Patent Application: US-58447004-A

Abstract:
method of treating conditions associated with raised activity of core 2 glcnac - t by administering an inhibitor of udp - glcnac : galβ1 , 3galnac - r β1 , 6 - n - acetylglucosaminyl transferase . diseases associated with raised activity of core 2 glcnac - t include inflammatory diseases , atherosclerosis , diabetic cardiomyopathy , cancers , including treatment or prevention of metastasis , or diabetic retinopathy .

Description:
compounds of the formula i can be extracted from a variety of plant species . reference is made in this respect , and by way of example only , to yoshikawa et al ( 55 ), sasheda et al ( 59 ), akhov et al ( 60 ), joshi and dev ( 61 ), ravikumar et al ( 56 ), vasil &# 39 ; eva and paseshnichenko ( 62 ), shimomura et al ( 57 ), sharma and sharma ( 63 ), petit et al ( 64 ), mimaki and sashida ( 58 ), and hostettman ( 65 ) and references therein ). these documents are all incorporated herein by reference . alternatively , they can be synthesised by conventional organic chemistry methods and techniques . reference in this respect is made to carbohydrate and steroid chemistry textbooks such as “ essentials of carbohydrate chemistry and biochemistry ” by thisbe k . lindhorst ( 2000 ) wiley , “ carbohydrates in chemistry and biology ” edited by beat ernst , gerald w . hart and pierre sinay ( 2000 ) wiley , “ essentials of carbohydrate chemistry ” by john f . robyt ( 1998 ) springer verlag , “ carbohydrate chemistry ” by hassan s . el khadem ( 1988 ), “ carbohydrate building blocks ” by mikael bols ( 1996 ), “ glycochemistry : principles , synthesis , and applications ” edited by p . g . wang and c . r . bertozzi ( 2001 ) marcel dekker , n . y . and “ carbohydrate chemistry ” by the royal society of chemistry staff ( 1989 ) crc press . the compounds of the present invention can be prepared from commercially available aglycones or by isolation of the aglycone or other precursor either from fenugreek seeds or from another plant source and subsequent chemical modification of the precursor . the skilled worker will for example be aware of many sources of spirostanol and furostanol aglycones such as diosgenin , yamogenin , tigogenin , neotigogenin , sarsapogenin , smilagenin , hecogenin , solasodine or tomatidine ( for example hostettman and references therein ( 65 )), specifically for methods of synthesis of spirostanol saponins having 2 , 4 branched oligosaccharide moieties , from diosgenin see du et al 2003 ( 73 ). this reference also makes further reference to the synthesis if other glycosylated steroids , for example from cholesterol . the methods disclosed can be used to synthesize compounds in which steroids are chemically glycosylated to form compounds of the formula i . further reference is made to li et al ( 66 ) for synthesis of a trisaccharide substituted spirostanol saponins , deng et al ( 67 ), for synthesis of a variety of tri and tetra saccharide substituted spirostanol saponins , li et al ( 68 ), yu et al ( 69 ), yu et al ( 70 ) for methods of synthesis of furostanol saponins and interconversion of spirostanol and furostanol saponins , yu and tao ( 71 ), cheng et al ( 72 ) and du et al ( 73 ). these references also provide information and further references on derivatisation of monosaccharide hydroxyalkyl groups . methods of synthesising galβ1 - 3 ( 6deoxy ) galnacα - conjugates are given in paulsen et al ( 48 ). these methods may be adapted by the skilled worker in combination with other methods referenced herein to synthesize other compounds of the formula i . bovine retinal capillary endothelial cells ( brec ) and pericytes ( brp ) were established from bovine retinas dissected from eyes of freshly slaughtered cattle as described previously ( 48 ). briefly , the isolated retinas were homogenised in serum - free minimal essential medium ( mem , gibco , paisley , uk ) and filtered through 85 μm nylon mesh . the trapped microvessels were digested with collagenase - dispase ( 1 mg / ml ) for 30 minutes ( brp ) and 90 