Patent Application: US-46544403-A

Abstract:
methods and devices for rapid screening of drug candidates , especially candidate agents for treatment of alzheimer &# 39 ; s disease and stroke are disclosed . the invention provides treatment compounds and a biosensor method and device which is particularly applicable to screening libraries of compounds .

Description:
the invention will now be described in detail by way of reference only to the following non - limiting examples and drawings . ad alzheimer &# 39 ; s disease app β - amyloid protein precursor caa cerebral amyloid angiopathy dmem dulbecco &# 39 ; s modified eagle &# 39 ; s medium dmpc dimyristoyl - l - a - phosphatidylcholine dmpe dimyristoyl - l - a phosphatidylethanolamine mpg dimyristoyl - l - α - phosphatidylglycerol dmps dimyristoyl - l - α - phosphatidylserine mts [ 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 5 -( 3 - carboxymethoxyphenyl )- 2 ( 4 - sulfophenyl )- 2h - tetrazolium smc smooth muscle cell suv small 100 nm unilamellar vesicles . we have examined the binding of aβ peptides both to synthetic lipid bilayers and to plasma membrane - enriched preparations derived from vascular smooth muscle cells ( smcs ), using surface plasmon resonance . we have found that the extent of binding of aβ to membranes correlates very well with the extent of aβ toxicity . importantly , we have demonstrated that aβ binding to synthetic lipids and intact smc membranes requires the presence of cholesterol , and that reduction of membrane cholesterol content with an inhibitor of cholesterol biosynthesis reduces aβ toxicity . our results strongly support the view that cholesterol - lowering drugs reduce aβ toxicity by reducing aβ - membrane binding . aβ peptides were synthesised using manual solid - phase boc ( n - tert - butocarbonyl ) amino acid synthesis . peptides were synthesized using manual solid - phase boc amino acid chemistry with in situ neutralisation . acylations were performed using 5 equivalents of the boc - protected amino acid , 4 . 9 equivalents of 2 -( 1h - benzotriazole - 1 - yl )- 1 , 1 , 3 , 3 - tetramethyluronium hexafluorophosphate , 5 equivalents of 1 - hydroxybenzotriazole and 7 . 5 equivalents of diisopropylethylamine in dimethyl formamide . each acylation was monitored using ninhydrin , and couplings were repeated if necessary . aβ peptides were cleaved from the resin using anhydrous hydrogen fluoride and p - cresol / p - thiocresol . hydrogen fluoride was then removed , and the peptide was dissolved in trifluoroacetic acid and precipitated with ether . peptide purification was achieved using an acetonitrile / water ( 0 . 01 % trifluoroacetic acid ) gradient on a reversed - phase preparative zorbax high performance liquid chromatography ( hplc ) column heated to 60 ° c . the purity (& gt ; 95 %) and identity of the peptide was analysed by analytical hplc , electrospray mass spectrometry and amino acid analysis . dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) and penicillin / streptomycin were purchased from gibco life technologies ( mulgrave , vic , australia ), and foetal bovine serum ( heat inactivated ) was purchased from commonwealth serum laboratories ( parkville , vic ., australia ). n - octyl - d - glucopyranoside , dimyristoyl - l - α - phosphatidylcholine ( dmpc ), dimyristoyl - l - α - phosphatidyl - dl - glycerol ( dmpg ), dimyristoyl - l - α - phosphatidylethanolamine ( dmpe ), dimyristoyl - l - α - phosphatidylserine ( dmps ) and d - cholesterol were purchased from sigma ( st louis , mo ., usa ). lovastatin was purchased from calbiochem ( sydney , nsw , australia ), and activated to its active open - ring form as previously described ( jakobisiak et al ., 1991 ). aβ 1 - 42 , aβ 1 - 40 , aβ 1 - 28 , aβ 17 - 42 and aβ 29 - 42 were dissolved in dimethyl sulfoxide ( dmso ) at a concentration of 2 mm . the peptide solutions were then sonicated ( 42 khz ) for 5 minutes , and centrifuged at 5 , 000 rpm for 1 minute at room temperature using a hermle z160m bench microfuge . solubilised peptides were immediately snap - frozen and mixed on a vortex mixer for 15 seconds . the peptides were then diluted into dmem for cell culture experiments , or in 0 . 