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Purpose: Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene. Migalastat, a pharmacological chaperone, binds to specific mutant forms of α-galactosidase A to restore lysosomal activity. Methods: A pharmacogenetic assay was used to identify the α-galactosidase A mutant forms amenable to migalastat. Six hundred Fabry disease–causing mutations were expressed in HEK-293 (HEK) cells; increases in α-galactosidase A activity were measured by a good laboratory practice (GLP)-validated assay (GLP HEK/Migalastat Amenability Assay). The predictive value of the assay was assessed based on pharmacodynamic responses to migalastat in phase II and III clinical studies. Results: Comparison of the GLP HEK assay results in in vivo white blood cell α-galactosidase A responses to migalastat in male patients showed high sensitivity, specificity, and positive and negative predictive values (≥0.875). GLP HEK assay results were also predictive of decreases in kidney globotriaosylceramide in males and plasma globotriaosylsphingosine in males and females. The clinical study subset of amenable mutations (n = 51) was representative of all 268 amenable mutations identified by the GLP HEK assay. Conclusion: The GLP HEK assay is a clinically validated method of identifying male and female Fabry patients for treatment with migalastat. Genet Med 19 4, 430–438.

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the gene that encodes α-galactosidase A and is characterized by pathological accumulation of globotriaosylceramide and globotriaosylsphingosine. Earlier, the authors demonstrated that oral coadministration of the pharmacological chaperone AT1001 (migalastat HCl; 1-deoxygalactonojirimycin HCl) prior to intravenous administration of enzyme replacement therapy improved the pharmacological properties of the enzyme. In this study, the authors investigated the effects of coformulating AT1001 with a proprietary recombinant human α-galactosidase A (ATB100) into a single intravenous formulation. AT1001 increased the physical stability and reduced aggregation of ATB100 at neutral pH in vitro, and increased the potency for ATB100-mediated globotriaosylceramide reduction in cultured Fabry fibroblasts. In Fabry mice, AT1001 coformulation increased the total exposure of active enzyme, and increased ATB100 levels in cardiomyocytes, cardiac vascular endothelial cells, renal distal tubular epithelial cells, and glomerular cells, cell types that do not show substantial uptake with enzyme replacement therapy alone. Notably, AT1001 coformulation also leads to greater tissue globotriaosylceramide reduction when compared with ATB100 alone, which was positively correlated with reductions in plasma globotriaosylsphingosine. Collectively, these data indicate that intravenous administration of ATB100 coformulated with AT1001 may provide an improved therapy for Fabry disease and thus warrants further investigation.

Duvoglustat HCl (AT2220, 1-deoxynojirimycin) is an investigational pharmacological chaperone for the treatment of acid α-glucosidase (GAA) deficiency, which leads to the lysosomal storage disorder Pompe disease, which is characterized by progressive accumulation of lysosomal glycogen primarily in heart and skeletal muscles. The current standard of care is enzyme replacement therapy with recombinant human GAA (alglucosidase alfa [AA], Genzyme). Based on preclinical data, oral co-administration of duvoglustat HCl with AA increases exposure of active levels in plasma and skeletal muscles, leading to greater substrate reduction in muscle. This phase 2a study consisted of an open-label, fixed-treatment sequence that evaluated the effect of single oral doses of 50 mg, 100 mg, 250 mg, or 600 mg duvoglustat HCl on the pharmacokinetics and tissue levels of intravenously infused AA (20 mg/kg) in Pompe patients. AA alone resulted in increases in total GAA activity and protein in plasma compared to baseline. Following co-administration with duvoglustat HCl, total GAA activity and protein in plasma were further increased 1.2- to 2.8-fold compared to AA alone in all 25 Pompe patients; importantly, muscle GAA activity was increased for all co-administration treatments from day 3 biopsy specimens. No duvoglustat-related adverse events or drug-related tolerability issues were identified., Pharmacological chaperones stabilize enzyme replacement in circulation, providing increased uptake of properly folded enzyme into tissues. In a phase 2a study, Kishnani et al. demonstrate a 2-fold increase in plasma and increased uptake in muscle of active recombinant human GAA following co-administration with duvoglustat HCl in patients with late onset Pompe disease.

