Patent Application: US-14128898-A

Abstract:
the present invention is directed to the use of glycosidase inhibitors for inhibiting the conversion of a pro - toxin to a toxin by a glycosidase enzyme . in particular , the present invention relates to the use of glycosidase inhibitors for the treatment of malaria , endotoxic shock or septic shock .

Description:
structural components of the malaria toxin which affect activity have been inferred from enzymatic and chemical treatments . tnf - induction by malaria toxin is unaffected ( 3 ) by treatment with dilute hono . this resistance to hono distinguishes the active portion of lipidic tnf - inducing malaria toxin from gpi anchors of proteins which are degraded by hono , owing to the presence of a non - n - acetylated hexosamine residue . glycoconjugates from parasites such as lipophosphoglycans attach to membrane via gpi anchors ( 16 ) and the glycan moiety of gpi - anchored proteins from parasites ( 27 ) may contain α - galactose residues . likewise , bacterial lipopolysaccharide ( lps ) contains α - galactose residues ( 20 ). α - d - galactosidase has also been shown to alter the antigenicity of an antigen in plasmodium falciparum culture medium ( 12 ) which induces tnf ( 23 ). mice bred at university college london (( cba × balb / c ) f1 or cba × c57b1 ) f1 ) were infected with p . yoelii ym or p . berghei anka . parasitized rbc ( 1 × 108 / ml ) in earle &# 39 ; s balanced salt solution ( bss ; life technologies ltd , paisley , u . k .) were incubated at 37 ° c . overnight , then disrupted by freezing and thawing , digested overnight with 250 μg pronase e ( sigma ) per mg of protein ( determined by biorad assay ), boiled , treated with 25 μg / ml polymyxin b agarose ( sigma ) to eliminate endotoxin , filtered through a 0 . 2 - μm filter ( sartorius ag , gottingen , germany ) and stored at 4 ° c . ( 24 ). tnf induction from thioglycollate - induced peritoneal macrophages and elisa assays for murine tnf were described previously ( 24 ). polymyxin b ( 5 μg / ml ) was included in all experiments with malaria toxin to exclude effects from contaminating lps . a more sensitive one - plate procedure for tnf production and assay was also used ( 5 ). cytotoxicity assays for tnf were done using l929 cells ( 23 ). green coffee bean α - d - galactosidase was obtained from 2 sources ( sigma , poole , dorset , uk and oxford glycosystems ltd , abingdon , oxford uk ). significant increase in tnf - induction in macrophages was reproducibly observed when more than a dozen different extracts of rbc infected with p . yoelii , and 3 with p . berghei ( but not uninfected rbc controls that did not induce tnf ) were pre - treated with α - d - galactosidase . a typical titration showing dose - dependent enhancement of tnf induction by one sample of malaria toxin treated with increasing amounts of α - galactosidase is shown ( fig1 ). the amount of tnf enhancement by any concentration of α - galactosidase depended on toxin potency , higher dilutions of toxin always showing greater enhancement . no tnf was induced from macrophages incubated with 2 u / ml enzyme only and no enhancement occurred with boiled enzyme , again excluding any contribution from contaminating lps , which is heat - stable . macrophages treated with 2 u / ml of enzyme for 1 hr at 37 ° c ., then stimulated with toxin or 2 or 10 ng / ml of lps , did not secrete more tnf than untreated cells . the α - galactosidase hydrolysis of p - nitrophenyl - α - d - galactopyranoside ( αpnp - gal ) is optimal at ph 6 . 0 ( 4 , 9 ) and undetectable at ph 8 . 0 ( 9 ), as we confirmed , but enhancement of tnf - induction by malaria toxin was greatest at ph 8 . 0 ( fig2 .). the tnf - inducing activity of lps was also enhanced with a similar ph profile to the toxin , indicating a homologous structure / function relationship . the degree of enhancement also varied with the concentration of lps , and at higher concentrations enhancement was also detectable at ph 6 . 0 . the most likely explanation for the lack of activity against αpnp - gal at ph 8 . 0 is the presence of an α - galactosidase activity which does not hydrolyse this substrate . this was supported by the observation of batch to batch variability and the fact that the stability of the tnf - enhancing activity differed from the αpnp - gal activity . although all enzyme preparations were first dialysed and adjusted to the same activity against αpnp - gal , batches from different sources enhanced to different degrees ( as illustrated in fig1 ). tnf - enhancing capacity deteriorated with storage at 4 ° c ., although activity against the αpnp - gal substrate did not . no differences were visible in a 10 % sds page mini - gel system ( pharmacia , uppsala , sweden ), using 4 . 5 μg of protein , between batches which did or did not enhance . apart from bsa , all preparations contained 3 bands of 33 kd , 29 kd and 27 kd ; 28 kd and 36 . 5 kd isoforms were previously described ( 4 ). no β - galactosidase activity was detectable against βpnp - gal at ph 6 . 0 , nor did 0 . 8 u / ml of β - galactosidase ( sigma , grade x from e . coli ), at any ph from 4 to 8 , affect malaria toxin or lps activity . it is commonly found that not all exoglycosidase activities can be monitored using a pnp surrogate substrate . for example , while the α -( 1 , 2 ), α -( 1 , 3 ) and α -( 1 , 6 ) mannosidase activity of jack bean a - mannosidase can be assayed using pnp - mannoside , the α -( 1 , 2 ) mannosidase activity from aspergillus phoenicis cannot and only natural substrates can be used ( 10 ). this property is normally referred to as the aglycon specificity of an exoglycosidase . 1 . bate , c . a . w ., and d . kwiatkowski . 1994 . a monoclonal antibody that recognizes phosphatidylinositol inhibits induction of tumor necrosis factor alpha by different strains of plasmodium falciparum . infect . immun . 62 : 5261 - 5266 . 2 . bate , c . a . w ., j . taverne , e . romβ , c . moreno , and j . h . l . playfair . 1992 . tnf induction by malaria exoantigens depends upon phospholipid . immunology 75 : 129 - 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