Patent Application: US-81599306-A

Abstract:
poly glycodendrimers can serve as multivalent inhibitors of selectin - mediated leukocyte recruitment regulating inflammatory response . disclosed are compounds and methods relating to functionalized branched glycopolymers . in embodiments , the compounds are capable of modifying cell adhesion events and inflammatory conditions . in a particular embodiment , a multi - arm peo polymer with sulfated lactose end groups can specifically inhibit interactions involving l - selectin . also disclosed are methods of synthetic preparation and use .

Description:
the invention may be further understood by the following non - limiting examples . introduction . the simultaneous presentation of saccharide epitopes on an appropriate macromolecular scaffold creates a multivalent display that amplifies the affinity of glycoside - mediated receptor targeting . in this regard , branched poly ( ethylene oxide ) ( peo ) polymers can provide useful scaffolds for in vivo blockade of selectin binding due to their defined molecular architecture , hydrophilicity , and availability of multiple surface reactive sites . moreover , the branched polymer structure also provides a mechanism for controlling accessibility , mobility , density , and supramolecular organization of pendant sugar epitopes , as additional elements that may facilitate the design of optimal selectin - binding antagonists with defined circulating half - life . herein , we report a simple strategy for the synthesis of 1st and 2nd generation dendrimer - like peo glycopolymers bearing sulfated β - lactose as potential l - selectin inhibitors . most current glycoconjugation strategies describe the use of an aliphatic or aromatic aglycone linker that carries an amine , thiol , thiourea , acid , or active ester as the reactive moiety . significantly , by using imidated lactose donors and a schmidt glycosidation coupling protocol , protecting group manipulations were minimized in the process of glycopolymer synthesis . three ( mn ˜ 5200 mu ) and four arm ( mn ˜ 5100 mu ) peo stars ( 1st generation ) carrying hydroxy end groups were synthesized by anionic polymerization using “ core - first ” methodology ( hou et al . 2003 , macromolecules 36 : 3874 - 3881 ; angot et al . 2000 , macromolecules 33 : 5418 - 5424 ). in addition , a dendrimer - like 2 nd generation peo star polymer ( 12 arm ) was synthesized based on a phosphazene core . this polymer consisted of a 1st generation of six peo arms , produced by an “ arm - first ” strategy onto the phosphazene core , followed by a 2nd generation of 12 hydroxyterminated peo branches polymerized directly onto the original six arm core ( mn ˜ 52 kd ) ( hou et al . 2003 , polymer 44 : 5067 - 5074 ). β - lactose octaacetate was selectively brominated at the anomeric center and subsequently activated to the imidate donor ( a ) as indicated in fig1 b . glycosylations of hydroxy - terminated peo star and dendrimer - like polymers by trichloroacetimidate glycosidation methodology , using bf 3 . oet2 as a lewis acid activator resulted in the covalent attachment of acetyl - protected lactose residues in high yield . zemplen conditions for deacetylation ( naome / meoh ) followed by lactose sulfation using an excess of so 3 . trimethylamine complex furnished target peo glycoclusters carrying terminal sulfated oligosaccharides 1c , 2c , and 3c ( see fig2 ). the efficiency , homogeneity , and degree of ligand ( lactose ) loading on the peo polymers were estimated by 1 h nmr spectroscopy , as well as by mass estimates obtained by maldi - tof and laser light scattering . moreover , ftir and sds - page analysis provided additional evidence of sulfated lactose units . specifically , the relative intensities of the anomeric h : ac : ch 3 signal ratio of 6 : 20 . 