Patent Application: US-201314405575-A

Abstract:
the present invention relates to an allosteric non - inhibitory chaperone of the lysosomal acid alpha - glucosidase for use in the treatment of a pathological condition characterized by a deficiency of the lysosomal acid alpha - glucosidase , to pharmaceutical composition thereof , to a method for increasing the activity of gaa in a subject and to a method for identifying an allosteric non - inhibitory chaperone for gaa .

Description:
fibroblasts from pd and fabry disease patients were derived from skin biopsies after obtaining the informed consent of patients . normal age - matched control fibroblasts were available in the laboratory of the department of pediatrics , federico ii university of naples . all cell lines were grown at 37 ° c . with 5 % co2 in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( invitrogen , grand island , n . y .) and 10 % fetal bovine serum ( sigma - aldrich , st louis , mo . ), supplemented with 100 u / ml penicillin and 100 mg / ml streptomycin . rhgaa ( alglucosidase , myozyme ) and rh - alpha - gal a ( agalsidase - beta , fabrazyme ) were from genzyme co , cambridge , mass ., usa . enzymes were prepared and diluted according to manufacturer instructions to nac , nas , nag , cys , ser , gly , 2 - mercaptoethanol , 4 - nitrophenyl - α - glucopyranoside ( 4np - glc ) nb - dnj and dgj were from sigma - aldrich . epigallo catechingallate ( cat . no . 93894 ) and resveratrol ( cat . no . 34092 ) were purchased from sigma - aldrich . the rabbit anti gaa primary antibody used for immunofluorescence and western blot analysis was purchased from abnova , heidelberg , germany ; the anti - beta - actin mouse monoclonal antibody was from sigma - aldrich . anti - rabbit and anti - mouse secondary antibodies conjugated to alexa fluor 488 or 596 were from molecular probes , eugene , oreg . ; hrp - conjugated anti - rabbit or anti - mouse igg were from amersham , freiburg , germany . labeling of rhgaa was performed using the alexa fluor 546 labeling kit ( molecular probes ) according to the manufacturer instructions . thermal stability scans of rhgaa were performed as described in [ flanagan et al . 2009 ]. briefly , 2 . 5 μg of enzyme were incubated in the absence and in the presence of nac and dnj , 10 mm and 0 . 1 mm , respectively , sypro orange dye , and sodium phosphate 25 mm buffer , 150 mm nacl , ph 7 . 4 or sodium acetate 25 mm buffer , 150 mm nacl , ph 5 . 2 . thermal stability scans were performed at 1 ° c ./ min in the range 25 - 95 ° c . in a real time lightcycler ( bio - rad ). sypro orange fluorescence was normalized to maximum fluorescence value within each scan to obtain relative fluorescence . melting temperatures were calculated according to ( niesen et al . 2007 ). the standard activity assay of rhgaa was performed in 200 μl by using 5 μg of enzyme at 37 ° c . in 100 mm sodium acetate ph 4 . 0 and 20 mm 4np - glc . the reaction was started by adding the enzyme ; after suitable incubation time ( 1 - 2 min ) the reaction was blocked by adding 800 μl of 1 m sodium carbonate ph 10 . 2 . absorbance was measured at 420 nm at room temperature , the extinction coefficient to calculate enzymatic units was 17 . 2 mm − 1 cm − 1 . one enzymatic unit is defined as the amount of enzyme catalyzing the conversion of 1 μmol substrate into product in 1 min , under the indicated conditions . the effect of different phs on the rhgaa stability was measured by preparing reaction mixtures containing 0 . 75 mg ml − 1 of enzyme in the presence of 50 mm citrate / phosphate ( ph 3 . 0 - 7 . 0 ) at a certain ph . after incubations at 37 ° c ., aliquots were withdrawn at the times indicated and the residual α - glucosidase activity was measured with the standard assay . to test the effect on the ph stability of rhgaa of chemical chaperons and of the other molecules , experiments were performed as described above by adding to the reaction mixtures the amounts of the different compounds indicated in the text . to study the rhgaa uptake and correction of gaa activity in pd fibroblasts , the cells were incubated with 50 micromol / 1 rhgaa for 24 hours , in the absence or in the presence of 10 mm nac . untreated cells or were used for comparison . after the incubation the cells were harvested by trypsinization and disrupted by 5 cycles of freezing and thawing . gaa activity was assayed by using the fluorogenic substrate 4 - methylumbelliferyl - alpha - d - glucopyranoside ( sigma - aldrich ) according to a published procedure [ porto et al , 2009 ]. briefly , 25 micrograms of protein were incubated with the fluorogenic substrate ( 2 mm ) in 0 . 2 m acetate buffer , ph 4 . 