02 m sodium phosphate buffer , ph 6 . 8 for biosensor experiments , to give a final concentration of 10 μm . to “ age ” aβ peptides , a process which increases the proportion of fibrillar oligomeric species ( jarrett and lansbury , 1992 ), peptides were incubated at 37 ° c . in a humidified atmosphere of 5 % co 2 for 5 days at a concentration of 100 μm . the concentration of amyloid fibrils was measured using the assay of klunk et al . ( 1999 ). aβ peptides were mixed with congo red ( cr ) in 0 . 02 m sodium phosphate buffer , ph 6 . 8 . the final concentration of peptide and cr was 10 μm . solutions of cr alone were also prepared in 0 . 02 m sodium phosphate buffer , ph 6 . 8 . the mixture was vortexed briefly and then incubated at room temperature for 15 min . the absorbances at 403 and 541 nm were measured using a biorad smartspec 3000 spectrophotometer . background absorbance values of buffer alone were subtracted from the values obtained from each sample . the concentration of aβ fibrils in each preparation was then determined using the formula [ a fibrils ]=( a 541nm aβ : cr solution / 4780 )−( a 403nm of aβ : cr solution / 6830 )−( a 403nm of cr solution / 8620 ) all preparations were prepared in triplicate , and the assay was conducted independently three times , with similar results being obtained in each experiment . vascular smcs from aortae of sprague - dawley rats were cultured in dmem supplemented with 10 % foetal bovine serum and penicillin / streptomycin at 37 ° c . in a humidified atmosphere of 5 % co 2 . vascular smcs were plated at a density of 10 4 cells / well in a 96 - well plate , or at a density of 10 6 cells / 75 cm 2 cell culture flask ( nunc , denmark ) in 20 ml culture medium , and grown to 80 % confluence , after which the medium was removed and replaced with fresh , serum - free medium with or without to μm lovastatin . the cells were then incubated for 72 h . the cells were then either used for the preparation of membranes , or incubated with aβ peptides ( 10 μm ) for cytotoxicity assay . in the latter case , aβ peptides were added to the culture medium and the cells incubated for a further 24 h . in control incubations , vehicle alone , ie lacking peptide , was added . cytotoxicity was determined using the celltiter 96 aqueous one solution cell proliferation assay kit ( promega corporation , madison , wis ., usa ) ( cory et al ., 1991 ). the f3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 5 -( 3 - carboxymethoxyphenyl )- 2 ( 4 - sulfophenyl )- 2h - tetrazolium ( mts ) reagent solution was added to the culture medium at a concentration of 10 % ( by volume ). the cells were then incubated for a further 2 h at 37 ° c ., and the absorbance of the samples read at a wavelength of 560 nm using a wallac victor 1420 plate reader . a crude plasma membrane preparation was prepared from vascular smcs by differential centrifugation ( hubbard et al ., 1983 ). cells were scraped off 10 × 75 cm 2 flasks using a cell scraper , and centrifuged in dmem at 3 , 000 rpm in a beckman coulter allegra 21r centrifuge at 4 ° c . for 3 min . the pellet was then washed with phosphate - buffered saline ( pbs ), added to 10 ml of stm buffer ( 0 . 25 m sucrose / 5 mm tris - hcl , ph 7 . 4 / 1 . 0 mm mgcl 2 ), and homogenized on ice using 10 up and down strokes in a 40 ml dounce - type glass homogenizer with a loose - fitting pestle . the homogenate was centrifuged at 1 , 100 rpm for 5 min . the supernatant fraction was saved and the pellet rehomogenized in 5 ml of stm buffer . the suspension was again centrifuged , and the first and second supernatant fractions combined , then centrifuged at 40 , 000 rpm for 2 h ( beckman l8 - m ultracentrifuge , 70 ti rotor , no brake ) and the resulting crude plasma membrane fraction resuspended in 1 . 0 ml of 0 . 02 m sodium phosphate buffer , ph 7 . 