9 : 3 for the three arm ( 1a ) derivative and an integration ratio of 7 . 95 : 27 . 97 : 8 for the four arm glycocluster 2a indicated complete glycosylation of the hydroxyl groups on the parent peo precursor . the increase in molecular weight was further corroborated using maldi - tof ( 1a : 6899 mu , 2a : 7524 mu ) and lls measurements , confirming quantitative functionalization . likewise , an nmr integration ratio of 3 . 9 : 13 . 7 : 3 , as well as maldi - tof , demonstrated a high degree of lactose conjugation (& gt ; 95 %) onto the dendritic peo scaffold of the 12 - arm , 2 nd generation , branched compound . subsequent deprotection followed by sulfation produced a highly charged sulfated glycodendron 3c ( observed seldi - tof : 61 . 8 kd , expected value ˜ 62 kd ). heparin can exhibit anti - inflammatory properties by mediating blockade of l - and p - selectins via sulfate - dependent interactions . in vitro observations have reported that sulfated esters can promote selectin binding when appropriately oriented on a lactose core . for example , a sulfated lactose derivative ( 6 , 6 ′- disulfo lactose ), lacking fucose and sialic acid residues , was superior to sle x as an in vitro inhibitor of l - selectin binding to glycam - 1 . see references 8 ( a , b , c ). we explored the capacity of sulfated branched star and dendrimer - like glycopolymers to limit inflammatory responses in vivo via presumed selectin - dependent blockade on the theory that selectin - glycoligand binding can be greatly amplified through multivalent presentation of oligosaccharide determinants . we tested this approach using our compounds in the context of an inflammatory condition , peritonitis . acute inflammation was induced in a mouse model by thioglycollate injection into the peritoneal cavity . potency was valence - dependent with 1c / 2c exhibiting little activity , while 3c ( 0 . 5 mg / mouse iv ) dramatically reduced neutrophil and macrophage recruitment by 86 and 60 %, respectively ( fig4 , p & lt ; 0 . 05 ). although heparin inhibited inflammatory cell recruitment to a similar degree , we believe that concurrent anticoagulant effects pose practical limitations on heparin &# 39 ; s clinical applicability . in contrast , 3c does not exhibit substantial anti - thrombin activity ( data not shown ). thioglycollate induced murine model of peritoneal inflammation . mice ( c57bl , male , 6 - 8 weeks old ) were injected intraperitoneally with 2 ml of 3 % thioglycollate broth . five minutes later , animals received intravenous injections of 0 . 2 ml sterile pyrogen - free saline ( s ) with and without heparin ( h ) or analogs ( 3c ) ( 0 . 5 mg / mouse with 0 . 2 ml saline ). mice were sacrificed after 3 hours and peritoneal cells harvested by lavage with 8 ml of ice - cold phosphate buffered saline ( pbs ) containing 3 mm edta . peritoneal cells were counted and cells stained for 30 minutes at 4 ° c . with fitc - conjugated rat anti - mouse gr - 1 mab diluted in pbs containing 2 . 