0 , for 60 minutes in incubation mixtures of 100 μl . the reaction was stopped by adding 700 μl of glycine - carbonate buffer , ph 10 . 7 . fluorescence was read at 365 nm ( excitation ) and 450 nm ( emission ) on a turner biosystems modulus fluorometer . protein concentration in cell homogenates was measured by the bradford assay ( biorad , hercules , calif .). to study gaa immunoreactive material , fibroblast extracts were subjected to western blot analysis . the cells were harvested , washed in phosphate - buffered saline , resuspended in water , and disrupted by five cycles of freeze - thawing . equal amounts ( 20 μg protein ) of fibroblast extracts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and proteins were transferred to pvd membrane ( millipore , billerica , mass .). an anti - human gaa antiserum was used as primary antibody to detect gaa polypeptides ; to detect β - actin , a monoclonal mouse antibody was used immunoreactive proteins were detected by chemiluminescence ( ecl , amersham , freiburg , germany ) to study the distribution of alexafluor546 labeleld gaa , pd fibroblasts grown on coverslips were fixed using methanol , permeabilized using 0 . 1 % saponin and locked with 0 . 01 % saponin , 1 % fetal bovine serum diluted in phosphate - buffered saline for 1 hour . the cells were incubated with the primary antibodies , with secondary antibodies in blocking solution and then mounted with vectashield mounting medium ( vector laboratories , burlingame , calif .). samples were examined with a zeiss lsm 5 10 laser scanning confocal microscope . authors used argon / 2 ( 458 , 477 , 488 , and 514 nanometers ) and hene1 ( 543 nanometers ) excitation lasers , which were switched - on separately to reduce crosstalk of the two fluorochromes . the green and the red emissions were separated by a dichroic splitter ( ft 560 ) and filtered ( 515 - 540 - nm bandpass filter for green and & gt ; 610 - nm long pass filter for red emission ). a threshold was applied to the images to exclude ˜ 99 % of the signal found in control images . animal studies were performed according to the european union directive 86 / 609 , regarding the protection of animals used for experimental purposes . the animals , mice model of pd [ raben et al , 1993 ], were allowed to drink 138 mm nac in water ad libitum ( 4 . 2 g / kg / day ) for 5 days . on day 3 they received a single rhgaa injection ( 100 mg / kg ) in the tail vein . on day 5 the animals were sacrificed and tissues were analyzed for gaa activity . previous studies have shown that changes of the physical environment , such as modifications of temperature and ph , induce perturbations in the conformation of lysosomal enzymes and affect their stability [ liebermann et al , 2007 ; shen et al , 2008 ]. resistance of wild - type enzymes to these physical stresses is commonly taken as an indicator to monitor the efficacy of pharmacological chaperones [ valenzano et al , 2011 ]. the effect of ph on rhgaa stability is shown in fig1 . at ph 5 . 0 rhgaa was stable for up to 24 hours . at non - lysosomal ph , either acidic ( 3 . 0 ) or neutral ( 7 . 0 , representative of non - lysosomal cellular compartments ), the enzyme was unstable and rapidly lost its activity with approximately 50 % residual activity after 4 hours and near complete inactivation ( less than 10 % residual activity ) after 16 hours . the stability of rhgaa at neutral ph was rescued by co - incubation with nac ( fig2 a ). the stabilizing effect of nac on rhgaa was dose dependent ( fig2 b ), and maintained even after 48 h of incubation ( fig2 c ). nac increased also the rhgaa thermal stability : at 10 mm concentration the melting temperature ( tm ) of rhgaa increased by 10 . 5 ± 0 . 5 ° c . ( tm 60 . 7 ± 0 . 5 ° c . vs 50 . 2 ± 0 . 1 ° c .) ( fig2 d ). to test whether the stabilizing effect of nac resulted from the sulfidryl group , the related amino acids n - acetyl serine ( nas ) and n - acetyl glycine ( nag ) were also tested . both compounds behaved as nac by inducing remarkable stabilization of rhgaa at ph 7 . 