Patent Application: US-30439502-A

Abstract:
method for enhancing in a mammalian cell the activity of an enzyme associated with gaucher disease by administering a competitive inhibitor of glucocerebrosidase in an amount effective to enhance the activity of the enzyme . preferred compounds for use in the method are imino sugars and related compounds . in particular , c8 - 12 - alkyl derivatives of n - alkyl - deoxynojirimycin , isofagomine compounds , and calystegine compoiunds are effective to enhance glucocerebrosidase activity .

Description:
in the work leading to the present application , the inventors tested a series of naturally occurring and chemically synthesized novel inhibitors for both in vitro inhibition of normal α - gal a and intracellular enhancement of a mutant α - gal a activity with fabry lymphoblasts to demonstrate that potent competitive reversible inhibitors of α - gal a are effective “ chemical chaperones ” which can stabilize the mutant enzyme and rescue it from degradation . applicants now have tested the chemical chaperone strategy with gaucher disease and g m1 - gangliosidosis , both of which belong to the lysosomal storage disorder family ( 26 - 27 ), to demonstrate that this therapeutic strategy of using potent competitive inhibitors as chemical chaperones to enhance the residual enzyme activity in the patient &# 39 ; s cells is not limited to fabry disease , and can be applied to gaucher disease and g m1 - gangliosidosis as examples of the principle which can be extended to to other lysosomal storage disorders as listed in table 1 . the structures of the inhibitors used in this invention are shown in fig1 . 1 - deoxynojirimycin ( dnj ) ( 1 ) was isolated from the roots of morus alba ( moraceae ) as described previously ( 28 ). 1 - deoxymannojirimycin ( manno - dnj ) ( 2 ) and 1 - deoxy - 3 , 4 - diepi - nojirimycin ( gulo - dnj ) ( 5 ) have been recently isolated from the barks of angylocalyx pynaertii ( leguminosae ). 1 - deoxy - 3 - epi - nojirimycin ( allo - dnj ) ( 3 ) was prepared by the microbial redox reaction at c - 3 of the n - benzyloxycarbonyl derivative of dnj as described previously ( 29 ) 1 - deoxygalactonojirimycin ( dgj ) ( 4 ) and 1 2 - dideoxy - galactonojirimycin ( 6 ) were prepared by the chemical epimerization of the 4 - oh group of 1 and fagomine , respectively , according to the literature procedure ( 30 ). α - homonojirimycin ( α - hnj ) ( 7 ), α - homomannojirimycin ( α - manno - hnj ) ( 8 ), and α - homoallonojirimycin ( α - allo - hnj ) ( 9 ) were isolated from the whole plant of aglaonema treubii ( araceae ) as reported previously ( 31 ). α - homogalactonojirimycin ( α - galacto - hnj ) ( 10 ) was prepared from 2 , 3 , 4 , 6 - tetra - o - benzyl - d - galactose by way of a wittig chain extension and a mercuricyclization according to the literature procedure ( 33 ). n - methyl - dgj ( 11 ) was prepared by treatment of 4 with 37 % hcho and 80 % formic acid according to the reference ( 34 ), and the n - ethyl ( 12 ), n - propyl ( 13 ), n - butyl ( 14 ) and n - hydroxyethyl ( 15 ) derivatives of 4 were prepared by treatment with the appropriate alkyl bromide and triethylamine in dmf . the reaction mixture of n - alkylation was evaporated in vacuo , and the residual syrup was resolved in meoh and applied to an amberlyst 15 column ( h + form ), washed with meoh , eluted with 0 . 5 m nh 4 oh , and concentrated . n - alkylated derivatives were finally purified by dowex 1 × 2 ( oh − form ) and amberlite cg - 50 ( n 4 + form ) chromatography with water as eluent . β - 1 - c - butyl - dgj ( 16 ) was isolated from adenophorae radix as described previously ( 35 ). 4 - epi - isofagomine ( 21 ), isofagomine ( 33 ) and its derivatives ( 34 - 37 ) were chemically synthesized as described previously ( 40 ). 2 , 5 - dideoxy - 2 , 5 - imino - d - mannitol ( dmdp , 27 ), 1 , 4 - dideoxy - 1 , 4 - imino - d - arabinitol ( dab , 28 ) were purified from derris malaccensis and morus alba , respectively ( 29 ). n - butyl - dnj ( 30 ), n - dodecyl - dnj ( 31 ), castanospermine ( 29 ) were from commercial sources . calystegine a 3 ( 17 ), calystegine a 5 ( 22 ), calystegine b 1 ( 23 ), calystegine b 2 ( 18 ), calystegine b 3 ( 24 ), calystegine b 4 ( 25 ), and calystegine c 1 ( 26 ) were prepared as published method ( 36 , 40a ), n - methyl calystegine a 3 ( 19 ) and n - methyl calystegine b 2 ( 20 ) were chemically synthesized . the structural characterization of the inhibitors is determined by mass spectrometry and 13 c - nmr , and some of the results are presented below . 