Effects of Orally Delivered Alpha-Galactosidase A on Gastrointestinal Symptoms in Patients With Fabry Disease

Fabry disease (FD) is an X-linked, multisystemic, life-threatening lysosomal storage disease due to an α-galactosidase A (AGAL) deficiency, leading also to gastrointestinal (GI) symptoms such as abdominal pain, bloating, and diarrhea.1Hilz M.J. et al.Dig Liver Dis. 2018; 50: 429-437Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar FD is treatable by life-long cost-intensive (∼250.000€ per year) enzyme replacement therapy (ERT) or chaperone therapy. However, a large proportion of patients still suffer from GI symptoms despite FD-specific therapy.1Hilz M.J. et al.Dig Liver Dis. 2018; 50: 429-437Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Non-FD patients with irritable bowel symptoms due to galacto-oligosaccharide intolerance benefit from FODMAP diet2Halmos E.P. et al.Gastroenterology. 2014; 146: 67-75Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar or nutritional supplements containing AGAL, an enzyme with amylase-like activity.3Ganiats T.G. et al.J Fam Pract. 1994; 39: 441-445PubMed Google Scholar, 4Di Stefano M. et al.Dig Dis Sci. 2007; 52: 78-83Crossref PubMed Scopus (34) Google Scholar, 5Hillilä M. et al.Scand J Gastroenterol. 2016; 51: 16-21Crossref PubMed Scopus (13) Google Scholar, 6Tuck C.J. et al.Am J Gastroenterol. 2018; 113: 124-134Crossref PubMed Scopus (28) Google Scholar Within the intestine, AGAL may break down complex galacto-oligosaccharides (such as raffinose, stachyose, or verbascose), probably reducing bowel symptoms by decreasing the colonic fermentation and thus gas production4Di Stefano M. et al.Dig Dis Sci. 2007; 52: 78-83Crossref PubMed Scopus (34) Google Scholar and changing the gut microbiota.7Aguilera-Correa J.J. et al.Sci Rep. 2019; 9: 12010Crossref PubMed Scopus (14) Google Scholar We report our experiences with 7 patients with FD (2 female patients) using commercially available oral dietary supplements containing a daily dose of 1800 U AGAL over 90 to 180 days. Furthermore, we support our data with a comprehensive biochemical characterization of the supplement. Detailed methods are provided within the Supplementary Materials. Seven patients with FD with GI symptoms ingested the commercially available source of 600 U orally delivered (od) AGAL 3 times a day (total intake: 1800 U) during 90 to 180 days and reported GI symptoms (questionnaire) once a month. Overall, intensity of abdominal pain decreased significantly after 8 weeks (P < .05), and after 12 weeks only 1 patient reported mild abdominal pain (Figure 1A). The frequency of diarrhea slightly decreased from 2.6 days/week to 0.5 day/week (P = .0934; Figure 1B). Although patients reported a general reduction of flatulence, no significant changes were observed (Figure 1C). None of the patients suffered from more severe GI symptoms during the observation, and all (especially the 2 treatment-naïve male patients) reported a general amelioration of GI symptoms and a better well-being. Patient 2 presented with constant increasing AGAL activities (up to 29% of the normal reference) (Figure 1D). Most other patients presented with a more heterogeneous AGAL response (Figure 1D–F). Lyso-Gb3 levels in dried blood spots remained stable for those under ERT and female patients without ERT (Figure 1G). Total plasma lyso-Gb3 levels decreased over time in patients 1 to 3 (Figure 1H). Analysis of the analogs revealed that lyso-Gb3 at m/z(-2), (+16), and (+50) decreased significantly in patient 1 and patient 2, and lyso-Gb3 at m/z(-28) in patient 3 (Supplementary Figure 1). Total plasma Gb3 mainly remained constant. An increase of Gb3(C24:1) was observed for ERT-naïve patient 7 (Supplementary Figure 1I). The potential absorption after intake of 1200 U AGAL was measured in a healthy male individual (39 years) revealing increasing plasma activities by at least 12% (r2 = 0.6040, P < .01) indicating a potential absorption. No adverse effects were reported by patients or the control after odAGAL intake. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of odAGAL revealed 2 dominant mannose-glycosylated bands (Supplementary Figure 2A), identified as α-galactosidase A (81.6 kDa; accession number: A0A117E2K7, 29% coverage; 18.8% similarity with human AGAL) and beta-glucosidase (93.3 kDa; accession number: A0A0U1ZXF9, 37% coverage) from Aspergillus niger. In vitro activity of odAGAL was comparable to agalsidase-alfa (Supplementary Figure 2B). Under neutral conditions, odAGAL was stable and half-life under lysosomal conditions was 135 minutes (Supplementary Figure 2C). In plasma, odAGAL was stable for >240 minutes (Supplementary Figure 2D). odAGAL activity was also concentration-dependent inhibited by 