0 ( fig3 a and 3b ) with no effect on the activity of the enzyme ( fig4 ). thus , these molecules , binding gaa at an allosteric sites that is different than the protein &# 39 ; s active site , belong to a new class of allosteric non - inhibitory chaperones . the non - acetylated homologs cys , ser , and gly and 2 - mercapto - ethanol , a structurally unrelated compound containing an sh group , did not prevent enzyme inactivation ( fig3 c - f ). these data indicate that the stabilizing effect was due to the presence of the acetyl group rather than to the sulfidryl group . thus , the — sh group may be substituted by different functionalities without abrogating the binding of the small - molecule to the enzyme . nac rescues mutated gaa in pd fibroblasts and transfected cos cells authors investigated the effect of nac in cultured fibroblasts from five pd patients ( pt ) carrying different mutations and with different phenotypes ( see table ii ). in these studies authors focused on nac as this molecule is already approved for clinical use and thus has a greater potential for clinical translation compared to nas and nag . * the genotype of patients was obtained as a routine diagnostic procedure to confirm the diagnosis of pompe disease . patients or their legal guardians provided their informed consent for the molecular analysis of the gaa gene . pt 2 on one allele has a deletion from amino acid residue 612 ( histidine ) to 616 ( aspartate ) and insertion of rgi ( arginine - glycine - isoleucine ); on the 5 second allele mutation arginine3751eucine . pt 4 : three mutations splicing c - 32 - 13t & gt ; g and p . s619n ( in cis ); on the second allele p . l5552p ** activity measured in fibroblasts and expressed as nmoles of 4 - methylumbelliferyl - alpha - d - glucopyranoside ( 4mu ) liberated / mg protein / hr ( control values 58 . 5 ± 28 . 1 nmoles 4mu / mg protein / hr ). nac enhanced the residual activity of mutated gaa in fibroblasts from patients 3 and 4 ( fig5 a ). patient 3 had the mutation l552p in association with a mutation causing aberrant splicing . patient 4 carried three mutations ( two , c .- 32 - 13t and p . s619n , on one allele , and the p . l552p mutation on the other allele ). of these mutations the p . l552p , has been previously reported to be responsive to dnj [ parenti et al , 2007 ; flanagan et al , 2009 ]. the response of individual mutations to nac was further evaluated by expressing a panel of mutated gaa gene constructs in cost cells , according to the methods reported in previous studies [ parenti et al , 2007 ; flanagan et a , 2009 ] ( fig5 b ). the mutated constructs were chosen to be representative of both imino sugar - responsive and non - responsive mutations , in order to compare the chaperoning profile of nac with that of imino sugars . the cells were transfected with the mutated constructs , incubated either in the presence or in the absence of 10 mm nac and harvested 72 hours after transfection . the mutations p . l552p , p . a445p , and p . y455f showed significant ( p & lt ; 0 . 01 and p & lt ; 0 . 05 for l552p and for a445p and y455f , respectively ), enhancement of gaa activity in the presence of nac . the increase in activity of the mutation pg377r was not statistically significant . the enhancement of enzyme activity in responsive mutations paralleled the increase in either 76 kda or in 70 kda active isoforms of gaa on western blot analysis . fig5 c shows western blots of cost cells over - expressing two of the responsive ( p . l552p , p . a445p ). the result of western blot analysis of a non - responsive ( p . g549r ) mutation is shown for comparison . for this latter mutation no change was seen in the amounts of the gaa active isoforms , already detectable in the absence of nac ( as previously reported in flanagan et al , 2009 ). these results suggest that nac has a different chaperoning profile compared to the active site - directed chaperones dgj and nb - dnj ( fig5 d ). authors have previously shown that chaperones enhance the efficacy of wild - type recombinant enzymes in pd and fabry disease [ porto et al , 2009 ; porto et al , 2011 ]. in pd fibroblasts the imino sugar nb - dnj enhanced rhgaa efficacy by approximately 1 . 3 to 2 - fold . this effect is of great interest as a possible strategy to improve ert efficacy in pd , and possibly in other lsds . authors tested whether the allosteric chaperone nac also show the same synergy . in fibroblasts from patient 3 , co - administration of rhgaa and nac ( 0 . 02 - 10 mm ) resulted in improved gaa activity with a dose - dependent effect ( fig6 a ). authors incubated five pd fibroblast cell lines with 50 microm rhgaa in the absence and presence of nac . the efficacy of rhgaa in correcting the enzymatic deficiency varied among the different cell lines , as already reported in previous papers [ cardone et al , 2008 ; porto et al , 2009 ], possibly due to individual factors implicated in the uptake and intracellular trafficking of the recombinant enzyme in each cell line . however , in all cell lines tested , co - incubation of rhgaa with nac resulted in an improved correction of gaa deficiency , with increases in gaa activity ranging from approximately 3 . 7 to 13 . 