1 - deoxynojirimycin ( dnj ) ( 1 ) hrfabms m / z 164 . 0923 [ m + h ] + ( c 6 h 14 no 4 requires 164 . 0923 ). 13 c - nmr ( 100 mhz , d 2 o ) δ ( ppm downfield from internal sodium 3 -( trimethylsilyl ) propionate ) 51 . 5 ( c - 1 ), 63 . 3 ( c - 5 ), 64 . 2 ( c - 6 ), 73 . 7 ( c - 2 ), 74 . 3 ( c - 4 ), 81 . 2 ( c - 3 ). 1 - deoxymannojirimycin ( manno - dnj ) ( 2 ) hrfabms m / z 164 . 0923 [ m + h ] + ( c 6 h 14 no 4 requires 164 . 0923 ). 13 c - nmr ( 100 mhz , d 2 o ) δ51 . 5 ( c - 1 ), 63 . 4 ( c - 5 ), 63 . 7 ( c - 6 ), 71 . 3 ( c - 4 ), 72 . 1 ( c - 2 ), 77 . 5 ( c - 3 ). 1 - deoxy - 3 - epi - nojirimycin ( allo - dnj ) ( 3 ) hrfabms m / z 164 . 0922 [ m + h ] + ( c 6 h 14 no 4 requires 164 . 0923 ). 13 c - nmr ( 100 mhz , d 2 o ) δ46 . 9 ( c - 1 ), 59 . 2 ( c - 5 ), 60 . 7 ( c - 6 ), 72 . 7 ( c - 2 ), 73 . 3 ( c - 4 ), 75 . 0 ( c - 3 ). 1 - deoxygalactonojirimycin ( dgj ) ( 4 ) hrfabms m / z 164 . 0921 [ m + h ] + ( c 6 h 14 no 4 requires 164 . 0923 ). 13 c - nmr ( 100 mhz , d 2 o ) δ51 . 9 ( c - 1 ), 61 . 7 ( c - 5 ), 64 . 2 ( c - 6 ), 70 . 9 ( c - 2 ), 72 . 1 ( c - 4 ), 77 . 9 ( c - 3 ). 1 - deoxy - 3 , 4 - diepi - nojirimycin ( gulo - dnj ) ( 5 ) hrfabms m / z 164 . 0921 [ m + h ] + ( c 6 h 14 no 4 requires 164 . 0923 ). 13 c - nmr ( 100 mhz , d 2 o ) δ46 . 9 ( c - 1 ), 56 . 9 ( c - 5 ), 63 . 8 ( c - 6 ), 68 . 3 ( c - 2 ), 72 . 0 ( c - 4 ), 73 . 0 ( c - 3 ). 1 , 2 - dideoxygalactonojirimycin ( 6 ) hrfabms m / z 148 . 0972 [ m + h ] + ( c 6 h 14 no 3 requires 148 . 0974 ). 13 c - nmr ( 100 mhz , d 2 o ) δ30 . 2 ( c - 2 ), 45 . 6 ( c - 1 ), 61 . 8 ( c - 5 ), 64 . 5 ( c - 6 ), 70 . 5 ( c - 4 ), 72 . 7 ( c - 3 ). α - homonojirimycin ( α - hnj ) ( 7 ) hrfabms m / z 194 . 1025 [ m + h ] + ( c 7 h 16 no 5 requires 194 . 1028 ). 13 c - nmr ( 100 mhz , d 2 o ) δ56 . 9 ( c - 5 ), 59 . 1 ( c - 1 ′), 59 . 7 ( c - 1 ), 64 . 8 ( c - 6 ), 74 . 4 ( c - 2 ), 74 . 9 ( c - 4 ), 77 . 1 ( c - 3 ). α - homomannojirimycin ( α - manno - hnj ) ( 8 ) hrfabms mn / z 194 . 1026 [ m + h ] + ( c 7 h 16 no 5 requires 194 . 1028 ). 13 c - nmr ( 100 mhz , d 2 o ) δ58 . 6 ( c - 5 ), 61 . 4 ( c - 1 ), 62 . 2 ( c - 1 ′), 63 . 9 ( c - 6 ), 71 . 4 ( c - 4 ), 71 . 6 ( c - 2 ), 74 . 7 ( c - 3 ). α - homoallonojirimycin ( α - allo - hnj ) ( 9 ) hrfabms m / z 194 . 1024 [ m + h ] + ( c 7 h 16 no 5 requires 194 . 1028 ). 13 c - nmr ( 100 mhz , d 2 o ) δ57 . 2 ( c - 5 ), 58 . 1 ( c - 1 ), 62 . 7 ( c - 1 ′), 63 . 5 ( c - 6 ), 72 . 0 ( c - 4 ), 72 . 1 ( c - 3 ), 72 . 2 ( c - 2 ). α - homogalactonojirimycin ( α - galacto - hnj ) ( 10 ) hrfabms m / z 194 . 1028 [ m + h ] + ( c 7 h 16 no 5 requires 194 . 1028 ). 13 c - nmr ( 100 mhz , d 2 o ) δ55 . 8 ( c - 5 ), 59 . 3 ( c - 1 ), 59 . 6 ( c - 1 ′), 64 . 5 ( c - 6 ), 71 . 8 ( c - 2 ), 71 . 9 ( c - 4 ), 73 . 8 ( c - 3 ). n - methyl - 1 - deoxygalactonojirimycin ( n - me - dgj ) ( 11 ) hrfabms m / z 178 . 1081 [ m + h ] + ( c 7 h 16 no 4 requires 178 . 1079 ). 13 c - nmr ( 100 mhz , d 2 o ) δ44 . 2 ( n - ch 3 ), 62 . 9 ( c - 1 ), 63 . 6 ( c - 6 ), 68 . 5 ( c - 2 ), 69 . 7 ( c - 5 ), 73 . 0 ( c - 4 ), 77 . 8 ( c - 3 ). n - ethyl - 1 - deoxygalactonojirimycin ( n - et - dgj ) ( 12 ) hrfabms m / z 192 . 1237 [ m + h ] + ( c 8 h 18 no 4 requires 192 . 1236 ). 13 c - nmr ( 100 mhz , d 2 o ) δ10 . 7 , 48 . 9 ( n - ethyl ), 57 . 8 ( c - 1 ), 63 . 2 ( c - 6 ), 65 . 0 ( c - 5 ), 69 . 9 ( c - 2 ), 73 . 0 ( c - 4 ), 77 . 9 ( c - 3 ). n - propyl - 1 - deoxygalactonojirimycin ( n - pr - dgj ) ( 13 ) hrfabms m / z 206 . 1392 [ m + h ] + ( c 9 h 20 no 4 requires 206 . 1392 ). 13 c - nmr ( 100 mhz , d 2 o ) δ13 . 9 , 19 . 2 , 57 . 2 ( n - propyl ), 58 . 6 ( c - 1 ), 63 . 3 ( c - 6 ), 65 . 5 ( c - 5 ), 69 . 9 ( c - 2 ), 73 . 0 ( c - 4 ), 77 . 9 ( c - 3 ). n - butyl - 1 - deoxygalactonojirimycin ( n - bu - dgj ) ( 14 ) hrfabms m / z 220 . 1546 [ m + h ] + ( c 10 h 22 no 4 requires 220 . 1549 ). 