5 % fbs . after washing three times with pbs , facscan analysis was performed as described by wang et al . ( 2002 ) and was used to sort , count and analyze the percentage of neutrophils and macrophages which were converted to the cell number using the formula n / m = total cell number x % of nim , respectively ( n : neutrophil ; m : macrophages ). the cells were gated ( facs ) expressing a high level of gr - 1 antigen ( n ) and mac - 1 ( m ). ( see additional ref : lagasse ; e ., weissman ; i . j . immunological meth . 1996 , 197 , 139 - 150 ). statistical differences among groups were analyzed by anova method using statview software . the ability of 3c to inhibit the adhesion of u937 cells to immobilized selectins was examined to determine whether the observed in vivo effect was mediated by presumed selectin blockade . 3c selectively blocked the adhesion of u937 cells to l - selectin in a dose - dependent manner ( ic 50 = 2 . 4 nm ), but did not exhibit anti - p - selectin or e - selectin activity . see fig5 . therefore , 3c is among the most potent l - selectin inhibitors yet reported ( see references 2a , 8 - 10 ). a partial explanation for this substantial biological activity can be that increased biological activity is a consequence of ligand presentation by dendrimeric scaffolds when compared to linear counterparts . despite significant anti - l - selectin activity , the observed in vivo activity was surprising since it has been recently reported that heparin &# 39 ; s anti - inflammatory activity in vivo is critically dependent on its ability to inhibit both l - and p - selectin mediated inflammatory cell adhesion ( wang et al ., 2002 ). indeed , a compound acting solely as a selective inhibitor of l - selectin is not anticipated to block in vivo leukocyte infiltration so completely . thus , it is likely that 3c , like heparin , blocks chemokine binding to the endothelium , which would further limit leukocyte extravasation . u937 cell binding to immobilized e -, l - or p - selectin . the ability of test compounds to competitively inhibit the adhesion of u937 cells to immobilized p -, l - or e - selectins was examined by coating each well of a 96 - well plate at 4 ° c . overnight with 8 μg / ml of protein a in 50 mm carbonate buffer , ph 9 . 4 . after blocking the plate with 10 % bsa in pbs , p - selectin ( 0 . 05 μg / well ), l - selectin ( 4 μg / well ) or e - selectin ( 1 μg / well ) chimeras were added . after 1 hr , wells were washed with blocking solution . u937 cells were grown in rpmi - 1640 medium ( containing 10 % fetal bovine serum ( fbs ), 100 u / ml penicillin and 100 μg / ml streptomycin in an atmosphere of 5 % co 2 in air and 100 % relative humidity . the cells were fluorescently labeled with 10 μm calcein am in rpmi 1640 medium containing 2 . 5 % fbs for 30 min at 37 ° c . the cells were collected by low speed centrifugation , washed three times with rpmi 1640 medium , and resuspended at a density of 2 × 10 6 cells / ml in medium without fbs . heparin and test compounds , or 10 mm sodium edta ( negative control ) were added at 50 μl / well , and then the fluorescently labeled u937 cell suspension ( 50 μl / well ) was added and incubated for 30 min at room temperature . non - adherent cells were removed by rinsing the plates three times with pbs , and the number of adherent cells was quantified by measuring the fluorescence intensity at 485 nm after cell with 2 % triton x - 100 in 0 . 1 m tris - hcl , ph 9 . 