minutes ( brec ) at 37 ° c . and filtered through a 53 μm nylon mesh . for growth of endothelial cells ( brec ), the digested microvessels were plated in gelatine coated tissue culture flasks and maintained in mem supplemented with 10 % pooled human serum , 2 mm glutamine , 100 iu / ml penicillin and 100 μg / ml streptomycin . for growth of pericytes ( brp ), the microvessels were plated in tissue culture flasks in growth medium supplemented with 10 % foetal calf serum . the cells were used at passage 2 - 3 . the cells were characterised using morphological criteria and by immunostaining with an antibody against factor viii related antigen and 3g5 - pericyte marker . the human leukocytic cell - line ( u937 ) was cultured in rpmi supplemented with 10 % foetal calf serum , 2 mm glutamine , 100 iu / ml penicillin and 100 μg / ml streptomycin . to investigate the potential of fenugreek to pharmacologically inhibit core 2 glcnac - t , enzyme activity was measured in leukocytes exposed to normal glucose ( 5 . 8 mm ) and high glucose ( 15 mm ) for 24 hours at 37 ° c . after incubation , the cells were lysed and frozen at − 20 ° c . until used for the measurement of core 2 glcnac - t . the activity of core 2 glcnac - t in cultured bovine retinal capillary pericytes ( brp ) and endothelial cells ( brec ) was also measured . core 2 glcnac - t immobilised on sepharose beads were used for this assay . for core 2 glcnac - t immunoprecipitation , as well as for western blots , a polyclonal antibody against core 2 glcnac - t was used . cells were lysed on ice in the following lysis buffer : 20 mm tris - hcl , ph 7 . 4 / 1 % triton x - 100 , 150 mm nacl , 1 mm edta , 1 mm egta , 0 . 2 mm sodium vandate , 1 mm pmsf 1 μg / ml aprotinin , 10 μg / ml leupeptin . the lysate was incubated at 4 ° c . for 20 minutes with constant agitation and insoluble material removed by centrifugation ( 14 , 000 g for 5 minutes at 4 ° c .). the clarified lysate was incubated with staphylococcal protein a - sepharose cl - 4b conjugated primary antibody for 2 hours with constant agitation at 4 ° c . the immunoprecipitates were washed with tris buffered saline ( 10 mm tris - hcl , ph 7 . 4 , 150 mm nacl ) containing 0 . 5 % triton x - 100 and used in the measurement of core 2 glcnac - t in the presence and absence of potential inhibitors . to measure core 2 glcnac - t activity , leukocytes were washed in pes , frozen and lysed in 0 . 9 % triton x - 100 at 0 ° c . the activity of core 2 glcnac - t was measured as described previously ( 41 ). briefly , the reaction was performed in a reaction mixture containing 50 mm 2 ( n - morpholino ) ethanesulfonic acid ( mes , sigma , dorset , uk ), ph 7 . 0 , 1 mm udp - 6 [&# 39 ; h ]- n - acetylglucosamine ( 16 , 000 dpm / nmol , nen life science products , hounslow , uk ), 0 . 1 m glcnac ( sigma , dorset , okla . ), 1 mm galβ1 - 3galnacα - p - nitrophenol ( sigma , dorset , uk ) as substrate , and 16 μl of cell lysate ( 100 - 200 μg protein ) for a final volume of 32 μl . after incubating the mixture for 1 hour at 37 ° c ., the reaction was terminated with 1 ml of ice - cold distilled water and processed on a c18 sep - pak column ( watersmillipore , watford , uk ). after washing the column with 20 ml of distilled water , the product was eluted with 5 ml of methanol . the radioactivity of the samples was counted in a liquid scintillation β - counter ( lkb - wallac , london , uk ). endogenous activity of core 2 glcnac - t was measured in the absence of the added acceptor . the specific activity was expressed as pmoles / h / mg of cell protein . in each case , the protein concentration was determined with biorad protein assay ( biorad , hertfordshire , uk ). adhesion of leukocytes to endothelial cells was examined by labelling with carboxyfluorescein ( molecular probe , uk ). the assay is well established ( 41 ). briefly , endothelial cells were grown to a confluent state in order to provide an endothelial cell surface for the adhesion of the carboxyfluorescein - labelled leukocytes ( u937 ). after treatment , the leukocytes were centrifuged ( 14 000 g for 1 minute ) and washed twice with serum - free rpml the cells were then resuspended in 1 ml of serum - free rpmi containing 50 μg / ml carboxyfluorescein . the cells were counted with a haemocytometer and a known number added to the endothelial cells . after 30 minutes incubation at 37 ° c ., non - adherent leukocytes were removed by washing with serum - free rpmi and the dishes fixed in 3 . 