13 c - nmr ( 100 mhz , d 2 o ) δ16 . 1 , 23 . 0 , 27 . 9 , 55 . 0 ( n - butyl ), 58 . 6 ( c - 1 ), 63 . 3 ( c - 6 ), 65 . 5 ( c - 5 ), 69 . 9 ( c - 2 ), 73 . 0 ( c - 4 ), 77 . 9 ( c - 3 ). n - hydroxyethyl - 1 - deoxygalactonojirimycin ( n - he - dgj ) ( 15 ) hrfabms m / z 208 . 1183 [ m + h ] + ( c 8 h 18 no 5 requires 208 . 1185 ). 13 c - nmr ( 100 mhz , d 2 o ) δ56 . 0 ( n — ch 2 —), 59 . 2 ( c - 1 ), 60 . 9 ( n — ch 2 ch 2 oh ), 63 . 7 ( c - 6 ), 66 . 4 ( c - 5 ), 69 . 7 ( c - 2 ), 73 . 3 ( c - 4 ), 77 . 8 ( c - 3 ). β - 1 - c - butyl - deoxygalactonojirimycin ( 16 ) hrfabms m / z 220 . 1543 [ m + h ] + ( c 10 h 22 no 4 requires 220 . 1549 ). 13 c - nmr ( 100 mhz , d 2 o ) δ16 . 1 , 25 . 0 , 29 . 6 , 33 . 5 ( c - butyl ), 61 . 1 ( c - 5 ), 61 . 8 ( c - 1 ), 64 . 2 ( c - 6 ), 71 . 8 ( c - 4 ), 74 . 9 ( c - 2 ), 77 . 9 ( c - 3 ). a was expressed from sf - 9 insect cells infected with a recombinant baculovirus encoding normal α - gal a gene and purified to homogeneity by concanavalin a - sepharose and mono q ( pharmacia lkb biotechnology , uppsala , sweden ) column chromatography according to the published methods ( 37 ). the enzyme activity was assayed with 2 mm p - nitrophenyl - α - d - galactoside as substrate in the presence of bovine serum albumin ( 3 mg / ml ) at ph 4 . 5 . the epstein - barr virus - transformed lymphoblast lines from a normal adult and a fabry patient with r301q mutation in α - gal a ( 38 ) were cultured in rpmi - 1640 medium ( nissui pharmaceutical co ., tokyo , japan ) supplemented with 10 % fetal calf serum ( fcs ) at 37 ° c . under 5 % co 2 . human fibroblasts from gaucher and g m1 - gangliosidosis patients were cultured in mccoy 5a medium supplemented with 10 % fcs at 37 ° c . under 5 % co 2 . cells were cultured in the presence or absence of inhibitor for 4 days . after being washed twice with phosphate - buffered saline ( pbs ), the cells were harvested and homogenized in 200 μl of h 2 o , and 10 μl of the supernatant obtained by centrifugation at 10 , 000 g was incubated at 37 ° c . with 50 μl of the substrate solution composed by 6 mm 4 - methylumbelliferyl α - d - galactoside ( 4 - mu - α - gal ) and 90 mm n - acetylgalactosamine in 0 . 1 m citrate buffer ( ph 4 . 5 ) for enzyme assay . one unit of intracellular enzyme activity was defined as one nmol of 4 - methylumbelliferone released per hour at 37 ° c . cells were cultured in the presence or absence of inhibitor for 5 days . after being washed twice with pbs , the cells were harvested and homogenized in 200 μl of h 2 o , and 10 μl of the supernatant obtained by centrifugation at 10 , 000 g was incubated at 37 ° c . with 50 μl of the substrate solution of 1 mm 4 - methylumbelliferyl β - d - galactoside ( 4 - mu - β - gal ) in 0 . 1 m citrate buffer ( ph 4 . 5 ) for enzyme assay . one unit of intracellular enzyme activity was defined as one nmol of 4 - methylumbelliferone released per hour at 37 ° c . cells were cultured in the presence or absence of inhibitor for 5 days . after being washed twice with pbs , the cells were harvested and homogenized in 200 μl of buffer i composed by 0 . 25 % sodium taurocholate , 0 . 1 % triton x - 100 and 0 . 1 m citrate buffer ( ph 5 . 2 ). the supernatant ( 10 μl ) obtained by centrifugation at 10 , 000 g was incubated at 37 ° c . with 50 μl of the substrate solution of 3 mm 4 - methylumbelliferyl β - d - glucoside ( 4 - mu - β - glu ) in the buffer i for determination of total β - glucosidase activity . the neutral β - glucosidase activity was determined by performing the same assay except pre - incubation of the enzyme solution with 3 mm conduritol b epoxide ( an irreversible inhibitor of acid β - glu ) at room temperature for 30 min . the glucocerebrosidase activity was determined by subtracting the neutral β - glucosidase activity from the total enzyme activity . one unit of intracellular enzyme activity was defined as one nmol of 4 - methylumbelliferone released per hour at 37 ° c . in vitro inhibition and intracellular enhancement of α - gal a in fabry lymphoblasts . the summary of ic 50 and selected k i values of dgj and its derivatives are shown in table 2 . dgj ( galacto - dnj ) was synthesized from d - glucose and found to be an extremely powerful inhibitor of coffee bean ( α - galactosidase ( 39 ). in the development of the present invention , both ic 50 and k i values of dgj toward human lysosomal α - gal a were calculated to be 0 . 