5 . raw data were converted to relative fluorescence intensity ( rfi ) for comparative purposes . general methods : all chemicals were reagent grade and used as supplied unless otherwise noted . solvents were dried and distilled before use . molecular sieves were activated at 350 ° c . for 3 h in vacuo . dichloromethane was distilled from cah 2 and stored over 4 å molecular sieves . all of the reactions were performed with oven - dried glassware under argon atmosphere and monitored by thin layer chromatography ( tlc ) on 250 - μm al pre - coated silica gel plates . detection was performed by examination under uv light ( 254 nm ) and by charring with 10 % sulfuric acid in water . flash chromatography was performed on silica gel ( mesh 200 - 425 ) with the appropriate solvents . size exclusion chromatography was performed with water as eluant . extracts were concentrated under reduced pressure at & lt ; 45 ° c . ( water bath ). 1 h nmr and 13 c nmr spectra were recorded on 300 mhz , 400 mhz or 600 mhz nmr spectrometers . for 1 h nmr and 13 c nmr spectra recorded in cdcl 3 , cd 3 od , d 2 o and dmso chemical shifts ( δ , delta ) are given in ppm relative to solvent peaks . coupling constants ( j ) are reported in hertz ( hz ). maldi - tof mass spectrometry data was performed using 2 , 5 - dihydroxybenzoic acid as the matrix . surface enhanced laser desorption / ionization time - of - flight mass spectrometry ( seldi - tof ms ) was carried out on sulfated 12 - stararm dendrimer 3c using sinapinic acid and “ cdc oligo ” matrix obtained from centre for disease control . bsa was used as an external standard for seldi - tof experiments . sample preparation for sds - page gel analysis was carried out by diluting 25 μl of sample solution with 25 μl stock solution of laemmli buffer solution ( 95 μl of laemmli buffer solution + 95 μl of mercaptoethanol ). a total amount of 50 μl was loaded on the gel and electrophoresis samples run in a 10 × tris / glycine / sds buffer solution . general procedure for synthesis of poly ( ethylene oxide ) glycopolymers ( 1a , 2a , 3a ). acetylated lactose imidate ( 1 . 5 eq / each oh ) was added to an ice - cooled solution of oh - terminated poly ( ethylene oxide ) dendrimer - like polymers containing 3 , 4 , 12 terminal arms dissolved in anhydrous ch 2 cl 2 . the lewis acid bf 3 . et 2 o ( 5 eq .) was added to the reaction mixture and stirred at 0 ° c . for 1 . 0 h followed by stirring at room temperature for 16 h . the reaction was quenched with diisopropylamine and concentrated and subjected to silica gel column chromatography using chcl 3 : meoh as eluent ( 0 %→ 10 %). the product was obtained as a white solid . all acetyl protected glycopolymers 1a , 2a , 3a were soluble in water . general procedure for deacetylation to give 1b , 2b , 3b . naome ( 1 . 5 eq / ac group ) was added via syringe to a solution of acetylated glycopolymers in dry meoh and the reaction mixture stirred at room temperature for 5 h . on completion , as determined by the disappearance of the starting compound on tlc , the reaction mixture was passed through an h + ion exchange column , filtered , and washed five times with meoh . the collected meoh fractions were pooled and concentrated to give an off - white residue , which was purified using a gel filtration column with water as eluant and freeze dried . general procedure for sulfation of glycopolymers 1c , 2c , 3c . the sulfur trioxide - trimethylamine complex was extensively washed with water to remove large amounts of trapped h 2 so 4 , followed by three washes each with ethanol and ch 2 cl 2 , filtration and vacuum drying . the so 3 . nme 3 complex was added to a solution of deprotected