Fabry disease is an X-linked lysosomal storage disorder (LSD) caused by mutations in the gene (GLA) that encodes the lysosomal hydrolase α-galactosidase A (α-Gal A), and is characterized by pathological accumulation of the substrate, globotriaosylceramide (GL-3). Regular infusion of recombinant human α-Gal A (rhα-Gal A), termed enzyme replacement therapy (ERT), is the primary treatment for Fabry disease. However, rhα-Gal A has low physical stability, a short circulating half-life, and variable uptake into different disease-relevant tissues. We hypothesized that coadministration of the orally available, small molecule pharmacological chaperone AT1001 (GR181413A, 1-deoxygalactonojirimycin, migalastat hydrochloride) may improve the pharmacological properties of rhα-Gal A via binding and stabilization. AT1001 prevented rhα-Gal A denaturation and activity loss in vitro at neutral pH and 37 °C. Coincubation of Fabry fibroblasts with rhα-Gal A and AT1001 resulted in up to fourfold higher cellular α-Gal A and ~30% greater GL-3 reduction compared to rhα-Gal A alone. Furthermore, coadministration of AT1001 to rats increased the circulating half-life of rhα-Gal A by >2.5-fold, and in GLA knockout mice resulted in up to fivefold higher α-Gal A levels and fourfold greater GL-3 reduction than rhα-Gal A alone. Collectively, these data highlight the potentially beneficial effects of AT1001 on rhα-Gal A, thus warranting clinical investigation.

Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency in alpha-galactosidase A (alpha-Gal A) activity and subsequent accumulation of the substrate globotriaosylceramide (GL-3), which contributes to disease pathology. The pharmacological chaperone (PC) DGJ (1-deoxygalactonojirimycin) binds and stabilizes alpha-Gal A, increasing enzyme levels in cultured cells and in vivo. The ability of DGJ to reduce GL-3 in vivo was investigated using transgenic (Tg) mice that express a mutant form of human alpha-Gal A (R301Q) on a knockout background (Tg/KO), which leads to GL-3 accumulation in disease-relevant tissues. Four-week daily oral administration of DGJ to Tg/KO mice resulted in significant and dose-dependent increases in alpha-Gal A activity, with concomitant GL-3 reduction in skin, heart, kidney, brain, and plasma; 24-week administration resulted in even greater reductions. Compared to daily administration, less frequent DGJ administration, including repeated cycles of 4 days with DGJ followed by 3 days without or every other day with DGJ, resulted in even greater GL-3 reductions that were comparable to those obtained with Fabrazyme. Collectively, these data indicate that oral administration of DGJ increases mutant alpha-Gal A activity and reduces GL-3 in disease-relevant tissues in Tg/KO mice, and thus merits further evaluation as a treatment for Fabry disease.

Genomic technologies such as DNA microarrays have been used to study biological processes involved in various normal and disease states; in addition, parallel transcriptional profiling methods hold a great deal of promise for the neurosciences. However, such experiments are technically more demanding and there are unique methodological difficulties for their use in the context of neurobiology and the study of central nervous system disorders.

Highly parallel gene expression profiling has the potential to provide new insight into the molecular mechanisms of complex brain diseases and behavioral traits. We review how gene expression profiling in various brain regions of inbred mouse strains has been used to identify genes that may contribute to strain-specific phenotypes. New data, which demonstrate the use of gene expression profiling in combination with behavioral testing to identify candidate genes involved in mediating variation in running wheel activity, are also presented. These and other studies suggest that a combination of gene expression profiling and more traditional genetic approaches, such as quantitative trait locus analysis, can be used to identify genes responsible for specific neurobehavioral phenotypes.

