Nutritional co-therapy with 1,3-butanediol and multi-ingredient antioxidants enhances autophagic clearance in Pompe disease.

Alglucosidase alpha is an orphan drug approved for enzyme replacement therapy (ERT) in Pompe disease (PD); however, its efficacy is limited in skeletal muscle because of a partial blockage of autophagic flux that hinders intracellular trafficking and enzyme delivery. Adjunctive therapies that enhance autophagic flux and protect mitochondrial integrity may alleviate autophagic blockage and oxidative stress and thereby improve ERT efficacy in PD. In this study, we compared the benefits of ERT combined with a ketogenic diet (ERT-KETO), daily administration of an oral ketone precursor (1,3-butanediol; ERT-BD), a multi-ingredient antioxidant diet (ERT-MITO; CoQ10, α-lipoic acid, vitamin E, beetroot extract, HMB, creatine, and citrulline), or co-therapy with the ketone precursor and multi-ingredient antioxidants (ERT-BD-MITO) on skeletal muscle pathology in GAA-KO mice. We found that two months of 1,3-BD administration raised circulatory ketone levels to ≥1.2 mM, attenuated autophagic buildup in type 2 muscle fibers, and preserved muscle strength and function in ERT-treated GAA-KO mice. Collectively, ERT-BD was more effective vs. standard ERT and ERT-KETO in terms of autophagic clearance, dampening of oxidative stress, and muscle maintenance. However, the addition of multi-ingredient antioxidants (ERT-BD-MITO) provided the most consistent benefits across all outcome measures and normalized mitochondrial protein expression in GAA-KO mice. We therefore conclude that nutritional co-therapy with 1,3-butanediol and multi-ingredient antioxidants may provide an alternative to ketogenic diets for inducing ketosis and enhancing autophagic flux in PD patients.

Glycogen storage in a zebrafish Pompe disease model is reduced by 3-BrPA treatment.

Pompe disease (PD) is an autosomal recessive muscular disorder caused by deficiency of the glycogen hydrolytic enzyme acid α-glucosidase (GAA). The enzyme replacement therapy, currently the only available therapy for PD patients, is efficacious in improving cardiomyopathy in the infantile form, but not equally effective in the late onset cases with involvement of skeletal muscle. Correction of the skeletal muscle phenotype has indeed been challenging, probably due to concomitant dysfunctional autophagy. The increasing attention to the pathogenic mechanisms of PD and the search of new therapeutic strategies prompted us to generate and characterize a novel transient PD model, using zebrafish. Our model presented increased glycogen content, markedly altered motor behavior and increased lysosome content, in addition to altered expression of the autophagy-related transcripts and proteins Beclin1, p62 and Lc3b. Furthermore, the model was used to assess the beneficial effects of 3-bromopyruvic acid (3-BrPA). Treatment with 3-BrPA induced amelioration of the model phenotypes regarding glycogen storage, motility behavior and autophagy-related transcripts and proteins. Our zebrafish PD model recapitulates most of the defects observed in human patients, proving to be a powerful translational model. Moreover, 3-BrPA unveiled to be a promising compound for treatment of conditions with glycogen accumulation.

Using human Pompe disease-induced pluripotent stem cell-derived neural cells to identify compounds with therapeutic potential.

Pompe disease (OMIM # 232300) is a glycogen storage disease caused by autosomal recessive mutations of the gene encoding alpha-1,4-glucosidase (GAA; EC 3.2.1.20). Despite the relatively effective employment of enzyme replacement therapy, some critical medical issues still exist in patients with this disease, including the persistence of abnormalities in the central nervous system (CNS), probably because of the inability of the recombinant GAA to pass through the blood-brain barrier. To address this issue, identification of more therapeutic agents that target the CNS of patients with Pompe disease may be required. In this study, we derived neuronal cells from Pompe disease-induced pluripotent stem cells (Pom-iPSCs) and proved that they are able to recapitulate the hallmark cellular and biochemical phenotypes of Pompe disease. Using the Pom-iPSC-derived neurons as an in vitro drug-testing model, we then identified three compounds, ebselen, wortmannin and PX-866, with therapeutic potential to alleviate Pompe disease-associated pathological phenotypes in the neurons derived from Pom-iPSCs. We confirmed that all three compounds were able to enhance the GAA activity in the Pom-iPSC-derived neurons. Moreover, they were able to enhance the GAA activity in several important internal organs of GAA-deficient mice when co-injected with recombinant human GAA, and we found that intraperitoneal injection of ebselen was able to promote the GAA activity of the GAA-heterozygous mouse brain. Our results prove the usefulness of Pom-iPSC-derived neuronal populations for identifying new compounds with therapeutic potential.