4 . total membrane cholesterol was determined using the amplex red cholesterol assay kit ( molecular probes , eugene , oreg ., usa ). the protein content of the membrane preparations was determined using the bicinchoninic acid ( bca ) assay using bovine serum albumin as standard . small 100 nm unilamellar vesicles ( suv ), containing dmpc , dmpg , dmps and dmpe , and cholesterol , were prepared in 0 . 02 m phosphate buffer ( ph 6 . 8 ) by sonication and extraction . briefly , 1 . 5 mg of total lipid was dissolved in 1 . 5 ml of chcl 3 : meoh ( 3 : 1 , v / v ). aliquots ( 408 μl ) were removed and evaporated under a stream of nitrogen , and the lipids further dried in vacuo overnight . the lipids were then resuspended in 600 μl of 0 . 02 m sodium phosphate buffer , ph 6 . 8 . the resulting lipid dispersion was sonicated in a bath type sonicator until clear , and then extruded 17 times through 100 nm pore diameter polycarbonate filters using liposofast apparatus ( avestin , ottawa , canada ) to obtain 100 nm swv . the mixed lipid vesicles contained 80 % ( w / w ), 60 % ( w / w ), 40 % ( w / w ), 30 % ( w / w ) or 0 % ( w / w ) cholesterol . the remaining lipid comprised a mixture of dmpc : dmpe : dmps : dmpg in a ratio of 75 : 20 : 2 . 5 : 2 . 5 ( by weight ). binding experiments were carried out with a biacore x analytical system ( biacore , uppsala , sweden ) using an l1 sensor chip ( biacore ), as supplied by the manufacturer . it will be appreciated that other types of biosensor may also be used , preferably provided that the chip surface is such that the bound lipid material is able to retain a bilayer or monolayer structure . the running buffer used for all experiments was 0 . 02 m sodium phosphate buffer , ph 6 . 8 ( phosphate buffer ). the washing solution was 40 mm n - octyl β - d - glucopyranoside . the regeneration solution was 10 mm sodium hydroxide . all solutions were freshly prepared , degassed and filtered through a 0 . 22 μm filter . the operating temperature was 25 ° c . the alkyl surface of the l1 chip was cleaned by an injection of 25 μl of non - ionic 40 mm octyl glucoside at a flow rate of 5 μl / min . suv ( 100 μl ) or vascular smc membranes ( 100 μl containing 0 . 33 mg protein ) were then immediately applied to the chip surface at a low rate of 5 μl / min . to remove any multilamellar structures from the synthetic lipid surface , 30 μl of 10 mm sodium hydroxide was injected at a flow rate of 50 gl / min , resulting in a stable baseline corresponding to the successful formation of an immobilized layer of suvs . peptide solutions were prepared at concentrations ranging from 0 . 5 to 10 μm . the solutions were injected over the lipid surface at a flow rate of 5 μl / min for 20 min . the peptide solution was then replaced by phosphate buffer and the peptide - membrane complex allowed to dissociate . the removal of the bound peptide and regeneration of the l1 chip surface , without removal of the synthetic lipid or vascular smc membrane layer , was achieved by an injection of sodium hydroxide ( 30 μl , 10 mm ) at a flow rate of 50 μl / min . the amount of binding in response units ( ru ) was fitted by a simple one - to - one langmuir reaction model ( morton et al ., 1995 ): using biaevaluation 3 . 0 software ( biacore ). the data obtained from five different binding experiments performed at five different concentrations of peptide were fitted globally to obtain the equilibrium association constant ( k a ). the accuracy of each fit was assessed by calculating a x 2 value for the data ( karlsson and failt , 1997 ). a lower x 2 value was taken as an indication of a better fit of the data . the binding was found to approach equilibrium after incubation with the peptide for 20 min . therefore for routine quantification of total binding to the membrane , the ru value obtained 20 min after addition of the peptide ( ru 20 ) was taken as an estimate of maximum binding at each concentration ( r eq ). scatchard plots were generated using biaevaluation 3 . 