Patent Application: US-48770400-A

Abstract:
a method of reducing brain damage resulting from seizures caused by an organophosphorus nerve agent includes administering to a patient a therapeutically effective amount of hu - 211 . the organophosphorus nerve agent may be gb , gd , ga or gf . the therapeutically effective amount of hu - 211 is in the range of about 48 mg to about 200 mg per day .

Description:
thirty - four male sprague - dawley rats ( crl : cd [ sd ]- br ; charles river labs , wilmington , mass . ), weighing between 250 - 300 g , were used . animals were housed individually in polycarbonate cages under conditions of constant temperature ( 21 ± 2 ° c .) and humidity ( 50 ± 10 %) using at least 10 complete air changes per hour of 100 % fresh air , and a 12 - hour light - dark cycle ( full spectrum lighting cycle with no twilight ). throughout the study , food and water were available ad libitum , except during the observation period , which began 1 . 5 hours prior to and ended 5 hours following soman administration . each rat was anesthetized with sodium pentobarbital ( 55 mg / kg , i . p .) and positioned in a stereotaxic apparatus ( david koif instruments , tujunga , calif .). three holes were drilled through the skull into which screw electrodes were placed , in accordance with the procedure recommended by braitman and sparenborg ( 1989 ) for electrocorticographic ( ecog ) recordings . electrodes were connected to a standard small - animal head - piece and secured by dental cement . on the morning of the sixth day following surgeries , animals were connected to an ecog recording system and allowed at least two hours to acclimate . baseline ecog activity and behavior were monitored for at least 15 min . following baseline recordings , animals were injected ( i . p .) with 125 mg / kg of the oxime hi - 6 . this was followed 30 min later by injection of 180 μg / kg soman ( 1 . 6 ld 50 s . c .) or sterile saline . within one min following soman or saline injection , animals were injected ( i . m .) with 2 mg / kg atropine methylnitrate ( amn ). hi - 6 and amn were administered to protect against the peripheral effects of soman . the treatment drug , hu - 211 ( pharmos ltd ., rehovot , israel ), was injected ( 25 mg / kg , i . p .) 5 min following the onset of seizures , as determined by ecog recordings . a non - soman , treatment drug control group received hi - 6 , saline , amn , as described above ; in addition , hu - 211 was administered 15 min following saline injection . the above paradigm yielded five treatment groups : ( 1 ) soman - injected positive controls , n = 13 ; ( 2 ) hu - 211 5 min post - onset , n = 8 ; ( 3 ) hu - 211 treatment drug controls , n = 3 ; ( 4 ) non - soman negative controls , n = 6 . ecog recordings were monitored for 5 hours following soman administration . additional recordings ( 30 min ) were obtained at 24 hours from surviving , animals . twenty - seven hours after soman / saline administration , rats were given a lethal injection of pentobarbital anesthesia ( 100 mg / kg , i . p .) and euthanatized , upon evidence of labored breathing , via transcardial perfusion with ice cold 4 % paraformaldehyde in 0 . 1m phosphate buffer ( pb , ph 7 . 4 ). brains were immediately excised and longitudinally divided into left and right hemispheres . alternate hemispheres ( left or right ) were postfixed by immersion in a second solution of ice cold 4 % paraformaldehyde in 0 . 1m pb for 4 - 6 hours . these hemispheres were subsequently sucrose - saturated ( 30 % sucrose in 0 . 1m pb , for 72 hours ) and coronally sectioned at 40 μm . serial sections either were collected directly onto polylysine coated slides for cresyl violet ( cv ) staining or cryoprotected ( watson et al ., 1986 ) and stored at − 20 ° c . pending immunocytochemical staining . the remaining hemispheres were paraffin processed , sectioned at 4 μm and stained with hematoxylin and eosin ( h & amp ; e ). immunocytochemistry requires strict attention to detail to achieve uniform staining at sufficient quality for semiquantitation and between - subject comparisons . this procedure employed a monoclonal antiserum , raised in mice , against microtubule - associated protein 2 ( map2 ) ( sigma chemical co ., st louis , mo . ), and utilized the avidin - biotin - peroxidase method of hsu et al . ( 1981 ); “ elite abc mouse kits ” were obtained from vector labs ( burlingame , calif .). for each subject , a single free - floating brain section ( bregma − 3 . 1 ± 0 . 2 and − 4 . 8 ± 0 . 