04 μm ( table 2 , fig3 a ). dnj ( 1 ) was a weak inhibitor of this enzyme with an ic 50 value of 830 μm , while other isomers such as manno - ( 2 ), allo - ( 3 ), and gulo - dnj ( 5 ) showed no appreciable inhibition even at 1000 μm . the deoxygenation at c - 2 of dgj ( 6 ) reduced its inhibitory potential over 6000 - fold . these results suggested to applicants that a galactosyl configuration of an imino sugar is preferable for the inhibition of α - gal a . α - hnj ( 7 ) was not an inhibitor of α - gal a , but α - manno - hnj ( 8 ) was a weak inhibitor of the enzyme . ( α - galacto - hnj ( 10 ) mimicking α - d - galactopyranose was first expected to be a more specific and potent inhibitor of α - gal a than dgj . from 1 h - nmr studies , the 3 j h , h - coupling constants ( j 2 , 3 = 9 . 8 hz , j 3 , 4 = 3 . 0 hz , j 4 , 5 = 2 . 6 hz ,) observed for α - galacto - hnj ( 10 ) clearly showed that this compound is predominantly in a chair conformation which maintained the ground - state structure of the substrate . however , insertion of a hydroxymethyl group to the α - anomeric position of dgj decreased the affinity for α - gal a by approximately 4 - fold . surprisingly , α - allo - hnj ( 9 ) showed a fairly potent inhibitory activity toward α - gal a , with an ic 50 value of 4 . 3 μm . from its structure , this compound could form two different conformations as shown in fig2 α - allo - hnj ( fig2 a ) vs . c - 2 epimer of ( α - galacto - hnj ( fig2 b ). the j h , h - coupling constants in compound 9 ( j 1 , 2 = 4 . 6 hz ; j 2 , 3 = 2 . 9 hz ; j 3 , 4 = 2 . 9 hz ; j 4 , 5 = 6 . 5 hz ) indicated that the conformation deviates from a chair form as a result of the 1 , 3 syn - diaxial interaction between the substituents at c - 2 and c - 4 ( fig2 b ). furthermore , the c - 5 carbon in the 13 c - nmr spectrum of 9 is observed as a broad signal , presumably due to “ wobble ” at c - 5 . the potent inhibitory activity of α - allo - hnj ( 9 ) toward α - gal a may be due to the partial stereochemical and conformational similarities between a flexible α - allo - hnj conformation ( fig2 c ) and a galactosyl cation ( fig2 d ), which has been presumed to be a transition state intermediate in the enzyme - catalyzed galactoside hydrolysis ( 40 ). the n - alkyl derivatives of dgj were studied for α - gal a inhibition because n - alkylation of dnj and α - hnj resulted in analogues with increased potency and substrate specificity on digestive α - glucosidases and processing α - glucosidase i ( 41 - 44 ), and n - alkylation of dnj and dgj increased inhibitory potential toward glucosyltransferase ( 45 , 46 ). however , n - alkylation of dgj markedly lowered its inhibitory activity toward α - gal a ( table 3 ), suggesting that modification of the imino group is not preferred for inhibition of α - gal a . the naturally occurring dgj derivative , β - 1 - c - butyl - dgj , has recently been isolated from adenophorae radix as a potent inhibitor of coffee bean ( α - galactosidase with an ic 50 value of 0 . 71 μm ( 35 ). the ic 50 value for α - gal a was determined to be 24 ∞ m . the inhibition mode of four potent inhibitors of α - gal a , dgj ( 4 ), ( α - galacto - hnj ( 10 ), α - allo - hnj ( 9 ) and β - 1 - c - butyl - dgj ( 16 ) were studied . lineweaver - burk plots indicated that they are competitive inhibitors of α - gal a ( fig3 ). the calculated ki values of dgj , α - galacto - hnj , α - allo - hnj and β - 1 - c - butyl - dgj were found to be 0 . 04 μm , 0 . 25 μm , 2 . 6 μm , and 16 μm , respectively . as shown in fig4 b , those dgj derivatives that showed high inhibitory activity toward α - gal a were tested for enhancement of intracellular α - gal a activity in r301q lymphoblasts . treatment with dgj at 100 μm for 4 days increased enzyme activity in r301q lymphoblasts by about 14 - fold reaching 49 % of normal . enzyme activity was increased 5 . 2 - fold , 2 . 4 - fold , and 2 . 