Patent Abstract:
glycans are identified which have high affinity for c . difficile toxin a . they share one of two saccharide backbones and may have additional side chains . the backbones are galactose - β1 - 3 n - acetyl - d - glucosamine - β - 1 - 3 - galactose - β - 1 - 4 - n - acetyl - d - glucosamine and galactose - α - 1 - 3 - galactose - β - 1 - 4 - n - acetyl - d - glucosamine . the ligands may be used therapeutically , prophylactically , and diagnostically .

Detailed Description:
it is a discovery of the present invention that certain oligosaccharides are able to bind to clostridium difficile toxin a with higher affinity than previously known for other substances . weaker binders are known in the art and have been found to alleviate , reduce , ameliorate , inhibit or prevent the biological effects of toxin a . the oligosaccharides which bind tighter can be used similarly . oligosaccharides of the invention can be used in the free form or can be bound or conjugated to larger polymers . for example , the oligosaccharides can be bound to non - absorbable polymers which do not leave the gastrointestinal lumen , but remain in the lumen until excreted . many such non - absorbable polymers are known in the art and any can be used as is convenient . for example , surface - treated , calcined , diatomaceous earth ( chromosorb - p ™) can be used as a solid support to which the oligosaccharides of the present invention can be bound or attached . for attachment , the product can be silylaminated and then chain - extended glycosylamide linkages can be coupled to the silylaminated product . see nilsson , 1997 . another polymer which can be used is polystyrene sulfonate . this polymer demonstrates binding to toxins a and b on its own . the oliogosaccharides of the present invention can be bound or coupled to this polymer and the affinity of the polymer enhanced . another polymer which can be used is cholestyramine , a cationic resin that has been used as a bile acid sequestrant . side chains which may be added to the oligosaccharide backbone according to the present invention include fucose , galactose , glucose , mannose , n - acetylneuraminic acid , n - acetyl - d - glucosamine , d - acetyl - d - glactosamine , and short chains of two or more identical or different moieties . other saccharides which can be used according to the invention include glusoamine , galactosamine , n - acetylmuramic acid , n - acetylneuraminic acid , rhamnose , 2 - deooxy - d - ribose , mannitol , inositol , gluconic acid , glucaric acid , gluconolactone , glucuronololactone , ascorbic acid , glucuronic acid , dehydroascorbic acid , fructose , xylose , and arabinose . oligosaccharides of the present invention can be administered per os or per anus to a mammal . the mammal may be a farm animal , such as an ostrich , chicken , turkey , cow , horse , pig . the mammal can be a companion animal such as a cat or a dog . the mammal can be a human . the oligosaccharides can be delivered before or after symptoms are noted . mammals which are at risk of developing c . difficile infection can be treated in a prophylactic mode . this will reduce the severity , delay , or prevent the development of symptoms . mammals are at elevated risk of infection if they are hospitalized , antibiotic treated , especially clindamycin treated , a transplant recipient , hiv - infected , or immunosuppressed . oligosaccharides of the present invention can also be used diagnostically . binding can be used to determine whether symptoms present in a mammal are caused by c difficile infection . pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically . proper formulation is dependent upon the route of administration chosen . for oral administration , the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art . such carriers enable the compounds of the invention to be formulated as tablets , pills , dragees , capsules , liquids , gels , syrups , slurries , suspensions and the like , for oral ingestion by a patient to be treated . pharmaceutical preparations for oral use can be obtained solid excipient , optionally grinding a resulting mixture , and processing the mixture of granules , after adding suitable auxiliaries , if desired , to obtain tablets or dragee cores . suitable excipients are , in particular , fillers such as sugars , including lactose , sucrose , mannitol , or sorbitol ; cellulose preparations such as , for example , maize starch , wheat starch , rice starch , potato starch , gelatin , gum tragacanth , methyl cellulose , hydroxypropylmethyl - cellulose , sodium carboxymethylcellulose , and / or polyvinylpyrrolidone ( pvp ). if desired , disintegrating agents may be added , such as the cross - linked polyvinyl pyrrolidone , agar , or alginic acid or a salt thereof such as sodium alginate . dragee cores are provided with suitable coatings . for this purpose , concentrated sugar solutions may be used , which may optionally contain gum arabic , talc , polyvinyl pyrrolidone , carbopol gel , polyethylene glycol , and / or titanium dioxide , lacquer solutions , and suitable organic solvents or solvent mixtures . dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses . pharmaceutical preparations which can be used orally include push - fit capsules made of gelatin , as well as soft , sealed capsules made of gelatin and a plasticizer , such as glycerol or sorbitol . the push - fit