glycopolymers ( 1b , 2b , 3b ) in dry dmf in molar excess ( 7 equivalents per hydroxyl group ) and the reaction mixture stirred at 60 ° c . for 3 days . an five equivalents of so 3 . nme 3 complex was then added and stirring continued at 60 ° c . for an additional 3 days . the reaction mixture was then filtered and the residue washed thoroughly with meoh / water ( 50 : 50 ). the reaction solution was concentrated and passed through a gel filtration column using meoh / water as the eluant . products 3b to 3c were dialyzed for 3 days , freeze dried , and then column purified . appropriate fractions were pooled and lyophilized to afford the sulfated glycopolymers as amorphous white solids . all compounds were characterized using 1 h and 13 c nmr , maldi - tof , laser light scattering , and sds - page gel electrophoresis . spectral data of 1a . 1 h nmr ( cdcl 3 , 400 mhz ): 5 . 30 ( d , 3h , j = 2 . 8 hz ), 5 . 14 ( t , 3h , j = 9 . 6 hz , j = 9 . 2 hz ), 5 . 08 - 5 . 03 ( m , 3h ), 4 . 92 ( dd , 3h , j = 3 . 2 hz , j = 3 . 6 hz ), 4 . 85 ( m , 3h ), 4 . 52 ( d , 3h , j = 8 . 0 hz ), 4 . 44 ( d , 3h , j = 7 . 6 hz ), 4 . 2 - 4 . 0 ( m , 12h ), 3 . 92 - 3 . 71 ( m , 6h ), 3 . 68 - 3 . 34 ( m , peo backbone ), 3 . 22 ( s , 6h , — ch 2 — o —), 2 . 08 - 1 . 91 ( 7 × 3 coch 3 ), 0 . 89 ( s , 3h , ch 3 ). 13 c nmr ( cdcl 3 , 400 mhz ): 164 . 1 ( coch 3 ), 164 . 0 ( coch 3 ), 163 . 8 ( coch 3 ), 163 . 7 ( coch 3 ), 163 . 4 ( coch 3 ), 163 . 3 ( coch 3 ), 162 . 7 ( coch 3 ), 94 . 7 ( c - 1 ′), 94 . 2 ( c - 1 ), 69 . 9 , 67 . 5 , 66 . 4 , 66 . 2 , 65 . 3 , 64 . 7 , 64 . 6 , 64 . 2 , 64 . 0 , 63 . 9 , 63 . 8 , 62 . 7 , 60 . 2 , 59 . 5 , 55 . 6 , 55 . 3 , 54 . 4 , 34 . 6 , 23 . 3 , 14 . 5 , 14 . 4 , 14 . 3 , 14 . 2 , 14 . 1 , 10 . 9 . maldi - tof : found 6899 . 8299 ( expected 7050 ). spectral data of 1b ( deprotected ) 1 h nmr ( d 2 o , 400 mhz ): 4 . 35 ( d , 3h , j = 8 . 4 hz ), 4 . 28 ( d , 3h , j = 7 . 6 hz ), 3 . 90 - 3 . 34 ( m , — och 2 ch 2 o skeleton ), 3 . 25 ( s , 6h , — ch 2 — o ), 3 . 16 ( t , j = 8 . 0 hz , j = 8 . 4 hz ), 0 . 87 ( s , 3h , ch 3 ). 13 c nmr ( d 2 o , 400 mhz ): 103 . 1 , 102 . 2 , 78 . 5 , 75 . 5 , 74 . 9 , 74 . 4 , 73 . 6 , 72 . 9 , 72 . 6 , 71 . 9 , 71 . 1 , 70 . 7 , 69 . 7 , 69 . 3 , 68 . 8 , 68 . 7 , 61 . 2 , 60 . 5 , 60 . 2 , 40 . 6 , 17 . 0 . maldi - tof : found 5909 . 2649 ( expected 6100 ). spectral data of 1c ( sulfated ) 1 h nmr ( d 2 o , 600 mhz ): 4 . 94 - 4 . 92 ( m , sugar ring protons ), 4 . 4 - 4 . 0 ( m , sugar ring protons ), 3 . 93 - 3 . 42 ( m , och 2 ch 2 o —), 3 . 26 ( s , 6h ), 3 . 1 - 3 . 04 ( m , sugar ring protons ), 0 . 78 ( s , ch 3 ). 13 c nmr ( d 2 o , 400 mhz ): 94 . 8 ( c - 1 ′), 93 . 2 ( c - 1 ), 71 . 1 , 70 . 8 , 69 . 4 , 69 . 0 , 68 . 7 , 68 . 4 , 67 . 6 , 67 . 1 , 65 . 5 , 64 . 1 , 63 . 2 , 62 . 9 , 62 . 4 , 62 . 2 , 61 . 2 , 60 . 8 , 59 . 9 , 34 . 1 , 10 . 5 . spectral data of 2a 1 h nmr ( cdcl 3 , 600 mhz ): 5 . 05 ( t , 4h , j = 10 . 2 hz ), 4 . 96 ( t , 4h , j = 8 . 4 hz , j = 8 . 4 hz ), 4 . 82 ( m , 4h ), 4 . 74 ( t , 4h , j = 7 . 8 hz ), 4 . 4 ( d , 4h , j = 8 . 4 hz ), 4 . 35 ( d , 4h , j = 7 . 8 hz ), 4 . 34 ( d , 4h , partly merged with h - 1 ), 4 . 1 - 3 . 92 ( m , 16h ), 3 . 8 - 3 . 72 ( m , 8h ), 3 . 7 - 3 . 38 ( m , — och 2 ch 2 o — skeleton ), 3 . 28 ( s , 8h , ch 2 — o ), 2 . 01 - 1 . 82 ( 7 × 4 coch 3 ). 13 c nmr ( cdcl 3 , 600 mhz ): 163 . 9 ( coch 3 ), 163 . 8 ( coch 3 ), 163 . 6 ( coch 3 ), 163 . 5 ( coch 3 ), 163 . 2 ( coch 3 ), 163 . 1 ( coch 3 ), 162 . 6 ( coch 3 ), 94 . 