0 - fold with respect to the activity of cells treated with rhgaa alone ( fig6 b ). the enhancing effect largely exceeded that due to the rescue of the native mutated enzyme ( patients 3 and 4 ) and was observed also in non - responsive cell lines ( patients 2 and 5 ). authors also observed an increase in the amount of fluorochrome - labelled gaa in the presence of the chaperone nac , compared to cells incubated with fluorescent gaa alone ( fig6 c ). by this approach only the fluorescent exogenous enzyme is detectable and variations in the intensity of fluorescence reflect only the effects on the recombinant enzyme . the combination of these results supports the concept that the enhancing effect of chaperones is directed towards the wild - type recombinant enzyme and is consistent with the data previously reported with the chaperone nb - dnj [ porto et al , 2009 ]. a western blot analysis ( fig6 d ) and the quantitative analysis of each band ( fig6 e ) showed increased amounts of gaa - related polypeptides in the cells treated with nac and rhgaa , compared to cells treated with the recombinant enzyme alone . the processing of rhgaa ( that is provided in commercial preparations for clinical use as 110 kda precursor ) into the mature active isoforms was also improved in the presence of nac . the analysis of the gaa band density showed a relative increase of the intermediate ( 95 kda ) and mature ( 76 - 70 kda ) gaa molecular forms in the presence of nac , compared to the 110 kda precursor . since the gaa precursor is converted into the active forms in the late - endosomal / lysosomal compartment [ wisselaar et al , 1993 ], this indicates improved lysosomal trafficking of the enzyme . other anti - oxidant drugs ( resveratrol , epigallo chatechingallate ) did not enhance rhgaa in pd cultured fibroblasts ( fig7 ). these results , together with the analysis of nac - gaa interaction and with the data in cell - free systems , exclude that the effect of nac is due to its anti - oxidant properties . authors also tested the combination of nac and rhgaa in a mouse model of pd [ raben et al , 1993 ]. mice were treated with a single injection of rhgaa at high doses ( 100 mg / kg ) in combination with oral nac for 5 days ( fig8 a ). mice treated with the recombinant enzyme alone were used as controls . forty - eight hours after rhgaa injection the animals were euthanized and gaa activity was assayed in different tissues . albeit not statistically significant , in all tissues examined ( liver , heart , diaphragm and gastrocnemium ) the combination of nac and rhgaa was superior to rhgaa alone in correcting enzyme activity ( fig8 b ). comparison of nac with the imino sugar chaperone nb - dnj , and specificity of nac &# 39 ; s effect to compare the effect of nac on thermal denaturation of rhgaa to that of the imino sugar dnj , authors performed thermal stability scans of rhgaa in the absence and in the presence of the two chaperones . both chaperones increased rhgaa thermal stability , with dnj causing the best shift in tm ( 65 . 9 ± 0 . 3 ° c .). this result is apparently in contrast with the data shown in fig7 , indicating that nac is superior to imino sugars in enhancing the efficacy of rhgaa in pd fibroblasts . the discrepancy of these results , however , may be explained by the lack of inhibitory effect of nac on the recombinant enzyme in cells . a corollary of the fact that nac and imino sugar chaperones interact with different protein domains , is that their effect may be cumulated . this might represent an additional advantage for the treatment of patients , in order to obtain the best stabilization of rhgaa and the highest synergy with ert . this hypothesis was supported by the results of thermal denaturation of rhgaa , showing the highest stability of the enzyme with the combination of nac and dnj ( tm = 75 . 9 ± 0 . 3 ) ( fig9 a ), and by studies in two pd cell lines ( from patients 2 and 4 ). these cells were incubated with rhgaa , with rhgaa plus either nac or nb - dnj , and with rhgaa plus the combination of the two chaperones . in both cell lines the combination of nac and nb - dnj resulted in the highest enhancement of gaa activity by rhgaa ( fig9 b ). an important concern on the use of pharmacological chaperones is the specificity of their effects and the possibility of interactions with other enzymes . gaa belongs to family gh31 of glycoside hydrolases , interestingly , this family was included in the gh - d superfamily of glycoside hydrolases together with families gh36 and gh27 [ ernst et al . 