7 % formalin in pbs . attached leukocytes were counted in 10 random high - powered fields (× 100 ) by fluorescence microscopy . the results were expressed as percentage of adherent leukocytes / field . brp and brec were plated in 3 cm tissue culture dishes and incubated in growth medium for 24 hours at 37 ° c . then the cells were incubated in fresh growth medium containing normal glucose ( 5 . 8 mm ) or elevated glucose ( 25 mm ) in the absence or presence of fenugreek sub - fractions . after 4 days incubation , the number of viable cells was counted using a haemocytometer and trypan blue and the results expressed as percentage of control ( 5 . 8 mm glucose ). after treatment , some of the cells were stored for measurement of core 2 glcnac - t activity . as shown in fig2 a , 24 hour exposure to elevated d - glucose significantly increases the activity of core 2 glcnac - t in human leukocytes ( u937 ). it has now been found that crude extract prepared from fenugreek seeds has the potential to inhibit glucose - induced activity of core 2 glcnac - t in human leukocytes ( fig2 b ) and leukocyte - endothelial cell adhesion ( fig2 c ). leukocyte - endothelial cell adhesion was measured by adding a known number of leukocytes stained with carboxyfluorescein to a monolayer of retinal capillary endothelial cells . the number of attached leukocytes was then counted under a fluorescence microscope using 10 - random fields . the results illustrated in fig3 were obtained by exposing human leukocytes ( u937 ) to elevated glucose for 24 hours . the cells were then lysed , incubated with crude fenugreek seed extract f1 and core 2 glcnac - t activity was measured after 30 minutes incubation . fenugreek seed extracts were obtained as follows ( see fig4 ). fenugreek seeds ( indian fenugreek seeds obtained as methi seeds from fudco , 184 ealing road , wembley , middlesex , uk ) were ground in a hammer mill and filtered through nylon mesh . 820 g of the dark - yellow powder obtained were defatted by continuous washing with hexane in a soxhlet apparatus for eight hours . then the plant material was dried and continuously extracted for 8 hours with ethanol . filtration to remove solid residues and concentration in vacuo of the ethanol yielded a semi - solid brown crude extract labelled f1 ( 65 g ). since this appeared to contain residual oil , 50 g of the crude extract f1 were shaken with cold hexane ( 500 ml ). the hexane soluble material was filtered off and the solvent removed to give f3 ( 15 . 4 g ), while the insoluble residue was collected on the filter paper and dried to give f2 ( 27 g ). normal phase silica - gel flash chromatography was now employed using a commercial kit ( biotage ). f2 ( 5 g ) was adsorbed onto silica - gel ( 5 g ) and packed into the sample barrel that was connected by short tubing to the main chromatography column ( 20 cm × 4 cm ) containing silica - gel kp - sil . the sample was eluted onto and through the column with a succession of solvents of increasing polarity consisting of varying mixtures of light petroleum ( 40 / 60 ), chloroform , methanol and acetone . eluting sub - fractions were examined by tlc and similar ones pooled to give seven main eluted sub - fractions f8 to f14 representing compounds of increasing polarity . the silica was removed and shaken with 100 % methanol , filtered and dried to give a residue labelled f15 . weights and approximate elution solvents for each sub - fraction are set out in table 2 . the potential of these purified sub - fractions to inhibit glucose - induced activity of core 2 glcnac - t in leukocytes was examined . firstly , it was demonstrated that sub - fraction f2 can inhibit glucose - induced core 2 glcnac - t activity in leukocytes ( fig5 ). further experiments demonstrated the presence of the inhibitor of core 2 glcnac - t in sub fractions f13 and f14 ( fig6 a and 6 b ). sub - fractions f9 and f13 were then analysed . an aqueous aliquot ( 0 . 