2 mm ) was removed from cryoprotectant and processed simultaneously , with neuroanatomically homologous brain sections from the other subjects , to minimize individual variations associated with tissue processing . sections were cleared of cryoprotectant solution by four rinses in 0 . 1m phosphate buffer ( pb ; ph 7 . 4 ; 15 min / rinse , with the exception of the first rinse which lasted 10 - 45 min ). starting with the second rinse in pb , all incubation times were strictly monitored to ensure uniformity . endogenous peroxidase activity was quenched by placing sections into 3 % h 2 o 2 in 0 . 05m tris buffer ( tb ; ph 7 . 6 ) for 5 min . all of the following steps were preceded by three rinses in 0 . 05m tb ( ph 7 . 6 ) unless otherwise indicated . to block nonspecific staining , sections were incubated in 0 . 05m tb containing 5 % normal horse serum and 0 . 1m d , l - lysine . sections were incubated for 18 hours at 4 ° c . in primary antiserum diluted 1 : 4000 for map2 with 0 . 05m tb containing 1 % normal horse serum . sections were incubated for 30 min at room temperature in biotinylated secondary antiserum ( i . e ., horse - antimouse ) diluted 1 : 200 in 0 . 05m tb containing 1 % normal horse serum and 1 % normal rat serum . sections were incubated in a solution containing the avidin - biotin peroxidasc complex ( diluted 1 : 50 in 0 . 05m tb ) for 20 min . each pair of sections was preincubated in 1 . 0 ml of freshly prepared ( i . e ., 20 - 25 min old ) 0 . 05 % 3 ′, 3 - diaminobenzidine tetrahydrochloride ( dab ; sigma chemical co ., st louis , mo .) solution for 5 min , at which time 33 μl of 0 . 3 % h 2 o 2 was added and immediately followed by vigorous agitation . sections remained in the resultant solution containing 0 . 048 % dab and 0 . 01 % h 2 o 2 for 2 . 5 min ± 3 sec . the reaction was stopped by two rinses in 0 . 05m tb ( for 5 and 15 min , respectively ). negative control sections were incubated in the same solutions for the same incubation times as the other brain sections , with the exception that the primary antibody solution ( anti - map2 or anti - gfap ) was replaced by a 0 . 05m tb solution containing 1 % horse serum without the primary antibody . sections were floated onto polylysine - coated slides , dried , dehydrated , cleared and mounted . morphometric image analysis of map2 immunohistochemistry was performed using a quantimet 600 image analysis system ( leica cambridge ltd ., cambridge , england ), equipped with an olympus bh - 2 biological microscope ( olympus optical co ., ltd ., tokyo , japan ). morphometric assessments of the cross - sectional areas of map2 - negative staining ( i . e ., necrosis [ ballough et al ., 1995 ; hicks et al ., 1995 ]) in the piriform temporal lobe , i . e ., piriform cortex and contiguous brain regions ( e . g ., endopiriform nuclei , amygdaloid nuclei and perirhinal cortex ), were performed according to the procedure of ballough et al . ( 1995 ). previous studies have shown that the deep piriform cortex and surrounding areas present the most clearly defined and easily quantifiable lesions of contiguous necrosis at 27 h following soman - induce seizures in rats ( ballough et al ., 1995 ). to standardize image comparisons between brain sections from each subject , the image analysis system was arbitrarily preset , by adjusting the hue , saturation and intensity of the binary image , to reflect a maximum contrast between map2 - negative ( necrotic ) and map2 - positive immunostaining on a typical soman - injected positive control section . for each brain section , piriform cortical map2 - negative immunostaining was interactively outlined , using a pointing device , and area determinations were calculated automatically by the image analysis system . the average of two measurements for each brain section was recorded . h & amp ; e stained brain sections ( bregma − 3 . 3 ± 0 . 2 and − 4 . 8 ± 0 . 2 mm ) were assessed for classical histopathological damage to the piriform cortex , amygdaloid complex , hippocampus and thalamus . damage was scored on a scale of 0 to 4 , where 0 = no histologic lesion , 1 = minimal damage ( 1 - 10 % neuronal loss ), 2 = mild ( 11 - 25 % neuronal loss ), 3 = moderate ( 26 - 45 % neuronal loss ), and 4 = severe (& gt ; 45 % neuronal loss ). for each brain region and each subject , map2 - negative area measurements were grouped according to treatment and compared using one - way analysis of variance ( anova ). in all cases , when the one - way anova f - test demonstrated significant differences between group means , the data were further analyzed using the student - newman - keuls ( snk ) multiple range test . regional damage ratings obtained from h & amp ; e - stained contralateral hemispheres were assessed using kruskal - wallis and mann - whitney nonparametric statistical analyses . in all cases , values for p & lt ; 0 . 