0 software ( biacore ). to measure aβ toxicity against vascular smcs , cultures of cells were treated with aβ peptides and analogues ( 10 μm ) and the amount of toxicity determined using the mts assay ( cory et al ., 1991 ). aβ1 - 40 , aβ1 - 42 , aβ29 - 42 and aβ17 - 42 were all found to cause significant toxicity , as shown in fig1 . aβ1 - 28 was less toxic , suggesting that the toxicity of aβ may be associated with the more hydrophobic c - terminal region of the peptide . aging the peptides by incubation for 5 days caused a significant increase in toxicity . previous studies ( e . g ., pike et al ., 1991 ) have shown that the aggregation of the aβ into fibrils may be important for the generation of toxic species . the present results support this conclusion . incubation of aβ peptides for 5 days significantly increased the proportion of fibrillar species as determined by a congo red binding assay , as shown in fig2 . in general , the amount of aβ toxicity approximately correlated with the proportion of fibrillar species . for example , there was a significant increase in cytotoxicity after aging aβ 1 - 42 ( p & lt ; 0 . 05 ; student &# 39 ; s t test ). in addition , aged aβ 1 - 28 , which formed fewer fibrillar species than the other aβ species tested , was significantly less toxic to vascular smc cultures . as previous studies have shown that aβ can interact directly with lipid membranes , we examined the possibility that the toxic effects of aβ were due to a direct interaction with the vascular smc membrane . to study the binding of aβ to lipids , biosensor technology was employed . initially a biosensor chip was coated with a synthetic lipid mixture containing 60 % cholesterol , 30 % dmpc , 8 % dmpe , 1 % dmps and 1 % dmpg , and sensorgrams obtained for aβ 1 - 42 and aβ 1 - 40 . these are shown in fig3 a and fig3 b respectively . the peptides bound to the lipid surface in a biphasic manner . the initial association of the peptide to the lipid surface was rapid . maximum binding was approached approximately 20 min after application of the peptide . the dissociation curve of the bound complex followed a similar biphasic pattern . the signal fell rapidly at the end of the injection period , since the peptide was no longer present and the buffer flow removed a large amount of weakly bound peptide . typically , the peptide sensorgrams did not return to zero until the biosensor was stripped with 10 mm naoh , indicating that a proportion of the peptide remained bound to the surface . the biphasic nature of the ab - lipid interaction was confirmed by a curve fitting analysis of the association and dissociation curves . a poor fit was obtained using the simplest one to one langmuir binding model ( morton et al ., 1995 ) ( x 2 for aβ1 - 42 = 1035 ; x 2 for aβ1 - 40 = 1200 ). however , a significantly improved fit was obtained using a competing reaction model ( karlsson and falt , 1997 ) ( x 2 for aβ1 - 42 = 400 ; x 2 for aβ1 - 40 = 370 ). scatchard plot analysis of the binding data was also consistent with a biphasic interaction for both aβ1 - 42 and aβ1 - 40 , as shown in fig3 c . a comparison of aβ peptides and analogues for their abilities to bind to the synthetic lipid mixture showed that there was good agreement between the extent of lipid binding , as shown in fig4 and the amount of toxicity caused by each peptide as shown in fig1 . for example , the more toxic c - terminal aβ fragments ( aβ 29 - 42 and aβ 17 - 42 ) bound more strongly than the less toxic n - terminal fragment ( aβ 1 - 28 ). the ratio of cholesterol to phospholipid in the synthetic lipid mixture was directly related to the extent of high - affinity lipid binding , as illustrated in fig5 . when a pure phospholipid mixture was used , very little binding was observed . however , at higher concentrations between 30 - 80 % cholesterol , the amount of binding increased . this increase in binding was also reflected by an increase in the equilibrium association constants determined after fitting the kinetic data from fig3 a and 3b to a competing reaction model . as shown in table 1 , both aβ 1 - 42 and aβ 1 - 40 peptides had a higher binding affinity for synthetic lipid mixtures containing a greater percentage of cholesterol . values of equilibrium association constants ( k a ) for the binding of aβ to synthetic lipid mixtures based on a competing reaction model of binding cholesterol : pl ratio peptide k a1 k a2 30 : 70 aβ1 - 42 1 . 