3 - fold by cultivation with α - galacto - hjn , α - allo - hjn and β - 1 - c - butyl - dgj at 100 μm , respectively , while weak inhibitors such as n - alkyl derivatives of dgj showed only a slight enhancement effect at 100 μm . the effectiveness of intracellular enhancement paralleled to the in vitro inhibitory activity ( fig4 a ), indicating that a potent inhibitor serves as an effective enhancer . the enzyme activity in r301q lymphoblasts was elevated with increasing inhibitor concentration in a certain range ( for dgj , 1 - 100 μm , fig5 ). α - galacto - hnj , α - allo - hnj , and β - 1 - c - butyl - dgj enhanced the α - gal a activity by 12 . 5 -, 3 . 9 -, and 6 . 3 - fold at 1000 μm , respectively . however , higher concentrations significantly reduced the enhancement effect , presumably causing inhibition of the enzyme activity . applicants confirmed that inclusion of dgj in the medium at 20 μm did not cause intracellular inhibition of globotriaosylceramide metabolism , indicating that intracellular dgj concentration appeared to be lower than the concentration normally required to inhibit the intracellular enzyme activity at that condition ( 24 ). intralysosomal enzyme activity may not be inhibited by α - galacto - hnj , α - allo - hnj , and β - 1 - c - butyl - dgj added in the culture medium at 1000 μm , because these compounds exhibited weaker inhibitory activity than dgj . although β - 1 - c - butyl - dgj was a less effective inhibitor of α - gal a than α - allo - hnj ( k i = 16 μm vs k i = 2 . 6 μm ), both enhancement effects were the same at 100 μm , and the effect of β - 1 - c - butyl - dgj at 1000 μm was higher than that by α - allo - hnj at the same concentration ( fig5 ). this suggested that the bioavailability of β - 1 - c - butyl - dgj may be better than α - allo - hnj , because increase of lipophilicity resulting from the c - alkylation at c - 1 of dgj may enhance the efficient transport across cell and er membranes . calystegine compounds are polyhydroxylated nortropane alkaloids . certain of these alkaloids exhibit potent inhibitory activities against glycosidases ( 47 ). the enzyme activity in r301q lymphoblasts was also elevated with increasing concentration of calystegine a 3 ( fig6 a ), calystegine b 2 ( fig6 b ), n - methyl - calystegine a 3 ( fig6 c ), and n - methyl - calystegine b 2 ( fig6 d ), respectively in a range of 100 - 1000 μm . the above results further supported applicants &# 39 ; therapeutic concept that potent competitive inhibitors can serve as efficient chemical chaperones to enhance intracellular mutant enzyme activity in cells derived from patients of fabry disease . according to this theoretical concept , more potent inhibitors serve as more powerful chemical chaperones . as shown in the following examples , this therapeutic strategy of using potent competitive inhibitors or substrate analogs is not limited to fabry disease , but also applicable to other lysosomal storage disorders and general hereditary disorders resulted from protein folding defects , such as , but not limited to , α 1 - antitrypsin deficiency , familial hypercholesterolemia , alzheimer &# 39 ; s disease , marfan syndrome , osteogenesis imperfecta , carbohydrate - deficient glycoprotein syndrome , and maroteaux - lamy syndrome . intracellular enhancement of β - galactosidase activity in fibroblasts from g m1 - gangliosidosis patients . g m1 - gangliosidosis is a progressive neurological disease caused by hereditary deficiency of lysosomal acid β - galactosidase ( β - gal ) which hydrolyses the terminal β - galactosidic residual of ganglioside g m1 and other glycoconjugates ( 27 ). three clinical forms are described as infantile type ( severe form ), juvenile type ( sub - severe type ), and adult onset type ( mild type ). no treatment is available for this disorder . applicants applied the strategy of using potent inhibitors as chemical chaperones to enhance intracellular mutant enzyme activity to human g m1 - gangliosidosis fibroblasts . human g m1 - gangliosidosis fibroblasts were cultured for 5 days with dgj ( 4 ) and 4 - epi - isofagomine ( 21 ) ( both are inhibitors of β - gal ) at 500 μm , and 50 μm , respectively ( fig6 ). the enhancement effect was not efficient with the fibroblasts from patients of infant type disease ( bgf - 1 and