05 were considered significant . all rats that received soman showed ecog evidence of sustained seizures and status epilepticus for several hours , as defined by the continued presence of high amplitude ( i . e ., greater than four - times baseline ) rhythmic spike or sharp wave activity . treatment with hu - 211 had no detectable effect on the strength or duration of seizures , as determined from visual observation of the ecog recordings . band spectral analysis of the ecog data should provide evidence of whether hu - 211 administration produced a less obvious shift in wave pattern . proconvulsive behavioral signs of soman intoxication included repetitive chewing , facial clonus , forepaw clonus , motor stereotypy , and wet - dog shakes . overt motor convulsions were characterized by rhythmic clonic jerks of both head and forepaws , rearing , salivation and straub tail . by all appearances , hu - 211 had no effect on proconvulsive or convulsive behavior . four rats from the soman - injected positive control group and one from the hu - 211 five - min post - onset group died prior to scheduled sacrifice and were deleted from the experiment . fig1 a - c are map2 immunohistochemical stains of the rat temporal lobe showing macroscopic lesions produced by soman and neuroprotection by treatment with hu - 211 at 5 minutes following onset of seizures . fig1 a is a non - soman control . fig1 b shows soman - induced lesions after status epilepticus ( se ). fig1 c shows neuroprotection produced by hu - 211 at 5 minutes following seizure onset , coincident with se . in fig1 a - c , bl denotes basal lateral amygdala , den denotes dorsal endopiriform nucleus and pir denotes piriform cortex . the black letters indicate damaged areas . in unlesioned brain regions , map2 - immunopositive staining was localized in neuronal perikarya , proximal dendrites and neuropil ( fig1 a ). it was not observed in areas composed of white matter except in small numbers of scattered neurons . these findings are consistent with reports by bernhardt and matus ( 1984 ) and matus ( 1994 ). immunocytochemical negative control sections for which non - immunized serum was used showed no map2 immunoreactivity . in brain regions exhibiting lesions resulting from soman - induced seizures , pronounced and clearly demarcated reductions in map2 immunostaining were observed ( fig1 b ). severe lesions were typified by a near total absence of map2 immunoreactivity . those brain regions that were most severely affected included the piriform cortex , entorhinal cortex , dorsal endopiriform nucleus and the laterodorsal thalamic nucleus ( fig1 b ). pronounced reductions in map2 immunostaining were often seen in the perirhinal cortex , amygdaloid complex ( i . e ., lateral , basolateral and posteriolateral cortical amygdaloid nuclei ) and midline thalamic nuclei ( e . g ., mediodorsal and ventromedial ). no reductions in map2 immunoreactivity were visually discernible in any of the hippocampal fields ; however , map2 loss was seen in the hilus of the dentate gyrus . in the piriform cortex and contiguous regions , a lesion ( i . e ., from the soman - injected positive control group ) that would be considered typical of those that were morphometrically measured presented the following : with the deep piriform cortex as a central focus , the lateral and ventral margins of the lesion directly abutted , but did not include , the primary olfactory neurons of layer 2 . these lesions often extended dorsally to include the perirhinal cortex , and non - contiguous lesions were occasionally seen in the dorsal frontal parietal cortex . medially , the area of damage consistently included the dorsal endopiriform nucleus and often included the lateral and basolateral amygdala . necrotic areas devoid of map2 immunoreactivity were surrounded by intensified immunostaining in the penumbra , which enhanced the contrast between the penumbra and necrotic border and facilitated delineation ( fig1 b ). neuroprotection by hu - 211 , administered 5 min post - onset of seizures , is clearly seen in fig1 c . histograms of mean cross - sectional areas of temporal lobe necrosis ( i . e ., map2 - negative immunostaining in the piriform cortex and contiguous regions ) are shown in fig2 . the cross - sectional area of map2 - negative immunostaining ( necrosis ) of the temporal lobes from soman - injected positive controls was 3 . 