8 × 10 5 6 . 9 × 10 4 aβ1 - 40 1 . 1 × 10 5 5 . 2 × 10 4 60 : 40 aβ1 - 42 2 . 0 × 10 8 4 . 8 × 10 7 aβ1 - 40 4 . 0 × 10 7 1 . 3 × 10 6 80 : 20 aβ1 - 42 2 . 7 × 10 8 6 . 9 × 10 7 aβ1 - 40 4 . 8 × 10 7 1 . 0 × 10 7 to determine whether the cholesterol content of the vascular smc membrane influences the binding of aβ , we prepared a plasma membrane - enriched fraction from vascular smooth muscle cells and applied this fraction to the biosensor chip . when applied at a concentration of 10 μm , both aβ1 - 40 and aβ1 - 42 bound to the membrane fraction , as shown in fig6 . the total amount of binding was approximately 10 % of that observed for a synthetic lipid mixture containing 60 % cholesterol and 40 % phospholipid , as shown in fig5 . the effect of lovastatin , a cholesterol biosynthesis inhibitor , on aβ binding to the membrane was also examined . cells were pretreated with lovastatin for 72 h and then a plasma membrane - enriched fraction was prepared . the protein composition of the membrane fractions was closely similar in the control ( 3 . 29 ± 0 . 15 mg / ml ) and lovastatin - treated groups ( 3 . 24 ± 0 . 03 mg / ml ), indicating that lovastatin treatment did not significantly alter the amount of total membrane protein . treatment of the cells with lovastatin was found to strongly decrease binding of both aβ1 - 40 and aβ1 - 42 to the membrane fraction , as shown in fig6 a and b ). after lovastatin treatment , the cholesterol content of the membrane fraction was reduced to approximately 55 % of that recovered from untreated cells ( fig6 panel b ). aβ1 - 40 and aβ1 - 42 binding to the plasma membrane fraction of lovastatin - treated cells was approximately 25 % of that achieved without lovastatin pretreatment . as indicated by their equilibrium association constants , both peptides displayed a higher binding affinity for untreated vascular smc membranes than for membranes treated with lovastatin . this is summarised in table 2 . to examine whether this decrease in binding might have any consequences for aβ toxicity , the amount of aβ toxicity was measured following lovastatin treatment of cells , using the mts assay . the results are shown in fig7 . lovastatin alone had little effect on the ability of vascular smcs to reduce mts . however , cells pretreated with lovastatin were more resistant than untreated cells to aβ toxicity , as the aβ1 - 40 and aβ1 - 42 - induced decrease in mts reduction was approximately 25 - 40 % lower in lovastatin - treated cells than in controls . we have demonstrated that cholesterol is required for the binding of aβ to synthetic lipid mixtures and to vascular smc membranes . our results also suggest that lowering plasma membrane cholesterol can decrease aβ toxicity . taken together , our results indicate that binding of aβ to the lipid component of the plasma membrane is required for aβ toxicity . analysis of the sensorgram data indicated that the binding of aβ to lipid membranes is more complicated than a simple one to one interaction . this conclusion was also supported by the scatchard plots , which were nonlinear ( i . e ., biphasic ). while several interpretations of these data are possible , one possibility is that aβ exists in multiple states , each of which binds to lipids with a different affinity . aging the peptides by incubation for days was found to increase aβ binding and to increase the concentration of amyloid fibrils . unaged aβ also contained some amyloid fibrils , suggesting that different oligomeric species have different affinities for lipid binding . for this reason , the data from