bgf - 6 ). however , the intracellular enzyme activities in fibroblasts established from patients diagnosed as juvenile and adult types disease were elevated to 9 - 53 % of normal ( fig7 b ). the residual enzyme activity in bgf - 7 was markedly increased 27 - fold by inclusion of compound 4 at 500 μm ( fig7 a ). these results indicate that compound 4 and 21 are powerful chemical chaperones for β - gal , and can he used as potential therapeutic agents for treatment of g m1 - gangliosidosis . gaucher disease is characterized by the accumulation of glucosylceramide ( glucocerebroside ) due to the deficient activity of lysosomal acid β - glucosidase ( glucocerebrosidase , β - glu ) ( 26 ). three types of gaucher disease have been identified : 1 ) type 1 ( adult - onset ), lack of primary central nervous system involvement ; 2 ) type 2 ( infantile - onset ), acute neuronopathic form of the disease with an early onset ; type 3 ( late - infantile / juvenile - onset ), subacute neuronopathic form . enzyme replacement therapy is effective only for type 1 disease . various natural and synthetic compounds were tested with human normal β - glu for inhibitory activity , and the ic 50 values are shown in table 3 . several potent inhibitors were found among calystegine compounds . calystegine b 2 ( 18 ) ( ic 50 value , 0 . 99 μm ), calystegine b 1 ( 23 ) ( 2 . 5 μm ), calystegine c 1 ( 26 ) ( 2 . 5 μm ), and calystegine a 3 ( 17 ) ( 3 . 1 μm ) were the best inhibitors in this class . castanospermine ( 29 ) is a known potent inhibitor for α - glucosidase , however , it also present fair inhibitory activity against β - glu ( 19 μm ). dnj ( 1 ) and n - butyl - dnj ( 30 ) were weak inhibitors for this enzyme , however , n - dodecyl - dnj ( 31 ) turned to be one of the most potent inhibitor with ic 50 at 0 . 05 μm . since dnj and n - butyl - dnj were moderate inhibitors of the enzyme , the high potency of this compound ( 31 ) is believed from the long alkyl chain in the molecular which is probably recognized by the recognition domain normally recognizing the ceramide part of the substrate . isofagomine ( ifg , 33 ) was reported as a potent inhibitor against almond β - galactosidase ( 40 ), and revealed as the most potent inhibitor among those tested with ic 50 value at 0 . 04 μm . modification of the imino group ( compounds 34 - 37 ) of ifg reduced inhibitory activity substantially . this result consistent with applicants &# 39 ; earlier finding with α - gal a in which alkyl modification of dgj nullified its inhibitory activity . noticeably , compound 37 which contains a 12 carbon chain in the backbone increased 30 - fold in its potency compared with compound 32 which contains a 4 carbon chain . combined with the result generated from dnj ( 1 ) and n - dodecyl - dnj ( 31 ), it is expected that n - dodecyl - ifg serves as a powerful inhibitor for human β - glu . in accordance with the invention , these inhibitors should be effective in enhancing activity of the defective enzyme associated with gaucher disease and treatment of the disorder . isofagomine and derivatives ifg ( 33 ) is the most potent inhibitor tested for β - glu in vitro . its intracellular enhancement activity was investigated with fibroblasts established from a gaucher patient with n370s / n370s genotype . the intracellular enzyme activity was increased 55 - 80 % by cultivation the cell with ifg added in the culture medium at 1 - 50 μm ( fig8 a ). higher than 50 μm concentration nullified the enhancement effect . the enhancement effect was monitored for 5 days . the residual enzyme activity in gaucher cells did not change on day 1 or 2 , however , the enzyme activity was elevated alter day 3 and increased more than 80 % at day 5 ( fig8 b ). this data demonstrated that ifg , a potent inhibitor of β - glu , also serves as an enhancer for residual β - glu in the cells derived from gaucher patients when a appropriate concentration is applied . effective concentrations are expected to be lower than those needed to inhibit the enzyme , but will be able to be determined through routine experimentation by those of skill in the art for gaucher disease and other disorders . ifg derivatives ( compounds 34 - 37 ) demonstrated