56 ± 0 . 75 mm 2 . hu - 211 reduced this necrotic cross - sectional area to 0 . 86 ± 0 . 69 mm 2 ( i . e ., a 75 . 8 % reduction ). the apparent elevation of map2 staining seen in fig1 c more than likely resulted from mrna - induced expression of map2 during the intense seizure activity ( ballough et al ., 1995 ). neither the non - soman injected negative control nor the hu - 211 drug treatment control groups showed evidence of brain damage as discerned from examination of map2 - immunostained sections . general histopathological assessments of h & amp ; e - stained brain sections from rats that received soman but not hu - 211 indicated that soman - induced seizure - related brain damage was bilaterally symmetrical and characterized by tissue necrosis , neuronal loss , chromatolysis , vacuolization . pyknosis and gliosis . the most severe brain damage was consistently observed in the piriform cortex , entorhinal cortex , dorsal endopiriform nucleus and the laterodorsal thalamic nucleus . pronounced damage was often seen in perirhinal cortex , amygdaloid complex ( i . e ., lateral , basolateral and posteriolateral cortical amygdaloid nuclei ), hippocampus ( i . e ., hippocampal fields ca1 and ca3 and dentate gyrus ), and midline thalamic nuclei ( e . g ., mediodorsal and ventromedial ). histopathological damage ratings for h & amp ; e - stained brain sections are based on the presence of necrotic neurons and / or the absence of a defined neuronal population ; shrunken neurons are considered the result of artifactual change . damage to the neuropil is progressively greater as ratings increase from “ mild ” to “ severe ,” and is characterized by increasingly severe malacia and hyalinization typical of necrosis . a statistically significant ( p = 0 . 03 ) drop in damage ratings was observed in the hu - 211 group treated at 5 min after onset of seizures ( 2 . 4 ± 0 . 38 ) compared with soman controls ( 3 . 8 ± 0 . 2 ). this represents a damage reduction from severe ( i . e ., from & gt ; 45 % neuronal loss in untreated soman - exposed groups ) to mild ( 11 - 25 % neuronal loss in the hu - 211 , 5 - min group ). damage ratings for hu - 211 controls and vehicle controls were all zero . the results presented here are significant in that hu - 211 reduced neuronal damage following exposure to soman without stopping established seizures . earlier reports had indicated that noncompetitive nmda antagonists such as ketamine and mk - 801 protect thalamic neurons from seizure - related brain damage without preventing seizure activity ( labruyere et al ., 1986 ; smith et al ., 1987 ; clifford et al ., 1989 , 1990 ). it was suggested by these investigators that the nmda antagonists may have prevented seizure - related damage by blocking nmda receptor ion channel complexes on the dendrosomal surfaces through which glutamate excitotoxicity is expressed ( clifford et al ., 1989 ). it is also possible that seizure activity in some brain areas can be maintained by other transmitter systems / receptors without nmda participation ( clifford et al ., 1989 ). in addition to nmda blocking activity , hu - 211 has been reported to block calcium influx and possess free radical scavenging activity ( biegon and bar joseph , 1995 ). for humans , the active dose of hu - 211 to reduce seizure - related brain damage caused by organophosphorus nerve agents is generally in the range of from about 48 mg to about 200 mg per day . however , it is evident to one of skill in the art that dosages would be determined by the attending physician , according to the method of administration , patient &# 39 ; s age , weight , counterindications and the like . administration of therapeutically effective amounts of hu - 211 as used herein encompasses oral , parenteral , intravenous , intramuscular , sub - cutaneous , transdermal , intratechal , rectal and intra - nasal administration . while the invention has been described with reference to certain preferred embodiments , numerous changes , alterations and modifications to the described embodiments are possible without departing from the spirit and scope of the invention as defined in the appended claims , and equivalents thereof . ballough g . p . h ., martin l . j ., cann f . j ., graham j . s ., smith c . d ., kling c . e ., forster j . s ., phann s ., and filbert m . g . 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