Patent Application: US-30687294-A

Abstract:
a method is disclosed for blocking the sec receptor and also inhibiting the neurotoxic effects of amyloid - β protein by treating sec receptor - bearing cells with a synthetic inhibitor peptide , e . g . peptide pb145 .

Description:
note : while the specification concludes with claims particularly pointing out and distinctly claiming the subject matter regarded as forming the present invention , it is believed that the invention will be better understood from the following preferred embodiments of the invention taken in connection with the accompanying drawings : fig1 is a graphical representation which shows the effect of amyloid - β 25 - 35 on mtt reduction in pc12 cells . the reduction in percent control response is plotted on the y - axis against the concentration ( logm ) of the amyloid - β 25 - 35 on the x - axis . fig2 is a graphical representation of the effect of amyloid - β 25 - 35 as shown in fig1 in comparison to corresponding effect of other regions of the amyloid - β protein , namely amyloid - β 1 - 40 , amyloid - β 1 - 16 and amyloid - β 1 - 28 in pc12 cells . fig3 is a bar graph which shows the percent inhibition of amyloid - β 25 - 35 neurotoxicity in the absence or presence of various peptides , namely peptide pb 96 , peptide pb 446 , control peptide pb 154 , α 1 - antitrypsin - elastase ( α 1 - at - elas ) and α 1 - at at various concentrations ( μm ) in pc12 cells . the amino acid sequences of pb 96 , pb 446 and pb 96 relative to the partial sequence of α 1 - at of which they are fragments are shown at the top of the bar graph . fig4 is a bar graph which shows the percent inhibition of amyloid - β 25 - 35 neurotoxicity in the presence of peptide pb145 or control peptide pb 154 at various concentrations ( μm ) in pc12 cells . the amino acid sequences of these two peptides compared to the partial sequence of α 1 - at are shown at the top of the bar graph . fig5 is a graphical representation which shows the toxic effect of amyloid - β 25 - 35 on other sec receptor - bearing cell types in u373mg cells and the inhibition of neurotoxicity in the presence of peptide pb145 . the reduction in percent control is plotted on the y - axis against the concentration ( logm ) of the amyloid - β 25 - 35 on the x - axis . in order to illustrate the invention in greater detail , the following specific laboratory examples were carried out ( although specific examples and details are thus illustrated herein , it will be appreciated that the invention is not limited to these specific examples or details ): the mtt assay ( 19 ) was used to monitor the neurotoxicity of amyloid - β peptide . this assay has been shown to correlate with the ldh assay and morphologic changes of neurotoxicity in the neuronal cell line pc 12 and in cortical neurons in primary culture ( 20 , 21 ). inhibition of mtt reduction is a specific , early indicator of amyloid - β - mediated cytotoxicity . the pc12 cell line was used because it is the most well characterized cell line of neuronal origin and because previous studies have shown there is abundant expression of sec receptor in this cell line . using 125 i peptide 105y there is specific , saturable binding . scatchard plot analysis predicts a k d ˜ 34 . 3 nm and ˜ 180 , 000 plasma membrane receptors per cell . first , the effect of amyloid - β 25 - 35 on mtt reduction in pc12 cells was examined ( fig1 ). pc12 cells were incubated for six ( 6 ) hours at 37 ° c . with amyloid - β 25 - 35 in several different concentrations as shown on the horizontal axis . mtt was added and the cells incubated at 37 ° c with amyloid - β 25 - 35 in several different concentrations as shown on the horizontal axis . mtt was added and the cells incubated at 37 ° c . for another two ( 2 ) hours . cell extraction buffer was added and the incubation continued at 37 ° c . overnight . cell extracts were then subjected to spectro - photometric analysis at 570 nm . results were reported as percent control response as determined by the difference between control cells and cells lysed prior to incubation with mtt . the results show a concentration - dependent decrease in mtt reduction in response to amyloid - β 25 - 35 . the point of half - maximal reduction is between 10 - 9 to 10 - 8 m which correlates well with the point of half - maximal saturation of the sec receptor . second , the specificity of the effect of amyloid - β 25 - 35 on mtt reduction in pc12 cells was examined ( fig2 ). the results show that amyloid - β 1 - 40 has an effect similar to that of amyloid - β 25 - 35 , but amyloid - β 1 - 16 and amyloid - β 1 - 28 have no effect . these data indicate that the effect is specific for the region corresponding to amyloid - β 25 - 35 . third , the possibility that the effect of amyloid - β 25 - 35 is mediated by the sec receptor was examined ( fig3 ). in this case pc12 cells were incubated with amyloid - β 25 - 35 in the absence or presence of peptide 96 or peptide 446 which compete for binding to the sec receptor . peptides 96 and 446 inhibit the effect of amyloid - β 25 - 35 , but negative control peptide 154 does not . pc12 cells were also incubated with α 1 - at - elastase complexes which compete for binding to the sec receptor . α 1 - at - elastase complexes inhibit the effect of amyloid - β 25 - 35 , but negative control native α1 - at does not . these data suggest that the effect of amyloid - β 25 - 35 is mediated by the sec receptor . however , for each of these peptides the effect is short - term and each has weak agonistic activity at high concentrations when used alone . fourth , the possibility that peptide pb145 inhibits the toxic effect of amyloid - β 25 - 35 was examined ( fig4 ). the effect is concentration - dependent . the effect is evident at concentrations as low as 0 . 33 μm . the effect is time - dependent , but lasts at least 24 hours . peptide pb145 did not have any neurotoxic activity by itself . fifth , the possibility that amyloid - β 25 - 35 is toxic to other sec receptor - bearing cell types and that pb145 inhibits its toxic effects in these other cell types was examined ( fig . 5 ). in this case a human glioblastoma cell line u373mg was used . the results show that amyloid - β 25 - 35 is also toxic to the u373mg cell line , and that its toxicity is completely inhibited by pb145 . the neuronal cell line pc12 and the human glioblastoma cell line u373mg used in the assays described herein are conventional cell lines well known to the person skilled in the art and readily available to the public . for example , both cell lines are available to the public from the american type culture collection , rockville , md ., under accession numbers atcc crl 1721 ( for pc12 ) and atcc htb 17 ( for u373mg ). in the mtt assay used herein , inhibition of cellular redox activity is measured by 3 - 4 , 5 - dimethylthiazol - 2 - yl !- 2 , 5 - diphenyltetrazolium bromide ( mtt ) by conventional procedures as described by hansen et al . ( 19 ). various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention . it is intended that all such other examples be included within the scope of the appended claims . 1 . perimutter d . h ., glover g . i ., rivetna m ., schasteen c . s ., fallon r . j . identification of a serpin - enzyme complex ( sec ) receptor on human hepatoma cells and human monocytes . proc natl aced sci usa 1990 ; 87 : 3753 - 3737 . 2 . joslin g ., griffin g . l ., august a . m ., adams s ., fallon r . j ., senior r . m ., perlmutter d . h . the serpin - enzyme complex ( sec ) receptor mediates the neutrophil chemotactic effect of α1 - antitrypsin - elastase complexes and amyloid - β peptide . j clin invest 1992 ; 90 : 1150 - 1154 . 3 . perlmutter d . h ., joslin g ., nelson p ., schasteen c ., adams s . p . fallon r . j . endocytosis and degradation of α1 - antitrypsin - protease complexes is mediated by the sec receptor . j biol chem 1990 ; 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533 : 315 - 320 . 10 . mattson m . p ., cheng b ., david d ., bryant k ., lieberburg i ., rydel r . e . β - amyloid peptides destabilized calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity . j neurosic 1992 ; 12 : 378 - 389 . 