scatchard analysis must be interpreted with caution . the binding may be biphasic ; however , more data may reveal a more complex interaction . for the same reason , affinity constants calculated from the competing reaction model , as reported in tables 1 and 2 , should be viewed as indicating an overall affinity of aβ for the lipid membrane , rather than as having any independent significance . there have been conflicting reports on the role of cholesterol in aβ toxicity . zhou and richardson ( 1996 ) reported that methyl - β - cyclodextrin - cholesterol protects cells from aβ - mediated toxicity . however , in this study , cholesterol was added exogenously , and the lipid composition of the plasma membrane was not analyzed . in contrast , wang et al . ( 2001 ) reported that methyl - β - cyclodextrin alone attenuated aβ toxicity by lowering cell cholesterol , and suggested that the effect of methyl - β - cyclodextrin - cholesterol might be related more to a loss of cellular cholesterol , rather than to the addition of exogenous cholesterol . our own studies strongly support this view , as well as providing a biochemical explanation for the decreased toxicity . there is good evidence that aβ - membrane binding is involved in aβ toxicity . aβ can bind to phospholipids ( waschuk et al ., 2001 ), and inhibition of electrostatic interactions between aβ and the negative phospholipids can inhibit aβ toxicity ( hertel et al ., 1997 ). we have found that bound aβ was easily removed from lipid membranes with sodium hydroxide , suggesting that electrostatic , rather than hydrophobic forces , are involved . this contrasts with the behaviour of many other peptides , which bind hydrophobically and penetrate deeply into the lipid bilayer ( mozsolits et al ., 2001 ). indeed , although cholesterol can bind aβ , presumably via hydrophobic interactions , ji et al . have shown that aβ - cholesterol binding inhibits fibril formation , and have speculated that cholesterol might be neuroprotective . however , in our studies , increased cholesterol was clearly associated with increased toxicity . therefore direct binding of aβ to cholesterol may not be involved in the aβ - membrane interaction in our system . aβ can also bind to sphingolipids and gangliosides ( ariga et al ., 2001 ; valdez - gonzalez et al ., 2001 ; mahouf et al ., 200 ). thus it is possible that cholesterol plays some other role , perhaps in the control of membrane fluidity . this concept fits in well with our finding that changing the cholesterol content changes the equilibrium association constant ( k a ) for binding , i . e . that the properties of the entire membrane are altered by cholesterol to increase the affinity of binding . the mechanism by which aβ binding to the cell membrane causes cell toxicity is still unclear . free radical production , lipid peroxidation and alterations in ion channel function have been implicated ( reviewed by small and mclean , 1999 ). if aβ binds directly to the lipid component of the membrane , then alterations in membrane fluidity may occur . chochina et al . ( 2001 ), using synaptic plasma membranes , have reported that aβ 1 - 40 can increase neuronal membrane fluidity , although in contrast to this kremer et al . ( 2001 ), using synthetic lipids , found that aβ can decrease membrane fluidity . certainly changes in membrane fluidity could affect the function of a variety of proteins on the cell surface , including ion channels . for example , alterations in fluidity are known to affect the sub - membrane localization and function of the nicotinic receptor ( baenziger et al ., 2000 ). we have found that the extent of aβ aggregation correlates with the vascular smc toxic response . although the process of aging increases the number of amyloid fibrils formed from aβ , as assessed using a cr binding assay , this does not prove that fibrils are the major toxic form of aβ . for example , even though aged aβ 1 - 28 failed to form fibrils , it was still toxic to vascular sm , albeit in a