2006 ]. the latter family includes lysosomal alpha - galactosidase a ( alpha - gal a ), that is defective in another lsd , fabry disease [ germain , 2010 ]. two preparations of recombinant human alpha - gal a ( rh - alpha - gal a ) have been approved for ert in fabry disease patients . to test if nac is active on this enzyme , authors incubated rh - alpha - gal a and 10 mm nac in 50 mm sodium citrate / phosphate buffer , ph 7 . 0 . nac had no effect on rh - alpha - gal a after 48 h ( fig1 a ). in addition , when authors incubated three fabry disease cell lines with rh - alpha - gal a , in the absence and in the presence of nac , and in the presence of the known chaperone dgj ( fig1 b ), nac had no enhancing effect on the correction of alpha - deficiency by rh - alpha - gal a . as expected and as shown in a previous study where the same cell lines were used [ porto et al , 2011 ] dgj largely enhanced the effects of rh - alpha - gal a . therapeutic strategies directed towards the rescue of defective mutant enzymes are an attractive alternative to therapies based on the supply of wild - type enzyme , such as ert , gene therapy and transplantation of hematopoietic stem cells . the rescue of the mutant enzyme may be obtained by various approaches . one is aimed at adjusting with small - molecule drugs the capacity of the cellular networks controlling protein synthesis , folding , trafficking , aggregation , and degradation , thus facilitating the exit of mutated proteins from the endoplasmic reticulum [ mu et al , 2008 ; powers et al , 2009 ; ong and kelly , 2011 ; wang et al , 2011 ]. alternatively , small - molecule drugs , so called pharmacological chaperones , may act directly on the defective enzymes , favoring the most stable conformation ( s ) of these proteins , and preventing their recognition and disposal by the endoplasmic reticulum associated quality control and degradation systems . albeit attractive and rapidly evolving towards clinical translation , some aspects of the biochemistry of pct are incompletely understood or require optimization . an important issue in this respect is the potential inhibition of target enzymes . according to a recent review all chaperones proposed or used for the treatment of lsds are reversible competitive inhibitors of the target enzymes , and may in principle interfere with the activity of these enzymes [ valenzano et al , 2011 ]. thus , treatment protocols based on the pulsed administration of chaperones ( that have a short plasma half - life ) to rescue mutant enzymes ( that in general have a longer half - life ) have been so far developed and tested in lsds mouse models [ khanna et al , 2010 ; benjamin et al , 2012 ]. another limitation of chaperones is that they are effective in rescuing only some disease - causing missense mutations , mainly located in specific enzyme domains , and are thus potentially effective only in a limited number of patients . for pd , it is possible to speculate that about 10 - 15 % patients may be amenable to pct with the imino sugar dnj [ flanagan et al , 2009 ]. these problems can be addressed by the identification of novel and allosteric non - inhibitory chaperones . in this study , authors have shown that nac and the related compounds nas and nag , have these features , being able to stabilize gaa without interfering with its activity and having a different chaperoning profile , compared to known chaperones . nac is a known anti - oxidant that was evaluated in the authors &# 39 ; laboratory , together with other related drugs ( resveratrol , epigallo catechingallate ) in pd fibroblasts for possible effects on rhgaa intracellular trafficking . the characterization of nac &# 39 ; s mechanism of action on rhgaa , however , indicated that molecular interactions with the enzyme , rather than the anti - oxidant effect , were responsible for rhgaa stabilization and that the other anti - oxidants studied did not stabilize the enzyme . this was somewhat surprising because nac is structurally very different from the imino sugars , the only known pharmacological chaperones of gaa so far , that resemble the natural substrates / products of the enzyme . authors showed that nac improved stability of gaa in response to physical stresses . for instance , increased resistance to ph variations is particularly interesting . compared to methods based on temperature denaturation , which are often used as a measure of the effects of chaperones , neutral ph may be more representative of some of the environmental conditions encountered by recombinant enzymes in plasma and in certain cellular compartments . it has been shown that ph induces conformational changes in lysosomal enzymes . this has been studied in detail for gba [ lieberman et al , 2007 ; lieberman et al , 2009 ]. gba stability and conformation were analyzed in