significant impact on enhancement of the intracellular enzyme activity in gaucher cells ( n370s / n370s ) cultivated with these compounds ( fig9 ). the residual enzyme activity was elevated 73 % ( compound concentration at 10 μm ) and 56 % ( 100 μm ) by compound 34 , 106 % ( 10 μm ) and 47 % ( 100 μm ) by compound 35 , and 50 % ( 10 μm ) and 54 % ( 100 μm ) by compound 36 , respectively the residual enzyme activity was increased 43 % by cultivation with compound 37 at 10 μm , however , decreased 53 % with the compound at 100 μm . although the inhibitory activity of the ifg derivatives was weaker than ifg , the intracellular enhancement activity of the ifg derivatives appears to be higher than ifg , since they achieved higher elevation of the mutant enzyme activity at lower concentrations . it is believed that the bioavailability of these compounds is significantly improved by the hydrophobic nature of the molecule , leading to easier crossing of cell and the er membranes , thereby increasing the intracellular concentration of these compounds . particularly , compound 37 at 100 μm decreased the residual activity , presumably intracellular concentration reached to the concentration required for inhibition . n - dodecyl - dnj n - dodecyl - dnj ( 31 ) is one of the most potent inhibitors of β - glu tested , and is believed to be recognized by the domain usually recognizing ceramide of the natural substrate . n - dodecyl - dnj also enhanced β - glu activity in fibroblasts derived from gaucher patient with n370s / n370s mutation . the enzyme activity increased 95 % by cultivation the cells with n - dodecyl - dnj at 0 . 5 μm for 5 days ( fig1 a ). the elevation of enzyme activity was dose - dependent between the concentrations of 0 . 05 - 0 . 5 μm added to the medium . however , n - dodecyl - dnj at higher than 1 μm nullified the enhancement effect . the time course of cultivation of the cells with n - dodecyl - dnj at 0 . 5 μm indicated that the residual enzyme activity increased after day 3 ( fig1 b ). since n - dodecyl - dnj and ifg are recognized by different recognition domains of the enzyme ( n - dodecyl - dnj , ceramide recognition domain vs . ifg , glucoside recognition domain ), a compound with a combination of n - dodecyl - dnj and ifg such as n - dodecyl - ifg is expected to be a powerful agent for enhancing residual enzyme activity in gaucher cells . calystegine compounds calystegine a 3 ( 17 ), calystegine b 1 ( 23 ), calystegine b 2 ( 18 ) and calystegine c 1 ( 26 ) exhibited potent inhibitory activity against β - glu and were tested for intracellular enhancement of β - glu activity with fibroblasts derived from gaucher patient with a genotype of l444p / l444p ( fig1 ). the residual enzyme activity in the patient &# 39 ; s cells was increased 230 %, 76 %, 126 % and 136 % by cultivation with calystegine b 2 , b 1 , a 3 and c 1 at 10 μm , respectively . the results indicate that these compounds also act as effective enhancers for gaucher fibroblasts . applicants have shown that i ) α - allo - hnj ( 9 ), α - hgj ( 10 ), β - 1 - c - butyl - dgj ( 16 ), calystegine a 3 ( 17 ), calystegine b 2 ( 18 ), n - metyl calystegine a 3 ( 19 ), and n - methyl calystegine b 2 ( 20 ) are able to effectively increase the intracellular α - gal a activity in fabry lymphoblasts by cultivation the cells with the above individual compound in concentration ranges of 10 - 1000 μm ; ii ) dgj ( 4 ) and 4 - epi - isofagomine ( 21 ) are able to effectively enhance the intracellular β - gal activity in g m1 - gangliosidosis fibroblasts by cultivation the cells with the above individual compound in concentration ranges of 50 - 500 μm ; iii ) calystegine b 2 ( 18 ), calystegine b 1 ( 23 ), calystegine a 3 ( 17 ), calystegine c 1 ( 26 ), n - dodecyl - dnj ( 31 ), isofagomine ( 33 ), n - butyl - isofagomine ( 34 ), n -( 3 - cyclohexylpropyl )- isofagomine ( 35 ), n -( 3 - phenylpropyl )- isofagomine ( 36 ) and n -[( 2e , 6z , 10z )- 3 , 7 , 11 - trimethyldodecatrienyl ]- isofagomine ( 37 ) are able to effectively enhance the intracellular β - glu activity in gaucher fibroblasts by cultivation the cells with the above individual compound in concentration ranges of 0 . 