cell cycle make it an attractive model for the study of genome-wide regulation of gene activity. Parallel identification of all of the genes in a genome that are coordinately regulated during such a process provides a conSummary sistent internal standard for comparison of gene activity over time and makes it possible to search statistically Progression through the eukaryotic cell cycle is known for candidate regulatory sequences. Although the cell to be both regulated and accompanied by periodic cycle‐dependent regulation of many individual genes fluctuation in the expression levels of numerous has been studied, comprehensive results for a genome genes. We report here the genome-wide characteriza- are likely to reveal novel functional and physical organition of mRNA transcript levels during the cell cycle of zation in coordinate gene regulation. These results also the budding yeast S. cerevisiae. Cell cycle‐dependent provide an opportunity to identify related genes in the periodicity was found for 416 of the 6220 monitored human genome that may be involved in cell cycle periodtranscripts. More than 25% of the 416 genes were specific roles. found directly adjacent to other genes in the genome One of the key mechanisms of gene regulation takes that displayed induction in the same cell cycle phase, place on the level of mRNA transcription. Availability of suggesting a mechanism for local chromosomal orga- complete sequence for the Saccharomyces cerevisiae nization in global mRNA regulation. More than 60% of genome has made it possible to quantitate mRNA tranthe characterized genes that displayed mRNA fluctua- script levels for virtually every yeast gene (DeRisi et al., tion have already been implicated in cell cycle period- 1997; Wodicka et al., 1997). In this study, commercially specific biological roles. Because more than 20% of available high-density oligonucleotide arrays were used human proteins display significant homology to yeast to quantitate mRNA transcript levels in synchronized proteins, these results also link a range of human yeast cells at regular intervals during the cell cycle. DNA genes to cell cycle period-specific biological func- oligonucleotide probes are directly synthesized on these tions.

Pompe disease is an inherited lysosomal storage disease that results from deficiency in acid alpha-glucosidase (GAA) activity, and is characterized by progressive accumulation of lysosomal glycogen in heart and skeletal muscles. Enzyme replacement therapy using recombinant human GAA (rhGAA) is the only approved treatment available for Pompe disease. While rhGAA provides some clinical benefits, the infused enzyme tends to be unstable at neutral pH/body temperature, shows insufficient uptake in key tissues, and can elicit immune responses that affect tolerability and efficacy. We have shown previously that oral pre-administration of the pharmacological chaperone AT2220 (1-deoxynojirimycin HCl, duvoglustat HCl) improves the pharmacological properties of rhGAA via binding and stabilization, leading to increased enzyme uptake and glycogen reduction in GAA knock-out (KO) mice. In this study we tested the effects of intravenous (IV) and subcutaneous (SQ) administration of co-formulated AT2220 and rhGAA (AT2220 + rhGAA) as an alternative to giving AT2220 orally prior to rhGAA IV. In rats, IV or SQ administration of co-formulated AT2220 + rhGAA increased the circulating half-life of rhGAA up to twofold, though the maximal rhGAA plasma levels achieved via the SQ route were significantly lower than those seen following IV administration. In GAA KO mice, four IV administrations of co-formulated AT2220 + rhGAA resulted in up to 2.5-fold greater enzyme uptake and glycogen reduction in disease-relevant tissues compared to rhGAA alone; histological staining confirmed reduced skeletal muscle glycogen. Interestingly, four SQ administrations of co-formulated AT2220 + rhGAA increased rhGAA uptake in GAA KO mice, which was not significantly different from those seen following four IV administrations of rhGAA alone. Collectively, these data highlight the potential effects of co-formulated AT2220 + rhGAA using IV or SQ administration, thus warranting further preclinical investigation.

Pompe disease is a lysosomal storage disease caused by deficiency of acid α-glucosidase (GAA) activity, and is characterized by impaired lysosomal glycogen catabolism, progressive skeletal muscle weakness, reduced cardiac function, and respiratory insufficiency. Recombinant human GAA (rhGAA, Genzyme) is the only approved enzyme replacement therapy (ERT) for Pompe, and is administered biweekly via intravenous infusion. While rhGAA does provide clinical benefit, it suffers from low stability at neutral pH/body temperature, shows modest tissue uptake and glycogen reduction, and can elicit immune responses that affect tolerability and efficacy. AT2220 (1-deoxynojirimycin HCl, duvoglustat hydrochloride) is a small molecule pharmacological chaperone that binds and stabilizes endogenous GAA in cells and tissues, resulting in increased lysosomal GAA activity. We hypothesized that AT2220 might also improve the pharmacological properties of exogenous rhGAA. In human plasma, AT2220 co-incubation increased the stability and prevented denaturation of rhGAA at neutral pH/ 37 °C for up to 24 h. In rats, a single oral administration of AT2220 followed 30 min later by intravenous bolus administration of rhGAA resulted in a dose-dependent increase of up to 2-fold in the circulating half-life of rhGAA. A similar effect was seen on the circulating half-life of rhGAA when administered via intravenous infusion. In mice lacking endogenous GAA, oral administration of AT2220 resulted in up to 2.5-fold greater rhGAA uptake and glycogen reduction compared to administration of rhGAA alone in disease-relevant tissues. Collectively, these data indicate that AT2220 increases the stability of rhGAA, and that greater enzyme activity and substrate turnover can be achieved in muscle when co-administered with rhGAA. Based on these findings, a Phase 2 study exploring AT2220 co-administration with rhGAA has been initiated.