11 . itagaki s ., mcgeer p . i ., akiyama h ., zhu s ., selkoe d . relationship of microgila and astrocytes to amyloid deposits of alzheimer &# 39 ; s disease . j neuroimmunology 1989 : 24 : 173 - 182 . 12 . rogers j ., luber - narod j ., styren s . d ., civin w . h . expression of immune system - associated antigens by cells of the human central nervous system : relationship to the pathology of alzheimer &# 39 ; s disease . neurobiol aging 1988 ; 9 : 339 - 349 . 13 . kurdowska a ., travis j . acute phase protein stimulation by α1 - antichymotrypsin - cathepsin g complexes : evidence for the involvement of interleukin - 6 . j biol chem 1990 ; 265 : 21023 - 21026 . 14 . joslin g ., witwer a ., adams s . p ., tollefsen d . m ., august a . m ., perlmutter d . h . cross competition for binding of α1 - antitrypsin ( α1 - at )- elastase complexes to the sec receptor by other serpin - enzyme complexes and by proteolytically modified α1 - at . j biol chem 1993 ; 268 : 1886 - 1893 . 15 . potempa j ., fedak o ., dubin a ., mast a ., travis j . proteolytic inactivation of α1 - antichymotrypsin . site of cleavage and generation of chemotactic activity . j biol chem 1991 ; 266 : 21482 - 21487 . 16 . tilg h ., vannier e ., vachino g ., dinarello c . a ., mier j . w . anti inflammatory properties of hepatic acute phase proteins : preferential induction of interleukin i ( il - 1 ) receptor antagonist over il - 1β synthesis by human peripheral blood mononuclear cells . j exp med 1991 ; 178 : 1629 - 1636 . 17 . arend w . p . interleukin 1 receptor antagonist : a new member of the interleukin 1 family . j clin invest 1991 ; 88 : 1445 - 1451 . 18 . goldgaber d ., harris h . w ., hia t ., maciag t ., donnelly r . j ., jacobsen j . s ., vilek m . p ., gajdusek d . c . interleukin 1 regulates synthesis of amyloid - β protein precursor mrna in human endothelial cells . proc natl acad sci usa 1989 ; 86 : 7606 - 7610 . 19 . hansen m . b ., nielsen s . e ., burg k . re - examination and further development of a precise and rapid dye method for measuring cell growth / cell kill . j immunol meth 1989 ; 119 : 203 - 210 . 20 . shearman m . s ., ragan c . i ., iversen l . l . inhibition of pc12 cell radox activity is a specific , early indicator of the mechanism of β - amyloid - mediated cell death . proc natl acad sci usa 1994 ; 91 : 1470 - 1474 . 21 . behl c ., davis j . b ., lesley r ., schubert d . hydrogen peroxide mediates amyloid - β protein toxicity . cell 1994 ; 77 : 817 - 827 . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 8 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 43 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 1 : ilealaglyargserleuasnproasnargvalthrleuargtyrasn151015lyspropheileleuvalleuphegluthrproglyasnserleuval202530pheleuglyargileserasnproalathrlys3540 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 11 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 2 : glyserasnlysglyalaileileglyleumet1510 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 31 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 3 : vallyspheasnlysprophevalpheleumetilegluglnasnthr151015lysserproleuphemetglylysvalvalasnprothrglnlys202530 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 20 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 4 : ilegluglnasnthrlysserproleuphemetglylysvalvalasn151015prothrglnlys20 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 37 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 5 : metserileproprogluvallyspheasnlysprophevalpheleu151015metilegluglnasnthrlysserproleuphemetglylysvalval202530asnprothrglnlys35 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 31 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 6 : vallyspheasnlysprophevalpheleumetilegluglnasnthr151015lysserproleuphemetglylysvalvalasnprothrglnlys202530 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 20 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 7 : ilegluglnasnthrlysserproleuphemetglylysvalvalasn151015prothrglnlys20 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 36 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 8 : serileproprogluvallyspheasnlysprophevalpheleumet151015ilegluglnasnthrlysserproleuphemetglylysvalvalasn202530prothrglnlys35