FEMALE FABRY PROJECT IN GERMANY TITLE Female Fabry patients in Germany - interdisciplinary medical care in German Fabry centers: when should ERT be started ? BACKGROUND Fabry disease (FD; OMIM #301500) is an X-linked (Xq22.1) inborn error of  glycosphingolipid catabolism resulting from deficient α-galactosidase A activity (GLA; 300644) due to mutations mainly in the GLA coding region. While former studies reported the incidence of FD to reach 1:40,000 to 1:120,000 [Ref. 1, 2], a recent newborn screening study in males revealed that the GLA mutation rate was ≈1:3,100 in their population [Ref. 3]. FD-specific manifestations result from the differential systemic accumulation of globotriaoslyceramide (Gb3) in the cellular lysosomes [Ref. 4]. Onset of first symptoms (acroparesthesias, angiokeratoma, abdominal pain, cornea verticillata and hypo- or hyperhidrosis) in affected hemizygous males with low or absent enzymatic GLA activity starts in early childhood. The persistent accumulation of Gb3/GL-3 in cells of different tissues comes along with an early onset of stroke, myocardial infarction, or renal failure, leading to a severely reduced life expectancy. Although X-chromosomal heredity and the X-lionization in females (mosaic pattern) can explain why FD-specific symptoms and manifestations are delayed and often milder in heterozygous affected females, lionization is not a sufficient model to explain observed bright variability of disease progression in females. Furthermore, it is unclear which affected females benefit from enzyme replacement therapy (ERT) and when ERT should be initiated. So far, large FD cohorts with well characterized phenotypes in clinical follow-ups are missing to validate the impact of ERT especially in affected females. PROJECT RATIONALE, HYPOTHESIS AND GOAL Due to the great variability in time and onset of disease progression in female Fabry patients it is currently still unclear, which female patient types would benefit from early ERT and when would be the optimal time to start therapy. The hypothesis is that female Fabry patients are treated to late with ERT (“therapeutic nihilism”). The project goal is to evaluate clinical symptoms and end-organ damage in a large cohort of untreated and treated female Fabry patients from 7 large Fabry centers in Germany to establish parameters which would allow and facilitate recommendation about adequate diagnostic and therapy start. Retrospective analysis and set up of a joint comprehensive data base is planned. STUDY DESIGN

• Multicenter study: German Fabry center network: Berlin, Hamburg, Koeln, Mainz, Muenster, Rostock, Wuerzburg
• Observational non-interventional study approach
• Retrospective data analysis of clinical phenotypes

DATABASE

• The interdisciplinary Fabry-centers in MUENSTER (n~150), WUERZBURG (n~180), BERLIN – Charité, Campus Virchow-Klinikum (n~40), KOELN (n~43), HAMBURG (n~14), ROSTOCK (n~28) and MAINZ (n~240) take care of approximately 695 FD patients (~50-60% females, n~382 females) by nephrologists, cardiologists, and neurologists, yearly.
• In the current project, it is envisaged to analyze the clinical phenotype of ~ 385 female Fabry patients.

DURATION OF THE STUDY Start of study: July 2014
Study duration: one year, until June 2015 EXPECTED RESULTS The retrospective, observational non-interventional study will give us the possibility to analyze clinical symptoms and end-organ damage in a large cohort of well-characterized untreated and treated female Fabry patients to identify parameters which would allow and facilitate recommendation about adequate diagnostic and therapy start. COORDINATION / STATISTICAL ANALYSIS Univ.-Prof. Dr. Dr. med. E. Brand, Dr. rer. nat. M. Lenders; Muenster FINANCIAL SUPPORT Shire International GmbH, 07/2014 – 06/2015 REFERENCES:

1. Meikle PJ, Hopwood JJ, Clague AE, Carey WF. Prevalence of lysosomal storage disorders. JAMA. 1999;281:249-254. 
2. Desnick RJ, Ioannou YA, Eng CM. Alpha-galactosidase A deficiency: Fabry disease. In: The metabolic bases of inherited disease, 8th edition. New York: McGraw-Hill, 2001:3733-3774. 
3. Spada M, Pagliardini S, Yasuda M, Tukel T, Thiagarajan G, Sakuraba H, Ponzone A, Desnick RJ. High incidence of later-onset fabry disease revealed by newborn screening. Am J Hum Genet. 2006;79:31-40. 
4. Zarate YA, Hopkin RJ. Fabry’s disease. Lancet. 2008;372:1427-1435.

I. Project title

Antibody-mediated inhibition of enzyme replacement therapy - relevance of dose?