5 ml ) of both subfractions f9 and f13 was extracted with 1 ml of dichloromethane , the aqueous phase was removed , filter - sterilised by filtration through 0 . 22 μm filter and used in the cell - based assay for core 2 glcnac - t activity . human leukocytes were exposed to elevated d - glucose ( 15 mm ) in the presence and absence of the aqueous phases of sub - fractions f9 and f13 , the results are presented in fig7 showing the presence of the core 2 glcnac - t inhibitor in the aqueous phase of sub - fraction f13 . the aqueous phase of sub - fraction f13 was purified by hplc into sub - fractions f18 . 7 - f41 . 1 coded by their hplc retention times . the aqueous phase of sub - fraction f13 was directly injected onto the hplc operating under reversed - phase conditions ( hewlett packard 1050 /\ 100 series ), separation was achieved with an octadecyl - bonded column with a methanol / water mobile phase , components eluted from the column were detected by a uv detector operating at a fixed wavelength of 22 nm , these components were revealed as peaks on the chromatographic trace from the mass spectrometer detector . the sub - fractions thus obtained were concentrated in vacuo to dryness , re - dissolved in phosphate buffered saline ( pbs ) and filter - sterilised . cell - based assays for core 2 glcnac - t activity were carried out and the results suggested the presence of core 2 glcnac - t inhibitor in sub - fractions f19 - f20 . 03 ( see fig8 and 9 ). subsequently larger amounts of the aqueous phase of sub - fraction f13 were purified similarly by hplc operating under reversed - phase conditions on a phenylbonded column with a methanol / water mobile phase into sub - fractions with retention times of 20 . 01 , 20 . 29 and 20 . 55 , which are equivalent to sub - fractions f19 . 13 , f19 . 37 and f19 . 44 above . cell based assays for core 2 glcnac - t activity confirmed the presence of the core 2 glcnac - t inhibitor in these sub - fractions f20 . 01 , f20 . 29 and f20 . 55 ( fig1 a ). the inhibition of core 2 glcnac - t by hplc purified sub - fraction f20 . 55 has been demonstrated using the cell - free assay system ( fig1 ). after exposing human leukocytes ( u937 ) to 15 mm glucose for 24 hours at 37 ° c ., the cells were lysed and then exposed to heated ( h , 100 ° c .) and non - heated ( nh ) sub - fraction f20 . 55 ( 1 : 500 dilution ). after 30 minutes exposure at 37 ° c ., the activity of core 2 glcnac - t was measured . as shown in fig1 , it was found that sub - fraction f20 . 55 directly inhibits core 2 glcnac - t in a cell - free assay . heating of sub - fraction f20 . 55 only slightly altered the level of core 2 glcnac - t inhibition . the core 2 glcnac - t inhibitor in the sub - fraction f20 . 55 has been identified through nmr analysis of a sample dissolved in cd 3 od . the following nmr experiments were performed : 1d proton , 2d dqf - cosy ( 1 h - 1 h correlation ) [ 8 hours ], 2d edited hsqc ( 1 h - 13 c one - bond correlation with multiplicity editing ) [ 22 hours ], 2d tocsy ( 1 h - 1 h relayed correlation ) [ 2 × 8 hours ]. 1 h and 13 c nmr data for the core 2 glcnac - t inhibitor in sub - fraction f20 . 55 is presented in tables 3 and 4 . crushed seeds ( 360 g , product of deep foods , inc ., union , n . j . 07083 , usa ) were extracted successively with heptane ( 2 × 700 ml ), acetone ( 4 × 600 ml ) and meoh ( 4 × 600 ml ) by boiling under reflux for 2 hrs each . the extracts were filtered and evaporated to dryness under vacuum and analyzed by lc / ms for the presence of furostanol saponins previously reported from this plant ( 55 , 74 , 75 ). the methanol extract ( 82 g , 22 . 