6 ( c - 1 ′), 94 . 2 ( c - 1 ), 69 . 8 , 66 . 4 , 66 . 2 , 65 . 2 , 64 . 6 , 64 . 1 , 63 . 9 , 63 . 8 , 63 . 5 , 62 . 6 , 60 . 1 , 55 . 6 , 54 . 4 , 47 . 2 , 39 . 4 , 25 . 1 , 16 . 2 , 14 . 4 , 14 . 3 , 14 . 2 , 14 . 1 , 14 . 0 . maldi - tof : found 7524 . 18 ( expected 7500 ). spectral data of 2b ( deprotected ) 1 h nmr ( d 2 o , 600 mhz ): 4 . 34 ( d , 4h , j = 7 . 8 hz ), 4 . 26 ( d , 4h , j = 7 . 8 hz ), 3 . 89 - 3 . 87 ( m , ring protons ), 3 . 81 - 3 . 78 ( dd , ring protons , j = 2 . 4 hz ), 3 . 75 ( d , ring protons , j = 3 . 0 hz ), 3 . 7 - 3 . 37 ( m , — och 2 ch 2 o — skeleton ), 3 . 32 ( s , 8h , — och 2 ), 3 . 18 - 3 . 15 ( m , ring protons ). 13 c nmr ( d 2 o , 600 mhz ): 103 . 1 , 102 . 3 , 78 . 5 , 75 . 5 , 74 . 9 , 74 . 4 , 72 . 9 , 72 . 6 , 71 . 1 , 70 . 7 , 69 . 7 , 68 . 8 , 68 . 7 , 61 . 2 , 60 . 2 , 45 . 2 . maldi - tof : found 6356 . 7333 ( expected 6300 ) spectral data of 2c ( sulfated ) 1 h nmr ( d 2 o , 600 mhz ): 4 . 96 ( d , 4h , j = 2 . 4 hz ), 4 . 9 ( d , 4h , j = 6 . 0 hz ), 4 . 64 - 4 . 62 ( m , ring protons ), 4 . 59 - 4 . 57 ( m , ring protons ), 4 . 38 ( dd , ring protons , j = 2 . 4 hz , j = 3 . 0 hz ), 4 . 34 ( t , ring protons , j = 5 . 4 hz , j = 4 . 2 hz ), 4 . 31 ( dd , ring protons , j = 5 . 4 hz , j = 4 . 8 hz ), 4 . 24 ( d , ring protons , j = 8 . 4 hz ), 4 . 21 ( d , ring protons , j = 7 . 8 hz ), 4 . 12 - 4 . 05 ( m , ring protons ), 3 . 97 - 3 . 85 ( m , ring protons ), 3 . 72 - 3 . 46 ( m , — och 2 ch 2 o — skeleton ), 3 . 36 ( s , 8h , ch 2 o —), 3 . 08 ( m , sugar ring protons ). 13 c nmr ( d 2 o , 400 mhz ): 94 . 7 , 93 . 3 , 71 . 2 , 70 . 8 , 69 . 4 , 69 . 1 , 68 . 7 , 68 . 5 , 67 . 2 , 65 . 4 , 64 . 2 , 63 . 8 , 63 . 2 , 62 . 6 , 62 . 3 , 60 . 5 , 59 . 9 , 45 . 8 . spectral data of 3a 1 h nmr ( cdcl 3 , 600 mhz ): 5 . 61 - 5 . 38 ( m , ring protons ), 5 . 26 - 5 . 23 ( m , ring protons ), 5 . 11 - 4 . 99 ( m , ring protons ), 4 . 87 - 4 . 66 ( m , ring protons ), 4 . 47 - 4 . 36 ( m , 24 anomeric h ), 4 . 2 - 3 . 92 ( m , ring protons ), 3 . 66 - 3 . 44 ( m , peo skeleton ), 3 . 42 ( s , peo skeleton ), 2 . 07 - 1 . 87 ( 7 × 12 coch 3 ), 0 . 9 ( s , 18h , ch 3 ). 13 c nmr ( cdcl 3 , 600 mhz ): 163 . 9 , 163 . 7 , 163 . 6 , 163 . 3 , 163 . 2 , 163 . 1 , 162 . 6 , 94 . 7 , 94 . 4 , 94 . 2 , 83 . 5 , 70 . 1 , 69 . 9 , 68 . 7 , 67 . 4 , 66 . 4 , 66 . 2 , 65 . 2 , 60 . 1 , 64 . 7 , 64 . 6 , 64 . 1 , 63 . 9 , 63 . 8 , 63 . 6 , 63 . 4 , 62 . 7 , 62 . 5 , 61 . 4 , 60 . 2 , 55 . 6 , 54 . 4 , 34 . 2 , 32 . 1 , 14 . 5 , 14 . 4 , 14 . 2 , 14 . 1 , 10 . 8 . maldi - tof : found 60254 ( expected 59500 ). spectral data of 3b ( deprotected ) 1 h nmr ( d 2 o , 600 mhz ): 4 . 54 - 4 . 48 ( m , ring protons ), 4 . 37 - 4 . 26 ( d , ring protons ), 4 . 16 - 3 . 88 ( m , ring protons ), 3 . 82 - 3 . 32 ( m , peo skeleton ), 3 . 12 ( s , peo skeleton ), 3 . 08 - 3 . 06 ( m , ring protons ), 0 . 81 ( s , ch 3 ). 13 c nmr ( d 2 o , 600 mhz ): 96 . 5 , 95 . 7 , 72 . 1 , 71 . 9 , 71 . 8 , 70 . 6 , 68 . 9 , 68 . 3 , 67 . 9 , 67 . 8 , 67 . 4 , 67 . 1 , 66 . 4 , 66 . 1 , 65 . 1 , 64 . 7 , 64 . 5 , 64 . 1 , 63 . 8 , 63 . 2 , 62 . 5 , 62 . 3 , 62 . 1 , 61 . 3 , 54 . 6 , 53 . 6 , 34 . 1 , 10 . 4 . maldi - tof : found 56132 . 266 ( expected approx 56 kd ). spectral data of 3c 1 h nmr ( d 2 o , 600 mhz ): 4 . 96 ( s , ring protons ), 4 . 84 ( d , ring protons , j = 6 . 0 hz ), 4 . 54 - 4 . 53 ( m , ring protons ), 4 . 36 - 4 . 28 ( m , ring protons ), 4 . 24 - 4 . 21 ( m , ring protons ), 4 . 19 ( d , ring protons ), 4 . 06 - 3 . 