1-desogygalactomijirimicin (Supplementary Figure 2E). Cellular odAGAL uptake in endothelial cells without endogenous AGAL was concentration- (Pearson r: 0.9945, P > .001; Supplementary Figure 2F) and mannose-6-phosphate receptor and mannose-receptor dependent (both P < .001) (Supplementary Figure 2F–H). Gb3 depletion was demonstrated using FD patient–derived urinary cells (Supplementary Figure 2I). Anti-drug antibodies (ADAs) from ADA-positive patients (no odAGAL supplementation) led only to weak odAGAL inhibitions (Supplementary Figure 2J). We report on the impact of odAGAL as a dietary supplement on GI symptoms in patients with FD. Our main outcomes were that nutritional supplement with odAGAL resulted in an improvement of GI symptoms and can result in an increase of AGAL activity in patients’ blood, demonstrating a potential uptake of functional enzyme via the gut, which was confirmed by comprehensive biochemical characterization and cell-culture experiments. GI symptoms can be treated by various concomitant medications, increasing the contractile force and accelerating intraluminal transit or inhibiting the proton pump,1Hilz M.J. et al.Dig Liver Dis. 2018; 50: 429-437Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar but should not be prescribed for long-term treatment due to side effects. According to current European FD recommendations, the presence of GI symptoms justifies the initiation of FD-specific therapy,8Biegstraaten M. et al.Orphanet J Rare Dis. 2015; 10: 36Crossref PubMed Scopus (178) Google Scholar but the clinical impact on these complaints is heterogeneous. The cost of oral dietary supplement containing odAGAL is currently ∼100€ per year. Even if only a moderate improvement of GI symptoms can be achieved, the 2500-fold lower costs compared with FD-specific treatment can justify an approach with the dietary supplement of odAGAL. Comparable effects on GI symptoms were observed in patients with irritable bowel syndrome treated with the FODMAP diet2Halmos E.P. et al.Gastroenterology. 2014; 146: 67-75Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar underlying either reduction of galacto-oligosaccharides or their proper digestion. Missing responses of flatulence might be explained by too low odAGAL dosages4Di Stefano M. et al.Dig Dis Sci. 2007; 52: 78-83Crossref PubMed Scopus (34) Google Scholar pointing toward a pharmacodynamic effect. Although preliminary, our results demonstrate an effect of the oral supplement on AGAL activities and support the patient-reported findings. Furthermore, our measurements of plasma lyso-Gb3 including the analogs revealed a significant decrease of total lyso-Gb3 at least in 3 patients. Our biochemical analyses demonstrated higher half-life of odAGAL compared with human recombinant agalsidase in human plasma and at pH neutral environments. Our longitudinal measurements after intake of 1200 U odAGAL revealed a slight AGAL activity increase in plasma in nonfasting conditions. Hence, the reduced half-life in acidic environment might require modification and encapsulation to pass the acidic stomach improving the efficiency of the oral AGAL. However, AGAL resorption seems not pivotal for beneficial effects on GI symptoms. odAGAL biochemically behaves in a similar manner as the recombinant human AGAL. As expected, only a slight recognition and inhibition by ADAs against odAGAL was observed in vitro, implying that odAGAL should also work in patients with ADAs without (allergic) side effects and without ADA-mediated activity inhibition. We conclude that oral administered AGAL might be a suitable and low-cost concomitant treatment approach to successfully improve GI symptoms in affected patients with FD. Future studies are now warranted to confirm our preliminary results. The measurement of enzymatic AGAL activity and plasma lyso-Gb3 levels from dried blood spots by Centogene AG, Rostock, Germany is gratefully acknowledged. We thank Samira Schiwek, Birgit Orlowski, and Anne Huster for their technical assistance. We are grateful to Waters Corp. for their continued scientific support and partnership in mass spectrometry. Malte Lenders, PhD (Conceptualization: Equal; Investigation: Equal; Methodology: Equal; Project administration: Equal; Resources: Supporting; Supervision: Equal; Writing – original draft: Equal). Michel Boutin, PhD (Formal analysis: Equal; Investigation: Equal; Writing – review & editing: Equal). Christiane Auray-Blais, PhD (Formal analysis: Equal; Investigation: Equal; Resources: Equal; Writing – review & editing: Equal). Eva Brand, MD, PhD (Conceptualization: Equal; Investigation: Equal; Project administration: Equal; Resources: Lead; Supervision: Lead; Writing – original draft: Equal). 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