7 % ( w / w ) of the seeds ) was found to contain the target compounds . the initial extraction of the seeds with heptane and acetone removed most of the less polar materials and improved subsequent chromatography . further de - fatting can be accomplished by partitioning the methanol extract between butanol and water . however , methanol extract contained relatively little polar material and an enriched saponin containing fraction can be obtained by a solid phase extraction using a styrenic resin such as diaion hp20 ( or sp207 , hp20ss , sp207ss , all available from sigma - aldrich ) resin without subjecting the extract to further de - fatting . the meoh extract ( cdxa - 13 - 132 - 1 , 81 . 2 g ) was dissolved in water - meoh ( 6 : 4 , 400 ml ) and loaded onto a diaion hp20 ( supelco diaion bp 20 , 350 g , 5 . 0 × 30 cm ) and eluted with water - meoh ( 4 : 6 , 600 ml ), meoh ( 2 l ), and acetone ( 2 l ). 250 ml fractions were collected . the fractions were analyzed by hplc and those with similar compositions were combined to produce 7 pools ( cdxa - 13 - 133 f1 to f7 ). the pool cdxa - 13 - 133 - f5 ( 22 . 5 g , 27 . 7 % w / w of the extract ) was found to contain the majority of the desired saponins . this pool ( 22 . 0 g ,) was chromatographed on normal phase silica ( 445 g , merck silica gel 60 , 70 - 230 mesh , 0 . 0763 to 0 . 200 mm , 5 . 0 × 30 cm ) and eluted with 3 l each of dichloromethane - meoh - water systems of following compositions : a ) 80 : 20 : 3 , b ) 75 : 25 : 3 , c ) 70 : 30 : 3 , and d ) 65 : 35 : 3 . 250 ml fractions were collected , analyzed by hplc and combined into 11 pools ( cdxa - 13 - 137 - f1 to f11 ). the fractions f6 and f7 were combined , dried ( 10 . 0 g , 45 %) and chromatographed on c8 silica ( 350 g , phenomenex luna c8 ( 2 ), 5 micron , 100 a , 5 . 0 × 28 cm ) and eluted with meoh - water systems of following compositions : 4 : 6 ( 800 ml ), b ) 5 : 5 ( 2 l ), c ) 55 : 45 ( 5 l ) 6 : 4 ( 1 l ), d ) 65 : 35 ( 1 l ), e ) 7 : 3 ( 1 l ), f ) 8 : 2 ( 1 l ) and meoh ( 1 l ). the fractions were analyzed by hplc and combined to give 29 pools ( cdxa - 13 - 138 - f1 to f29 ). 250 ml fractions were collected . fractions f13 to f16 were dried ( 1 . 155 g , 11 . 6 %) and purified by reverse phase hplc using a gilson semi preparative hplc system consisting of a uv / vis detector model 155 , pump model 321 , and liquid handler model 215 . five peaks were collected , p1 to p5 , ( fig . ** 1 to 5 ) and were identified by comparison of 1 h , 13 c nmr and mass spectral data with those reported in the literature for trigoneoside iva , its 25 ( s ) isomer — glycoside f . a further similar compound , compound 3 was detected . this compound has not been previously described . nmr spectra were recorded in d 5 pyridine . the proton spectra were recorded on a varian inova vxrs - 300 instrument at 300 mhz and the carbon spectra were recoded on a varian inova 400 instrument at 100 mhz . mass spectra were recorded on a finnigan lcq deca instrument in apci mode . peak 1 , trigoneoside iva : white solid ( 90 mg , 0 . 025 % w / w of the seeds ). 1 h nmr ( pyridine - d5 , 400 mhz , δ ): 0 . 90 ( 3h , s , 18 - h 3 ), 1 . 04 ( 3h , d , j = 6 . 8 hz , 27 - h 3 ), 1 . 07 ( 3h , s , 19 - h 3 ), 1 . 34 ( 3h , d , j = 6 . 8 hz , 21 - h 3 ), 1 . 79 ( 3h , s , j = 6 . 0 hz , rha - 6 ″- h 3 ), 3 . 88 ( 1h , m , 3 - h ), 4 . 09 ( 2h , m , 16 - h 2 ), 4 . 84 ( 1h , d , j = 7 . 6 hz , glc - 1 ′″- h ), 4 . 97 ( 1h , overlapped , glc - 1 ′- h ), 5 . 16 ( 1h , d , j = 7 . 6 hz , glc - 1 ′″- h ), 5 . 29 ( 1h , d like , 6 - h ), 6 . 29 ( 1h , br s , rha - 1 ″- h ). peak 2 , compound c / protodioscin : white solid ( 120 mg , 0 . 033 %). 1 h nmr ( pyridine - d5 , 400 mhz , δ ): 0 . 90 ( 3h , s , 18 - h 3 ), 1 . 04 ( 3h , d , j = 6 . 8 hz , 27 - h 3 ), 1 . 07 ( 3h , s , 19 - h 3 ), 1 . 34 ( 3h , d , j = 6 . 8 hz , 21 - h 3 ), 1 . 66 ( 3h , s , j = 6 . 0 hz , rha - 6 ′″- h 3 ), 1 . 79 ( 3h , s , j = 6 . 0 hz , rha - 6 ″- h 3 ), 3 . 88 ( 1h , m , 3 - h ), 4 . 09 ( 2h , m , 16 - h 2 ), 4 . 84 ( 1h , d , j = 8 . 0 hz , glc - 1 ′″- h ), 4 . 