further example of the method of the present invention , cell death was induced in cultured slices treated with the toxic fragment 25 - 35 of the amyloid β - protein . experiments were performed as in example 2 , with the difference that the culture medium contained small quantities of l - glutamic acid ( cultures were healthier in its presence ). toxic β - amyloid peptides were added to cultured hippocampal tissue explants subjected to 100 % culture medium exchange . amyloid peptides induced cell death in hippocampal slices . however , addition of 30 - 90 μm of pd098059 for 2 hours prior to the addition of toxic peptides prevented cell death . the purpose of the above description and examples is to illustrate some embodiments of the present invention without implying any limitation . as will be recognized by those of skill in the art based on the teachings herein , numerous changes and modifications may be made to the above - described invention without departing from its spirit or scope as defined in the appended claims . accordingly , this detailed description of preferred embodiments is to be taken in an illustrative , as opposed to a limiting sense . all patents and publications cited in the bibliographic citation below are incorporated by reference in their entireties . abdel - hamid , k . m . and tymianski , m . mechanisms and effects of intracellular calcium buffering on neuronal survival in organotypic hippocampal cultures exposed to anoxia / aglycemia and excitotoxins . j . neurosci ., 17 , 3538 , 1997 . adayev t . estephan r . meserole s . mazza b . yurkow e j . banerjee p . externalization of phosphatidylserine may not be an early signal of apoptosis in neuronal cells , but only the phosphatidylserine - 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