Improved efficacy of a next-generation ERT in murine Pompe disease.

Pompe disease is a rare inherited disorder of lysosomal glycogen metabolism due to acid α-glucosidase (GAA) deficiency. Enzyme replacement therapy (ERT) using alglucosidase alfa, a recombinant human GAA (rhGAA), is the only approved treatment for Pompe disease. Although alglucosidase alfa has provided clinical benefits, its poor targeting to key disease-relevant skeletal muscles results in suboptimal efficacy. We are developing an rhGAA, ATB200 (Amicus proprietary rhGAA), with high levels of mannose-6-phosphate that are required for efficient cellular uptake and lysosomal trafficking. When administered in combination with the pharmacological chaperone AT2221 (miglustat), which stabilizes the enzyme and improves its pharmacokinetic properties, ATB200/AT2221 was substantially more potent than alglucosidase alfa in a mouse model of Pompe disease. The new investigational therapy is more effective at reversing the primary abnormality - intralysosomal glycogen accumulation - in multiple muscles. Furthermore, unlike the current standard of care, ATB200/AT2221 dramatically reduces autophagic buildup, a major secondary defect in the diseased muscles. The reversal of lysosomal and autophagic pathologies leads to improved muscle function. These data demonstrate the superiority of ATB200/AT2221 over the currently approved ERT in the murine model.

Duvoglustat HCl Increases Systemic and Tissue Exposure of Active Acid α-Glucosidase in Pompe Patients Co-administered with Alglucosidase α.

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.

Suppression of mTORC1 activation in acid-α-glucosidase-deficient cells and mice is ameliorated by leucine supplementation.

Pompe disease is due to a deficiency in acid-α-glucosidase (GAA) and results in debilitating skeletal muscle wasting, characterized by the accumulation of glycogen and autophagic vesicles. Given the role of lysosomes as a platform for mTORC1 activation, we examined mTORC1 activity in models of Pompe disease. GAA-knockdown C2C12 myoblasts and GAA-deficient human skin fibroblasts of infantile Pompe patients were found to have decreased mTORC1 activation. Treatment with the cell-permeable leucine analog L-leucyl-L-leucine methyl ester restored mTORC1 activation. In vivo, Pompe mice also displayed reduced basal and leucine-stimulated mTORC1 activation in skeletal muscle, whereas treatment with a combination of insulin and leucine normalized mTORC1 activation. Chronic leucine feeding restored basal and leucine-stimulated mTORC1 activation, while partially protecting Pompe mice from developing kyphosis and the decline in muscle mass. Leucine-treated Pompe mice showed increased spontaneous activity and running capacity, with reduced muscle protein breakdown and glycogen accumulation. Together, these data demonstrate that GAA deficiency results in reduced mTORC1 activation that is partly responsible for the skeletal muscle wasting phenotype. Moreover, mTORC1 stimulation by dietary leucine supplementation prevented some of the detrimental skeletal muscle dysfunction that occurs in the Pompe disease mouse model.

Are evoked potentials in patients with adult-onset pompe disease indicative of clinically relevant central nervous system involvement?

