Patent Application: US-201013384127-A

Abstract:
the present invention relates to the medical field , in particular to the use of natural organic polyanions , i . e . humic substances , hss , in the treatment of neurodegenerative diseases , such as prion disease , alzheimer &# 39 ; s and parkinson &# 39 ; s disease .

Description:
the authors have extracted humic substances from agricultural soil and have separated and purified humic or fulvic acids in accordance with the protocol indicated in example 5 . to determine whether ha and fa substances can cure scgt1 cells of scrapie infection , the authors exposed the cells to increasing concentration of ha and fa . materials and methods — after exposure for 1 week to an increasing concentration of ha or fa ( 1 , 2 , 5 , 10 and 20 μg / ml ), scgt1 cells ( schatzl et al ., 1997 ) were harvested and lysis was performed by lysis buffer ( 0 . 25 - 1 ml 20 mm tris , ph 8 . 0 , containing 100 mm nacl , 0 . 5 % nonidet p - 40 , and 0 . 5 % sodium deoxycholate ) to obtain a total protein concentration of 0 . 1 mg / ml measured by the bicinchoninic acid assay ( pierce ). subsequently samples were incubated with 2 μg of proteinase k ( boehringer mannheim ) for 1 h at 37 ° c . digested samples were then mixed with equal volumes of 2 × sds sample buffer . all samples were boiled for 10 min prior to sds - polyacrylamide gel electrophoresis . after electrophoresis , western blotting was performed . blocked membranes were incubated with primary d18 monoclonal antibody ( to detect mouse prp ) at 1 : 1 , 000 dilution in pbst overnight at 4 ° c . after incubation with primary antibody , membranes were washed and incubated with horseradish peroxidase - labeled secondary antibody ( amersham life sciences ), diluted 1 : 5 , 000 in pbst for 45 min at rt , and washed again . after chemiluminescent development with enhanced chemiluminescence ( ecl ) reagent ( amersham ) for 1 min , blots were exposed to ecl hypermax film ( amersham ). since prp sc is proteinase k resistant , this is a rapid diagnostic test to evaluate the presence of prion in infected cells . results — after 1 week , the treatment with ha and fa compounds caused the disappearance of prp sc from scgt1 cells in a dose dependent manner without affecting cell viability ( fig3 ). these compounds have a half maximal effective concentration ( ec 50 ) of 7 . 8 μg / ml and 12 . 3 μg / ml for ha and fa , respectively . from these data , it is clear that the most potent compounds with respect to eliminating prp sc were humic acids . the concentration of humic substances required to eliminate & gt ; 95 % of preexisting prp sc was 20 μg / ml for both compounds . the potency of both hss compounds in eliminating prp sc seems dependent on their molecular weight . in fact , ha and fa have a molecular weight of 4 , 000 da and 1 , 500 da , respectively . the preceding results demonstrate the potent ability of hss compounds to clear prp sc from scgt1 cells . to explore whether these compounds could be used as a potential therapeutic for treatment of prion disease , we tested whether they were cytotoxic for scgt1 cells , using as criteria cell growth , morphology , and viability as measured by trypan blue staining none of the compounds was cytotoxic to scgt1 cells after exposure for 1 wk at concentrations up to 20 μg / ml ( fig4 ). encouraged by their success in reversing the accumulation of prp sc in scgt1 cells under non - cytotoxic conditions , the authors tested the anti - prion activity of hss substances using an in vitro amyloid conversion assay for prions . this test represents a useful tool to simulate the aggregation kinetics of the prion protein . the presence of drug - compounds binding prp c could have an effect on the kinetic of fibrils formation . the lag phase corresponds to the time prior the fibrils formation . stronger is the effect of a drug longer is the lag phase . in this experiment , the authors observed that hss compounds strongly inhibit the aggregation propensity of moprp ( 89 - 231 ). in particular , they observed that the lag phase of moprp ( 89 - 231 ) is longer in the presence of 20 μg / ml of either ha or fa . materials and methods — to monitor the fibril formation the authors performed the thioflavin t ( tht ) assay . tht fluorescence has been monitored at an emission wavelength of 485 nm and an excitation wavelength of 450 nm . during the time course of amyloid formation , a solution of tht , 20 - fold more concentrated than the final protein concentration , in phosphate buffered saline has been added to aliquots of 10 μg recombinant prp at room temperature , 25 ° c . and 37 ° c . in situ , fluorescence will be monitored in a 96 - well fluorescence plate reader ( 450 nm excitation and 485 nm emission ). tht fluorescence intensity has been read automatically every minute with shaking between measurements . for the screening of hss compounds different concentration of ha and fa ( 5 - 10 - 20 μg / ml ) has been added to the moprp solutions ( 50 μg / ml ). for this experiment the authors used two types of recombinant mouse prion protein ( accession number : np — 035300 ): one including residues from 89 to 231 ( moprp ( 89 - 231 )) and the other including residues from 23 - 231 ( moprp ( 23 - 231 )). the first one is the canonical prp fragment found in amyloid plaque during prion disease , whereas the second one is the mature physiological prion protein . results — anti prion propensity of hss has been evaluated considering the time required to the recprp solutions to form fibrils . the time prior to the fibrilization is called lag phase . in fig5 a ) we can observe the thioflavin t ( tht ) assay with the lag phase of moprp ( 89 - 231 ) with () and without (□) the presence of 20 μg / ml humic substances ( ha or fa ). in the presence of a concentration of hss ≧ 20 μg / ml we observed a significant longer lag phase in comparison with the control ( fig5 b ). this test supports our findings that ha and fa act as anti - prion agent both in vivo and in vitro . to start to elucidate the mechanism of action of hss on the prp , the authors investigate the effect of ha and fa on the secondary structure of recmoprp ( 89 - 231 ) and recmoprp ( 23 - 231 ) using : ( i ) far - uv circular dichroism ( cd ), ( ii ) adsorption assay using western blot and bca ( pierce ) analysis of the supernatant solutions after ultracentrifugation , ( iii ) elisa . in the absence of ha or fa , the spectra moprp ( 89 - 231 ) and moprp ( 23 - 231 ) have a double minimum at 222 and 208 nm , characteristic of α - helical structure , typical of prp c . interestingly , the addition of ha or fa to the protein provokes a decrease in negative ellipticity ( fig6 ). in particular , the effect is stronger in presence of ha both for moprp ( 89 - 231 ) ( fig6 a ) and moprp ( 23 - 231 ) ( fig6 c ). moreover , time - dependent transition of moprp ( 23 - 231 ) in the presence of ha was observed ( fig6 d ). changes in molar ellipticity could be related to two hypotheses : ( a ) they are due to conformational changes of the secondary structure ( i . e . loss of α - helical content ) or ( b ) partial protein precipitation . to demonstrate that changes in molar ellipticity are due to the precipitation of the prp in the presence of hss , the authors measured adsorption of 20 μg of moprp ( 89 - 231 ) and moprp ( 23 - 231 ) in the presence of 1 μg / ml to 20 μg / ml of ha and fa . no prion protein was detected by western blot and bca ( pierce ) analysis of the supernatant solutions after ultracentrifugation ( 100 , 000 g ) at hss concentration up to 5 μg / ml ( fig7 ). finally , the authors measured the propensity of hss on the binding with moprp ( 23 - 231 ) and fc_huprp ( 23 - 230 ) using the method of competitive elisa . for both proteins authors observed a significant decrease in absorbance due to the competitive effect of ha versus the binding site of the antibody . the authors &# 39 ; results suggest that ha could prp specifically ( fig8 ). materials and methods — extraction and purification of hs was carried on the basis of the procedures published by international humic substances society ( ihss ) and sequi et al . ( sequi et al ., 1986 ), both previously reported ( r . s . swift , 1996 ) with the following amelioration in order to optimize the analytical efficiency . briefly , hs were extracted from 2 mm - sieved soil sample with 0 . 1 m naoh ( 1 : 5 wt / vol ). the suspension was left overnight under a n 2 atmosphere with constant shaking . after a slower centrifugation at 13 , 000 rpm to remove the bulky material , the extract was centrifuged at 24 , 000 rpm and filtrated through a 0 . 45 μm nitrocellulose filter . the filtrate was then acidified until ph 2 with h 2 so 4 to precipitate humic acids . after centrifugation the supernatant was collected , and the pellet ( humic acids , ha ) resuspended with 0 . 5 naoh and stored . the supernatant was fed on a column packed with polyvinylpyrrolidone ( pvp ), previously equilibrated in 0 . 01 m h 2 so 4 . the eluate ( the non - retained , non - humified fraction ) was discarded , while the brown - coloured retained fraction ( fulvic acids , fa ), was subsequently eluted with 0 . 5 m naoh . both fractions were passed through h + exchanging resin to remove metal ions and adjusted to ph 7 . the organic carbon content of the ha and fa fraction were measured by wet oxidation method ( it . min . lex n . 248 oct . 21 st 1999 ). in conclusion , the present invention surprisingly demonstrate that non - cytotoxic concentrations of naturally occurring humic ( ha ) and fulvic ( fa ) substances can rapidly eliminate prp sc from chronically infected scgt1 cells . furthermore , the amyloid seeding assay ( asa ) of moprp ( 89 - 231 ) and moprp ( 23 - 231 ) showed a considerably longer lag phase in the presence of increasing concentration of has and fas . moreover , the interaction between recmoprp c and ha , fa using far uv circular dichroism and elisa assays is shown . adjou , k t , et al ., j gen virol 84 ( 2003 ), 2595 - 2603 . ar { hacek over ( c )} on i ., et al ., 2006 environmental chemistry letters 4 , 191 - 194 . bertsch , u ., k . f . winklhofer , et al . 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