capsules can contain the active ingredients in admixture with filler such as lactose , binders such as starches , and / or lubricants such as talc or magnesium stearate and , optionally , stabilizers . in soft capsules , the active compounds may be dissolved or suspended in suitable liquids , such as fatty oils , liquid paraffin , or liquid polyethylene glycols . in addition , stabilizers may be added . all formulations for oral administration should be in dosages suitable for such administration . for buccal administration , the compositions may take the form of tablets or lozenges formulated in conventional manner . the active agents may also be formulated in rectal compositions such as suppositories or retention enemas , e . g ., containing conventional suppository bases such as cocoa butter or other glycerides . the above disclosure generally describes the present invention . all references disclosed herein are expressly incorporated by reference . a more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only , and are not intended to limit the scope of the invention . previous work showed that of c . difficile toxin a binds to carbohydrates that contain gal alpha 1 - 3gal beta 1 - 4glcnac . since unsubstituted gal alpha 3gal beta 4glcnac beta sequences are not found in human tissues ( due to suppression of the gene coding for the human enzyme gal beta 3 - transferase ,) we reasoned that the actual physiologic target of toxin a must represent another oligosaccaharide . accordingly , our study was undertaken to identify other oligosaccharide structures bound by toxin a . binding to gal alpha 3gal beta 4glcnac beta - terminated glycosphingolipids of rabbit erythrocytes has also been demonstrated by teneberg et al . additional binding - active glycosphingolipids included galnac beta 3gal beta 4glcnac beta 3gal beta 4glc beta 1cer , galnac alpha 3gal beta 4glcnac beta 3gal beta 4 glc beta 1 cer , galnac alpha 3 ( fuc alpha 2 ) gal beta 4glcnac beta 3gal beta 4glc beta 1 cer , and glcnac beta 3 gal beta 4 glcnac beta 3 gal beta 4 glc beta 1 cer . to define the carbohydrate binding specificity of c . difficile toxin a , we utilized a glycan microarray constructed by using standard robotic microarray printing technology containing amine functionalized glycans coupled to an amino - reactive glass slide at the consortium for functional glycomics ( see website at functionalglycomics . org ). this array comprises over 260 synthetic and natural glycan sequences representing major glycan structures of known glycoproteins and glycolipids spotted in multiple replicates . microarrays were printed as described in blixt et al . purified clostridium difficile toxin a was applied to the slide at 200 micrograms / ml and incubated under a microscope cover - glass in a humidified chamber for 30 to 60 min . unbound material was washed away , and bound toxin a was detected with mouse anti - c . difficile toxin a monoclonal antibody , pcg4 ( 10 ug / ml ; available from genetex ). secondary goat anti - mouse antibody - alexa488 ( 10 ug / ml ) was then applied . following washing , fluorescence signals were analyzed using a confocal scanner ( scanarray 5000 , perkinelmer ). image analyses were carried out by using imagene image analysis software ( biodiscovery , el segundo , calif .). data were analyzed and plotted using microsoft excel software . c . difficile toxin a has previously been documented to recognize a carbohydrate antigen , lewis ×( galb1 - 4 [ fuca1 - 3 ] glcnacb ) ( infection and immunity 59 : 73 - 8 , 1991 ) and ( galb1 - 3galb1 - 4glcnacb ) ( biochem cong . 8 : 466 - 71 , 1997 ). please refer to fig1 . binding to these carbohydrate structures was demonstrated in our system , albeit weak , with fluorescence ( fl ) signals of 122 / 218 and 172 respectively . however , our analysis also demonstrated a number of more complex structures , with greater relative affinities ; gala1 - 3galb1 - 4 ( fuca1 - 3 ) glcnacb -( fl 1814 ), gala1 - 3 ( fuca1 - 2 ) galb1 - 4 ( fuca1 - 3 ) glcnacb -( fl 789 ), galnaca1 - 3 ( fuca1 - 2 ) galb1 - 4 ( fuca1 - 3 ) glcnacb -( fl 327 ), and galnaca1 - 3 ( fuca1 - 2 ) galb1 - 4glcnacb -( fl 188 ). each of these oligosaccharides bound toxin more strongly than the previously identified structures . these results support the importance of alpha 2 or 3 linked fuc and also demonstrate that galnaca1 - 3 can substitute for galb1 - 3 on the galb1 - 3galb1 - 4glcnacb backbone . the array binding study also identified a completely new and previously unappreciated oligosaccharide motif bound by c . difficile toxin a . galb1 - 3 ( fuca1 - 4 ) glcnacb1 - 3galb1 - 4glcnacb -( fl 29098 ), neuaca2 - 3galb1 - 3 ( fuca1 - 4 ) glcnacb1 - 3galb1 - 4 ( fuca1 - 3 ) glcnacb -( fl 14911 ), and neuaca2 - 3galb1 - 3glcnacb1 - 3galb1 - 4glcnacb -( fl 4435 ) were also identified . this supports the core galb1 - 3glcnacb1 - 3galb1 - 4glcnacb - backbone as a significantly stronger target of toxin a . the strongest signals were associated with galb1 - 3 ( fuca1 - 4 ) glcnacb1 - 3galb1 - 4glcnacb -, and neuaca2 - 3galb1 - 3 ( fuca1 - 4 ) glcnacb1 - 3galb1 - 4 ( fuca1 - 3 ) glcnacb - supporting the important contribution of alpha linked fucose to binding affinity . 1 . larson h e , price a b , honour p , borriello s p . clostridium difficile and the aetiology of pseudomembranous colitis . lancet 1978 ; 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