83 ( m , ring protons ), 3 . 71 - 3 . 38 ( m , peo backbone ), 3 . 52 ( s , peo backbone ), 1 . 23 - 1 . 02 ( m ), 0 . 82 ( s , ch 3 ). 13 c nmr ( d 2 o , 600 mhz ): 94 . 6 , 93 . 1 , 71 . 3 , 70 . 7 , 69 . 3 , 69 . 0 , 68 . 7 , 68 . 5 , 67 . 1 , 66 . 9 , 65 . 3 , 64 . 2 , 63 . 762 . 1 , 61 . 4 , 60 . 3 , 59 . 8 , 34 . 1 , 10 . 4 . seldi - tof : found 61852 . 9386 ( expected approx 62 . 6 kd ). demonstration of functional activity in the in vivo context of abdominal aortic aneurysm abdominal aortic aneurysms ( aaas ) affect 2 - 9 % of the population and are the 10th leading cause of death in white men over age 65 . the disease can be characterized by thinning of the extracellular matrix in the aortic media with associated destruction of elastin , loss of smooth muscle cells , and transmural infiltration of inflammatory cells . certain molecules such as mmps , chemokines , proinflammatory mediators can be upregulated . l - selectin dependant leukocyte - endothelial cell interactions can play an important role in the genesis of the inflammatory response , which leads to aortic aneurysm formation . we have synthesized a multivalent glycodendrimer , designated sr - 12 , whose anti - inflammatory activity is mediated , at least in part , by an anti - l - selectin effect ( ic 50 2 . 4 nm ). we hypothesize that l - selectin blockade via multivalent , glycodendrimers will suppress the formation and / or growth of abdominal aortic aneurysms by inhibition of leukocyte recruitment to the vascular wall . suppression of experimental abdominal aortic aneurysms ( aaas ) in mice by treatment with a polyethylene oxide glycopolymer antagonist of l - selectin was evaluated . the twelve arm multivalent heparinoid compound , sr - 12 ( see fig1 c ) suppresses experimental aaa in mice . a statistically significant reduction in aortic diameter was observed in the group treated with compound sr - 12 . see fig6 and the table below . in this example , an elastase infusion model was used in mice ( strain c57bl / 6 , n = 20 ). the mice were subjected to aortic elastase perfusion to induce experimental aortic aneurysms ( thompson and baxter , 1999 ). mice received 0 . 5 mg of compound sr - 12 ( also referred to as 3c ) or 0 . 2 ml saline iv each day for the first 5 days after elastase infusion . mice were sacrificed at 14 days . aortic diameters ( ad ) prior to and immediately after elastase infusion , as well as at 14 days are reported . the effectiveness of glycodendrimer sr - 12 in limiting aaa formation and growth is determined . the frequency , size , and growth rate of aortic aneurysms is determined in both elastase infusion and angiotensin ii ( ag ii ) murine models of aaa formation . immunohistochemical studies are performed to characterize cellular and structural changes and mmp - 2 and mmp - 9 expression is examined by rt - pcr and gel zymography . the capacity of sr - 12 to limit chemokine binding to surface bound heparan sulfate and abrogate chemokine specific cell activation is defined . the ability of sr - 12 to bind rantes , mip - 1alpha , mcp - 1 , and sdf - 1 and limit their interaction with surface bound heparan sulfate is determined by a surface plasmon resonance binding assay . both kinetic rate constants and equilibrium constants can be characterized . additionally , the ability of sr - 12 to abrogate chemokine mediated cell activation is studied . the ability is assessed of a carrier compound , sodium n -[ 8 -( 2 - hydroxybenzoyl ) amino ] caprylate ( snac ), to