PURPOSE: Pompe disease is a multisystem autosomal recessive glycogen storage disease. Autoptic findings in patients with classic infantile and late-onset Pompe disease have proven that accumulation of glycogen can also be found in the peripheral and central nervous system. To assess the functional role of these pathologic findings, multimodal sensory evoked potentials were analyzed. METHODS: Serial recordings for brainstem auditory, visual, and somatosensory evoked potentials of 11 late-onset Pompe patients were reviewed. Data at the onset of the enzyme replacement therapy with alglucosidase alfa were compared with follow-up recordings at 12 and 24 months. RESULTS: Brainstem auditory evoked potentials showed a delayed peak I in 1/10 patients and an increased I-III and I-V interpeak latency in 1/10 patients, respectively. The III-V interpeak latencies were in the normal range. Visual evoked potentials were completely normal. Median somatosensory evoked potentials showed an extended interpeak latency in 3/9 patients. Wilcoxon tests comparing age-matched subgroups found significant differences in brainstem auditory evoked potentials and visual evoked potentials. CONCLUSIONS: We found that the majority of recordings for evoked potentials were within the ranges for standard values, therefore reflecting the lack of clinically relevant central nervous system involvement. Regular surveillance by means of evoked potentials does not seem to be appropriate in late-onset Pompe patients.

Animal models for lysosomal storage disorders.

The lysosomal storage disorders (LSD) represent a heterogeneous group of inherited diseases characterized by the accumulation of non-metabolized macromolecules (by-products of cellular turnover) in different tissues and organs. LSDs primarily develop as a consequence of a deficiency in a lysosomal hydrolase or its co-factor. The majority of these enzymes are glycosidases and sulfatases, which in normal conditions participate in degradation of glycoconjugates: glycoproteins, glycosaminoproteoglycans, and glycolipids. Significant insights have been gained from studies of animal models, both in understanding mechanisms of disease and in establishing proof of therapeutic concept. These studies have led to the introduction of therapy for certain LSD subtypes, primarily by enzyme replacement or substrate reduction therapy. Animal models have been useful in elucidating molecular changes, particularly prior to onset of symptoms. On the other hand, it should be noted certain animal (mouse) models may have the underlying biochemical defect, but not show the course of disease observed in human patients. There is interest in examining therapeutic options in the larger spontaneous animal models that may more closely mimic the brain size and pathology of humans. This review will highlight lessons learned from studies of animal models of disease, drawing primarily from publications in 2011-2012.

Renal magnesium wasting, hypomagnesemic hypocalcemia, hypocalciuria and osteopenia in a patient with glycogenosis type II.

We describe a patient with late-onset glycogenosis type II with renal magnesium wasting, hypomagnesemic hypocalcemia, hypocalciuria and osteopenia. He was admitted to our hospital for evaluation of lower limb weakness and mild deterioration of liver function. Serum magnesium and calcium were low with low-to-normal levels of PTH in the patient. Echocardiogram revealed marked concentric hypertrophy of the left ventricle. An X-ray film of his spine showed a thoracic (Th12) vertebral compression fracture. Bone mineral density of the lumbar spine L2-L4 showed a reduced value. Kidney, liver and muscle biopsies were performed. These were found to have histologic features consistent with glycogenosis type II. In addition, accumulation of PAS-positive material in the cytoplasmic vacuoles of the tubular epithelium was present only in the distal tubules. An oral magnesium supplement was useful in helping to correct the hypomagnesemia, despite the presence of renal magnesium wasting in our patient. Magnesium supplement was also sufficient to maintain normal serum calcium concentrations. However, the hypocalciuria persisted in our patient despite correction of hypomagnesemia. In conclusion, the consistent association between the glycogen accumulation in distal tubules, renal magnesium wasting, hypomagnesemic hypocalcemia and hypocalciuria, in the absence of other identifiable reasons, suggests a cause-and-result relationship. Also, the combination of renal magnesium wasting, hypomagnesemia and hypocalciuria is a picture similar to that of Gitelman's syndrome in our patient. The glycogen accumulation in distal tubules may cause renal magnesium wasting and hypocalciuria through tubular injury. Therefore, we may speculate that the present case has glycogenosis type II-associated Gitelman's-like syndrome.