97 ( 1h , overlapped , glc - 1 ′- h ), 5 . 90 ( 1h , br s , rha - 1 ′″- h ), 5 . 32 1h , d like , 6 - h ), 6 . 45 ( 1h , br s , rha - 1 ″- h ). peak 3 , compound 3 : white solid ( 30 mg , 0 . 008 %). 1 h nmr ( pyridine - d5 , 400 mhz , δ ): 0 . 89 ( 3h , s , 18 - h 3 ), 1 . 06 ( 3h , s , 19 - h 3 ), 1 . 34 ( 3h , d , j = 6 . 4 hz , 21 - h 3 ), 1 . 66 ( 3h , s , j = 6 . 0 hz , rha - 6 ′″- h 3 ), 1 . 79 ( 3h , s , j = 6 . 0 hz , rha - 6 ″- h 3 ), 3 . 88 ( 1h , m , 3 - h ), 4 . 84 ( 1h , d , j = 8 . 0 hz , glc - 1 ′″- h ), 4 . 97 ( 1h , overlapped , glc - 1 ′- h ), 5 . 32 1h , d like , 6 - h ), 5 . 90 ( 1h , br s , rha - 1 ′″- h ), 6 . 45 ( 1h , br s , rha - 1 ″- h ). peak 4 , glycoside f : white solid ( 120 mg , 0 . 033 %). 1 h nmr ( pyridine - d5 , 400 mhz , δ ): 0 . 90 ( 3h , s , 18 - h 3 ), 1 . 00 ( 3h , d , j = 6 . 4 hz , 27 - h 3 ), 1 . 06 ( 3h , s , 19 - h 3 ), 1 . 35 ( 3h , d , j = 6 . 4 hz , 21 - h 3 ), 1 . 79 ( 3h , s , j = 6 . 0 hz , rha - 6 - h 3 ), 3 . 88 ( 1h , m , 3 - h ), 3 . 97 ( 2h , m , 16 - h 2 ), 4 . 84 ( 1h , d , j = 7 . 6 hz , glc - 1 ′″- h ), 4 . 97 ( 1h , overlapped , glc - 1 ′- h ), 5 . 16 ( 1h , d , j = 7 . 6 hz , glc - 1 ′″- h ), 5 . 29 ( 1h , d like , 6 - h ), 6 . 29 ( 1h , br s , rha - 1 ″- h ). shatavarin iv ( fig1 ) isolated from asparagus racemosus ( 56 ) and protodioscin from tribulus terrestris ( but also isolatable from fenugreek as compound c of ( 55 )) were both supplied by chromadex inc . 2952 s . daimler st . santa ana calif . protodioscin was also isolated from the above preparation of fenugreek as peak 2 conforming to published nmr spectra of protodioscin biological activity of trigoneoside iva , glycoside f , protodioscin and shatavarin iv heart lysate from bb rats were incubated in the presence , and absence of 20 ng / ml of each compound . after 1 h incubation at 37 ° c ., the activity of core 2 glcnac - t was measured , and expressed as pmoles / h / mg protein . the results are the mean of 3 - 5 separate experiments . the results are shown in fig1 a trigoneoside iva , its 25 ( r ) isomer glycoside f and shatavarin iv are highly active inhibitors of core 2 glcnac - t in cell free assays , whilst protodioscin , in which the glucose at the 4 position is replaced by rhamnose , is not active . human leukocytes ( u937 cells ) were exposed to 8 pg / ml human recombinant tnf - alpha in the presence and absence of 20 ng / ml of the test compound . after 24 h incubation , the activity of core 2 glcnac - t was measured , and expressed as pmoles / h / mg protein . the results are shown in fig1 b . trigoneoside iva , and glycoside f are highly active inhibitors of core 2 glcnac - t in cell free assays , whilst protodioscin is not active . it has been found that elevated glucose levels significantly increase the activity of core 2 glcnac - t in cultured bovine retinal vascular cells , namely capillary pericytes ( brp ) and capillary endothelial cells ( brec ) ( fig1 ). near confluent cultures were exposed to normal glucose ( n , 5 . 8 mm ) and high glucose ( g , 15 mm ) for 24 hours at 37 ° c . the cells were lysed and the activity of core glcnac - t measured in cell lysates . it has further been demonstrated that fenugreek seed extract has the potential to reverse glucose - induced toxicity ( fig1 ) in cultured bovine retinal capillary pericytes ( brp ) and endothelial cells ( brec ). cells were exposed to normal ( n , 5 . 8 mm ) and high 15 glucose ( g , 25 mm ) in the presence ( n - f , g - f ) and absence ( n , g ) of the fenugreek seed extract . after 4 days incubation , the number of viable cells was determined using a haemocytometer and trypan blue exclusion . it was found that fenugreek seed extract indeed reverses glucose - induced toxicity in cultured bovine retinal capillary pericytes and endothelial cells . however , it is not established yet whether fenugreek seed extract reverses glucose - induced toxicity by normalising the activity of core 2 glcnac - t . this protection of retinal vascular cells fenugreek seed extract is significant , because