facilitate effective biodistribution of sr - 12 when both are administered orally . effective plasma levels and the circulatory half - life of sr - 12 are measured in mice after oral delivery either alone or in association with a carrier compound . measured plasma levels and half - lives are compared to those achieved by intravenous delivery of the glycopolymer . all references throughout this application , for example patent documents including issued or granted patents or equivalents ; patent application publications ; unpublished patent applications ; and non - patent literature documents or other source material ; are hereby incorporated by reference herein in their entireties , as though individually incorporated by reference , to the extent each reference is at least partially not inconsistent with the disclosure in this application ( for example , a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference ). any appendix or appendices hereto are incorporated by reference as part of the specification and / or drawings . where the terms “ comprise ”, “ comprises ”, “ comprised ”, or “ comprising ” are used herein , they are to be interpreted as specifying the presence of the stated features , integers , steps , or components referred to , but not to preclude the presence or addition of one or more other feature , integer , step , component , or group thereof . separate embodiments of the invention are also intended to be encompassed wherein the terms “ comprising ” or “ comprise ( s )” or “ comprised ” are optionally replaced with the terms , analogous in grammar , e . g . ; “ consisting / consist ( s )” or “ consisting essentially of / consist ( s ) essentially of ” to thereby describe further embodiments that are not necessarily coextensive . the invention has been described with reference to various specific and preferred embodiments and techniques . however , it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention . it will be apparent to one of ordinary skill in the art that compositions , methods , devices , device elements , materials , procedures and techniques other than those specifically described herein can be applied to the practice of the invention as broadly disclosed herein without resort to undue experimentation . all art - known functional equivalents of compositions , methods , devices , device elements , materials , procedures and techniques described herein are intended to be encompassed by this invention . whenever a range is disclosed , all subranges and individual values are intended to be encompassed as if separately set forth . this invention is not to be limited by the embodiments disclosed , including any shown in the drawings or exemplified in the specification , which are given by way of example or illustration and not of limitation . the scope of the invention shall be limited only by the claims . rele s m et al ., j am chem soc . 2005 jul . 27 ; 127 ( 29 ): 10132 - 3 . dendrimer - like peo glycopolymers exhibit anti - inflammatory properties . feng x — s et al ., j am chem soc . 2005 ; 127 : 10956 - 10966 . toward an easy access to dendrimer - like poly ( ethylene oxide ) s . rele s m , iyer s s , baskaran s , chaikof e l ; j org chem . 2004 dec . 24 ; 69 ( 26 ): 9159 - 70 . design and synthesis of dimeric heparinoid mimetics . gillies e r , dy e , frechet j m , szoka f c . biological evaluation of polyester dendrimer : poly ( ethylene oxide ) “ bow - 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