Fabry disease is caused by more than 800 different mutations in the GLA gene that encodes the lysosomal enzyme α-galactosidase A (α-Gal A), resulting in accumulation of globotriaosylceramide (GL-3) and end-organ damage. Migalastat HCl (1-deoxygalactonojirimycin HCl, AT1001, GR181413A) is an investigational pharmacological chaperone that selectively binds and stabilizes α-Gal A, leading to increased cellular levels and greater lysosomal activity. Previously, a “research” human embryonic kidney (HEK) cell-based in vitro assay was developed to identify migalastat-responsive mutant forms of αGal A. The FACETS study (NCT00925301) compared migalastat to placebo in subjects with GLA mutations categorized as “amenable” to migalastat based on the research in vitro assay available at study initiation. The primary endpoint of this study was the percent change from baseline in kidney interstitial capillary (IC) GL-3 inclusions (responder analysis, 50% reduction threshold), which was not met. Meanwhile, a GLP-validated assay was created (“GLP HEK assay”) and testing of all mutant forms was completed before unblinding of the 6-month data. Approximately 90% of mutations remained in the same category of “amenable” or “non-amenable” using the same predefined in vitro criteria. However, 15 of 67 subjects enrolled in the study express mutant forms of α-Gal A considered to be nonamenable based on the GLP HEK assay, including 8 subjects with mutation R342Q. These 15 subjects may not be sufficiently responsive to migalastat, and may have impacted the previously reported study results. To explore the effect of migalastat in subjects with GLP HEK amenable mutations only, a post hoc analysis of the change from baseline (difference) in kidney IC GL-3 inclusions was conducted using an ANCOVA model with covariate adjustment for the baseline value and treatment-by-baseline interaction. Analysis of the quantitative difference and inclusion of baseline values in the analysis is the most appropriate measure of an effect on kidney IC GL-3 as many values were low at baseline. This analysis was first applied to all subjects in themITT population (n = 30 per group). Themean change from baseline in the migalastat group was a decrease of 0.22 ± 0.11 inclusions per capillary (IPC) (-66 inclusions per 300), and the placebo group showed an increase of 0.06 ± 0.09 IPC (+18 per 300); p = 0.052. The data were also analyzed for subjects with GLP HEK amenable mutations only (n = 25 migalastat, n = 21 placebo). The change from baseline in the migalastat group was a decrease of 0.31 ± 0.12 IPC (-93 per 300), while the placebo group showed an increase of 0.10 ± 0.13 IPC (+30 per 300); p = 0.0024. These findings were observed in the presence of a significant treatmentby-baseline interaction. This analysis demonstrates a measurable drug effect on change from baseline in kidney IC GL-3 inclusions during the first 6 months of treatment with migalastat, which is more pronounced in subjects with GLP HEK amenable mutations and higher baseline values. Additional data will be presented to support use of the GLP HEK assay to identify Fabry disease subjects with mutations that are most likely to demonstrate a drug effect with migalastat HCl.

Pompe disease is an inherited lysosomal storage disease that results from a deficiency in acid alpha-glucosidase (GAA) activity, and is characterized by progressive accumulation of lysosomal glycogen primarily in heart and skeletal muscles. Recombinant human GAA (rhGAA, Genzyme) is the only approved enzyme replacement therapy for Pompe, and is administered biweekly via intravenous infusion. While rhGAA provides clinical benefit, drawbacks as low stability at neutral pH/body temperature, modest tissue uptake and glycogen reduction, and immune responses affect tolerability and efficacy. AT2220 (1-deoxynojirimycin; duvoglustat HCl) is a pharmacological chaperone that selectively and reversibly binds and stabilizes endogenous GAA, facilitating proper folding and trafficking to lysosomes. AT2220-010 is an open-label, single ascending dose, fixed-sequence, two-period, dose-finding study comprised of four cohorts of 4–6 subjects with Pompe disease. Study objectives: (1) evaluate the safety of single ascending doses of AT2220 co-administered with rhGAA, and (2) characterize the pharmacokinetic interaction of AT2220 and rhGAA on activity and protein levels in plasma, muscle, and peripheral blood mononuclear cells, and on urine Hex four levels. Period 1, subjects receive rhGAA alone, then Period 2 they receive a single oral dose of AT2220 with rhGAA. Safety data from each completed dose cohort is reviewed by a Data Safety Monitoring Board. An innovative muscle biopsy technique, which requires specialized investigator training, is used to sample quadricep muscle either on Day 3 or 7 of each period. This unique crossover study design allows for meaningful evaluation of safety and pharmacokinetic data in the rare disease field by allowing each subject to serve as their own control and reducing variability as opposed to parallel designs requiring larger sample sizes for statistical comparisons.