significantly less pronounced manner than the other peptides . aβ is probably secreted as a monome , and subsequently aggregates into soluble oligomers or fibrils ( podlisny et al ., 1998 ). it is likely that the levels of soluble oligomeric species of aβ are also increased by the process of aging . a recent study by lambert et al . ( 1998 ) found that small , low molecular weight oligomers of aβ 1 - 42 are several orders of magnitude more potent as neurotoxins than high molecular weight fibrillar species of aβ 1 - 42 . therefore more work is needed to define the precise nature of the toxic form of aβ , and to ascertain the mechanism of toxicity . there is some epidemiological evidence that lowering blood cholesterol levels may be of benefit for alzheimer &# 39 ; s disease . in at least one population - based study , the incidence of ad has been found to be higher in individuals with higher cholesterol levels ( roher et al ., 1999 ). the ε4 allele of the apolipoprotein e gene is known to be a risk factor for alzheimer &# 39 ; s disease ( bales et al ., 1997 ; corder et al ., 1993 ), and demented individuals who are homozygous for the ε4 allele have been reported to have higher plasma cholesterol levels than do normal elderly controls ( czech et al ., 1994 ). this idea is also supported by the observation that ad - like pathology is less severe in app transgenic mice which have been treated with a cholesterol - lowering drug ( refolo et al ., 2001 ). indeed , two retrospective studies using cholesterol - lowering statins have reported dramatic decreases in the risk of developing ad ( jick et al ., 2000 ; wolozin et al ., 2000 ). cholesterol could have more than one role in the pathogenesis of ad . a number of studies have shown that cholesterol is also important for the regulation of aβ production ( mizuno et al ., 1998 ). high cholesterol uptake can increase aβ deposition in transgenic mice ( refolo et al ., 2000 ; sparks et al ., 1994 ). cholesterol depletion can inhibit the generation of aβ in hippocampal neurons ( simons et al ., 1998 ), and this effect may be mediated by app secretases ( kojiro et al ., 2001 ). thus it is possible that inhibition of cholesterol biosynthesis as a therapeutic strategy for ad may have a dual beneficial role , not only decreasing ad production in the brain , but also decreasing the toxic consequences of aβ accumulation . as cholesterol biosynthesis inhibitors have few major toxic side effects , this approach , coupled with other therapeutic strategies such as the use of secretase inhibitors ( nunan and small , 2000 ) aβ immunization ( schenk et al ., 1999 ) or acetylcholine esterase inhibitors such as galantamine reminyl ® could become the treatment of choice . it will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding , various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification . references cited herein are listed on the following pages , and are incorporated herein by this reference . alvarez , a ., alarcoón , r ., opaza , c ., campos , e . o ., muñoz , f . j ., calderón , f . h ., dajas , f ., gentry , m . k ., doctor , b . p ., de mello , f ., inestrosa , n . c . ( 1998 ) stable complexes involving acetylcholinesterase and amyloid - β peptide change the biochemical properties of the enzyme and increase the neurotoxicity of alzheimer &# 39 ; s fibrils . the journal of neuroscience , 18 ( 9 ): 3213 - 3223 ariga , t ., kobayashi , k ., hasegawa , a ., kiso , m ., ishida , h ., and miyatake , t . ( 2001 ) characterization of high - affinity binding between gangliosides and amyloid β - protein . arch . biochem . biophys . 388 , 225 - 230 avdulov , n . a ., chochina , s . v ., igbavboa , u ., o &# 39 ; hare , e . o ., schroeder , f ., cleary , j . p ., and wood , w . g . ( 1997 ) lipid binding to amyloid β - peptide aggregates : preferential binding of cholesterol a j . neurochem . 68 , 2086 - 2091 baenziger , j . e ., morris , m l ., darsaut , t . e ., and ryan , s . e . 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