damage to retinal vascular cells is a hallmark of early diabetic retinopathy . diabetic retinopathy in humans is mainly a vascular disease , primarily affecting the capillaries . the first ultrastructural and microscopic changes reported are retinal capillary basement membrane thickening and pericyte degeneration , both of which compromise the integrity of the capillary wall . pericyte degeneration leaves lightly stained compartments in the basement membrane sheath called pericyte “ ghosts ”. damage to both pericytes and endothelial cells leads to the formation of acellular capillaries . medicaments comprising the compounds of the formula i described herein can be administered by oral or parenteral routes , including intravenous , intramuscular , intraperitoneal , subcutaneous , transdermal , airway ( aerosol ), rectal , vaginal and topical ( including buccal and sublingual ) administration . for oral administration , the compounds of the invention will generally be provided in the form of tablets or capsules , as a powder or granules , or as an aqueous solution or suspension . tablets for oral use may include the active ingredients mixed with pharmaceutically acceptable excipients such as inert diluents , disintegrating agents , binding agents , lubricating agents , sweetening agents , flavouring agents , colouring agents and preservatives . suitable inert diluents include sodium and calcium carbonate , sodium and calcium phosphate , and lactose , while corn starch and alginic acid are suitable disintegrating agents . binding agents may include starch and gelatine , while the lubricating agent , if present , may be magnesium stearate , stearic acid or talc . if desired , the tablets may be coated with a material , such as glyceryl mono stearate or glyceryl distearate , to delay absorption in the gastrointestinal tract . capsules for oral use include hard gelatine capsules in which the active ingredient is mixed with a solid diluent , and soft gelatine capsules wherein the active ingredients is mixed with water or an oil such as peanut oil , liquid paraffin or olive oil formulations for rectal administration may be presented as a suppository with a suitable base comprising , for example , cocoa butter or a salicylate . formulations suitable for vaginal administration may be presented as pessaries , tampons , creams , gels , pastes , foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate . for intramuscular , intraperitoneal , subcutaneous and intravenous use , the compounds of the invention will generally be provided in sterile aqueous solutions or suspensions , buffered to an appropriate ph and isotonicity . suitable aqueous vehicles include ringer &# 39 ; s solution and isotonic sodium chloride . aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives , sodium alginate , polyvinylpyrrolidone and gum tragacanth , and a wetting agent such as lecithin . suitable preservatives for aqueous suspensions include ethyl and n - propyl p - hydroxybenzoate . the fenugreek seed extracts and core 2 glcnac - t inhibitors of the present invention may also be presented as liposome formulations . in general a suitable dose will be in the range of 0 . 01 to 10 mg per kilogram body weight of the recipient per day of the core 2 glcnac - t inhibitor , preferably in the range of 0 . 2 to 1 . 0 mg per kilogram body weight per day . the desired dose is preferably presented once daily , but may be dosed as two , three , four , five , six or more sub - doses administered at appropriate intervals throughout the day . these sub - doses may be administered in unit dosage forms , for example , containing 10 to 1500 mg , preferably 20 to 1000 mg , and most preferably 50 to 700 mg of active ingredient per unit dosage form . 1 . colley k . j ., “ golgi localization of glycosyltransferases : more question than answers ”, glycobiology 7 , 1 - 13 ( 1997 ) 2 . varki a ., “ biological roles of oligosaccharides : all of the theories are correct ”, glycobiology 3 , 97 - 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