Pompe disease is an inherited lysosomal storage disease that results from a deficiency in acid alpha-glucosidase (GAA) activity, and is characterized by progressive accumulation of lysosomal glycogen primarily in heart and skeletal muscles. Recombinant human GAA (rhGAA, Genzyme) is the only approved enzyme replacement therapy for Pompe, and is administered biweekly via intravenous infusion. While rhGAA provides clinical benefit, drawbacks as low stability at neutral pH/body temperature, modest tissue uptake and glycogen reduction, and immune responses affect tolerability and efficacy. AT2220 (duvoglustat HCl) is a pharmacological chaperone which selectively and reversibly binds and stabilizes endogenous GAA, facilitating proper folding and trafficking to lysosomes. AT2220-010 is an open-label, single ascending dose, fixed-sequence, two-period, dose-finding study comprised of four cohorts of 4–6 subjects with Pompe disease. During the first period, subjects receive their regularly scheduled intravenous infusion of rhGAA 20 mg/kg alone. During the second period they receive a single oral dose of AT2220 with rhGAA. The objectives of the study are (1) evaluate the safety of single ascending doses of AT2220 (50 mg, 100 mg, 250 mg, and 600 mg) and (2) characterize the pharmacokinetic interaction of AT2220 on rhGAA. Preliminary plasma, peripheral blood mononuclear cells, and muscle GAA activity, total GAA protein, and AT2220 concentrations as well as safety data including urine Hex 4 concentrations are available for the first and second cohorts. Anti-GAA antibodies are also being evaluated.

Parkinson’s disease (PD) may share an etiological basis with Gaucher disease, as GBA1 mutations lead to Gaucher disease when homozygous, and are associated with increased risk for PD when heteroor homozygous. Neuronopathic forms of Gaucher disease and PD also share the therapeutic challenge of targeting the central nervous system (CNS). Pharmacological chaperones (PC) are orally-available, small molecules that represent an innovative approach to specifically increase the activity of target enzymes. AT2101 is a PC developed to enhance the enzyme deficient in Gaucher disease, glucocerebrosidase (GCase). The accumulation of α-synuclein in the CNS is a hallmark of PD. We have successfully utilized AT2101 for proof-of-concept studies in which administration of AT2101 prevented the accumulation of α-synuclein in the brain of two murine PD models that overexpress human α-synuclein. We reasoned that the CNS exposure and other properties of AT2101 could be further improved while maintaining a good safety profile. For instance, AT2101 inhibits targets other than GCase and has a relatively long lysosomal half-life. An assessment of nearly 200 analogs of AT2101 led to the identification of several new PCs with superior characteristics, including greater CNS penetration, increased potency for enhancement of enzyme activity, accelerated efflux from both tissues and lysosomes, and improved target specificity. These new PCs are currently under investigation in models of Parkinson’s and Neuronopathic Gaucher disease.

The fluorescence intensity from randomly oriented Rhodobacter (Rb.) sphaeroides photosynthetic reaction centers at 77 K increases in an applied electric field. This is ascribed to a net reduction in the rate of the initial electron transfer reaction which competes with fluorescence due to the effect of the field on the free energy of charge separation. Quantitative analysis of the fluorescence change in an electric field indicates that the difference in permanent dipole moment between the ground and initially formed excited state is greater than between the ground and emitting state.

The change in absorbance (hole spectrum) of the primary electron donor (P870) in bacterial photosynthetic reaction centers has been studied at 1.5–2.1 K following narrow-band excitation at several wavelengths within the P870 absorption band. The hole width is very large, suggesting a homogeneous linewidth on the order of 200–300 cm−1. Possible interpretations of this highly unusual result, including ultra-fast excited-state decay, are discussed.