neutral and in acidic ph environments , and in complex with the pharmacological chaperone ifg . changes in ph resulted in different conformations of the enzyme , with small but critical differences in two loops localized at the mouth of active site . ifg binding favored the most stable conformations of the enzyme [ lieberman et al , 2007 ]. in cell - free assays nac prevented the loss of gaa activity as a function of ph and increased the enzyme thermal stability . in cost cells overexpressing mutated gaa incubation with nac resulted in increased residual gaa activity for four of the seven mutations studied . remarkably , the chaperoning profile of nac showed differences compared to that of nb - dnj and dgj . the mutation p . a445p , previously reported as non - responsive to imino sugar chaperones , appeared to be responsive to nac . this may translate into an expansion of the number of chaperone - responsive mutations , and should be further investigated in large - scale studies , like that performed in 76 different variants of the gaa gene [ flanagan et al , 2009 ]. it may be envisaged that preliminary screenings in vitro on a number of chaperones would allow personalization of treatment protocols aimed at obtaining the greatest beneficial effect in different pd patients . in these regards , the identification of nac and derivatives , which are structurally very different from the other known pharmacological chaperones identified in pd is quite promising . in fact , other molecules , whose chaperoning activity cannot be simply inferred from their structure , may be effective in several lsd , thereby opening new and wider opportunities for the identification of novel therapeutic drugs . nac also increased the efficacy of recombinant gaa , in particular rhgaa , in correcting the enzyme defect in pd fibroblasts . compared to the effect of nac , and of chaperones in general , on the mutated enzymes , this effect holds greater promise for the cure of patients affected by pd , and possibly of other lsds . it should be considered that , while the enhancement of endogenous defective enzymes by chaperones in most cases resulted in minor changes in terms of residual activity ( likely with a modest impact on patients &# 39 ; outcome ), the synergy of these drugs with ert caused ( at least in cellular systems ) remarkable increases of specific activity . in this study co - administration of nac and recombinant gaa , in particular rhgaa , resulted in complete correction of the enzymatic defect . a synergy between chaperones and ert has already been described using known chaperones in pd and fabry disease [ shen et al , 2008 ; porto et al , 2009 ; porto et al , 2011 ; benjamin et al , 2012 ]. the molecular bases of this synergy , however , are still incompletely understood . the enhancing effect of chaperones on ert may imply that a substantial fraction of the recombinant enzymes , during their traffic to lysosomes , is prone to degradation and is not able to reach its final destination . for the recombinant human gba , used for ert in gaucher disease , it was suggested that inability to recover most of the infused recombinant enzyme in the target tissues was due to losses occurring during transit to the lysosome [ xu et al , 1996 ; shen et al , 2008 ]. it has also been speculated that factors related to purification steps , body temperature and the neutral ph of blood , may result in stress for the enzyme during its transit through the circulation and tissue fluids , and lead to greater susceptibility to the action of proteases or denaturation . in a recent study on the co - administration of rh - alpha - gal a in the murine model of fabry diseases it has been shown that incubation of the recombinant enzyme in blood results in decreased stability [ benjamin et al , 2012 ]. in principle , the enhancing effect of chaperones on recombinant enzymes may be due to stabilization of the enzyme in the cell medium , to improved uptake by the cells , or to stabilization of the enzyme intracellularly , either through the endocytic pathway or within the lysosomal compartment . the present results showing an enhancing effect of nac on the mutant enzyme in cultured fibroblasts and in cost cells over - expressing mutated enzymes would favor the hypothesis that , at least in part , the stabilization occurs intracellularly . the present results support a synergy between chaperones and recombinant enzymes and have important clinical implications and may translate into improved clinical efficacy of ert , as shown in in vivo experiments in pd mice . ernst h a , et al . j mol biol 2006 may 12 ; 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