05 - 100 μm . applicants earlier disclosed in u . s . application ser . no . 09 / 087 , 804 a method for treatment of fabry disease by administration of potent competitive inhibitors of α - gal a to enhance the intracellular α - gal a activity in the fabry lymphoblsts . the mechanism underlying this treatment is believed to be that the competitive inhibitors serve as chemical chaperones to induce / ensure the proper ( native ) conformation of the mutant protein for a smooth escape from the er quality control system , thus accelerate the maturation and transport leading to increase of the intracellular enzyme activity . in the present application , applicants further demonstrate the correlation between in vitro inhibition and intracellular enhancement with a series of inhibitors for α - gal a . the results clearly show that more potent competitive inhibitors serve as more powerful enhancers for the mutant enzyme . in the present application , it is demonstrated that the method for treatment of fabry disease by administration of potent competitive inhibitor of the defective enzyme can be applied to g m1 - gangliosidosis and gaucher disease , both diseases belong to lysosomal storage disorder family , and potent inhibitors for β - gal or β - glu can effectively enhance the intracellular enzyme activities in the fibroblasts established from patients of g m1 - gangliosidosis or gaucher disease , respectively . since the lysosomal storage disorders share the same biological and biochemical pathogenic mechanisms , this method of using potent competitive inhibitors of the defective enzyme is expected to be applicable for the treatment of other lysosomal storage disorders listed in table 1 . recent study on glycosidase inhibitors showed that conventional type of iminosugars having a nitrogen atom replacing the ring oxygen of a sugar such as 1 - deoxynojirimycin are more potent and selective inhibitors for α - glycosidases , whereas 1 - n - iminosugars having a nitrogen atom at the anomeric position of the pyranose ring are more potent and selective for β - glycosidases ( 40 ). we expect that conventional type of iminosugars ( nojirimycin type ), e . g ., 1 - deoxy - nojirimycin , 1 - deoxy - galactonojirimycin , 1 - deoxy - iduronojirimycin , 1 , 2 - dideoxy - 2 - n - acetamido - nojirimycin , 1 - deoxymannonojirimycin , 1 - deoxyfuconojirirmycin , 2 , 6 - dideoxy - 2 , 6 - imino - sialic acid , and 1 , 2 - dideoxy - 2 - n - acetamido - galactonojirimycin ( fig1 ), which have a ground - state structure of the substate of a detective enzymne , e . g ., α - glucosidase , α - galactosidase , α - l - iduronidase , α - n - acetylglucosaminidase , α - mannosidase , α - l - fucosidase , α - n - acetyl - neuraminidase , and α - n - acetylgalactosaminidase , are potent inhibitors and powerful enhancers for treatment of pompe disease , fabry disease , hurler - scheie disease , sanfilippo disease , α - mannosidosis , fucosidosis , sialidosis and schindler - kanzaki disease , respectively . we also expect that 1 - n - iminosugars , e . g ., isofagomine , 4 - epi - isofagomine , 2 - n - acetamido - isofagomine , 6 - carboxy - isofagomine , and 2 - hydroxy - isofagomine ( fig1 ), which have a ground - state structure of the substrate of a defective enzyme , e . g ., β - glucosidase , β - galactosidase , β - n - acetylglucosarninidase , β - glucuronidase , and β - mannosidase , are potent inhibitors and powerful enhancers for treatment of gaucher disease , g m1 - gangliosidosis , krabbe disease , morquio disease , tay - sachs disease , sandohoff disease , sly disease , and β - mannosidosis . a summary of potent competitive inhibitors which are expected to effectively enhance the mutant enzyme activity associated with lysosomal storage disorders is presented in table 4 . 36 . asano n , kato a , matsui k , watson a a , nash r j , molyneux r j , hackett l , topping j , winchester b ( 1997 ) the effects of calystegines isolated from edible fruits and vegetables on mammalian liver glycosidases . glycobiology 7 , 1085 - 8 37 ishii , s ., kase , r ., sakuraba , h ., fujita , s ., sugimoto , m ., tomita , k ., semba , t ., and suzuki , y . 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