Determination of Fabry disease patients’ specific anti-drug antibody status against recombinant alpha-galactosidase A and impact on biomarker levels

II. Principle investigator

Univ.-Prof. Dr. Dr. med. Eva Brand, phone: 0049251-8348746, mail: Eva.Brand@ukmuenster.de

Dr. rer. nat. Malte Lenders, phone: 0049251-8348104, mail: Malte.Lenders@ukmuenster.de

University Hospital Münster, Internal Medicine D, Albert-Schweitzer-Campus 1, Building A1, 48149 Muenster, Germany,

III. Project duration

28 months

IV. Background

Fabry disease (FD; OMIM #301500) is an X-linked (Xq22.1) inborn error of glycosphingolipid catabolism resulting from deficient alpha-galactosidase A activity (GLA; 300644) due to mutations mainly in the GLA coding region. While former studies reported the incidence of FD to reach 1:40,000 to 1:120,000 [Meikle et al.1999, Desnick et al. 2001], a recent newborn screening study in males revealed that the GLA mutation rate was ≈1:3,100 [Spada et al. 2006]. FD-specific manifestations result from the differential systemic accumulation of globotriaoslyceramide (Gb3 or GL-3) in the cellular lysosomes [Zarate et al. 2008]. Onset of first symptoms (acroparesthesia, angiokeratoma, abdominal pain, cornea verticillata and hypo- or hyperhidrosis) in affected hemizygous males with low or absent enzymatic GLA activity starts in early childhood. The persistent accumulation of Gb3/GL-3 in cells of different tissues leads to an early onset of stroke, heart or renal failure, and cardiac arrhythmia leading to a severely reduced life expectancy.

Enzyme replacement therapy (ERT) with recombinant alpha-galactosidase A (i.e agalsidase-alfa/-beta) has been shown to be effective in reducing intracellular Gb3 and lyso-Gb3 levels resulting in an improvement of clinical outcomes. However, intravenous infusion of recombinant ERT may cause antibody formation with neutralizing effects on ERT especially in male FD patients. A positive antibody status is associated with an impaired therapeutic effect on Gb3 reduction and consequently impaired clinical outcome of affected patients (Linthorst et al. 2004; Rombach et al. 2012; Lenders et al. 2016).

V. Project rationale

So far only small patient groups have been analyzed for antibody-mediated ERT inhibition and limited data about the capacity to trigger drug-neutralizing antibody production in humans receiving agalsidase-alfa or agalsidase-beta infusions are available. To date, it is unclear if neutralizing properties of antibodies against agalsidase-alfa are similar to those against agalsidase-beta (and vice versa).  In addition, the clinical impact of a supersaturation of ADA titers is unclear. Furthermore, it remains elusive if a dose escalation to supersaturate ADA titers might also trigger further antibody production. However, recent studies demonstrate that even in patients with ADAs the total dose of infused enzyme has a beneficial effect on the biochemical response (i.e. lyso-Gb3) (Arends et al. 2018), which is probably due to the supersaturation of ADAs in affected patients (Lenders et al. 2018).

The project rationale is to determine the neutralizing ADA status in ERT-treated male Fabry patients from several Fabry centers in Germany in order to

1. identify affected patients at risk,

2. analyze if the individual infused ERT dose is sufficient to supersaturate ADA titers,

3. analyze if neutralizing properties of antibodies against agalsidase-alfa are similar to those against agalsidase-beta (and vice versa)

4. analyze if an approved dose escalation (switch from agalsidase-alfa (0.2 mg/kg) to agalsidase-beta (the dosage of agalsidase-beta will be the recommended dose of 1 mg/kg BW according to the SmPC) triggers further ADA formation,

5. analyze the effect of neutralizing antibodies on plasma lyso-Gb3 levels, as a marker for the biochemical response.

VI. References

Arends M, Biegstraaten M, Wanner C, Sirrs S, Mehta A, Elliott PM, Oder D, Watkinson OT, Bichet DG, Khan A, Iwanochko M, Vaz FM, van Kuilenburg ABP, West ML, Hughes DA, Hollak CEM. Agalsidase alfa versus agalsidase beta for the treatment of Fabry disease: an international cohort study. J Med Genet. 2018 Feb 7. pii: jmedgenet-2017-104863

Desnick RJ, Ioannou YA, Eng CM. Alpha-galactosidase A deficiency: Fabry disease. In: The metabolic bases of inherited disease, 8th edition. New York: McGraw-Hill, 2001: 3733-3774.

Meikle PJ, Hopwood JJ, Clague AE, Carey WF. Prevalence of lysosomal storage disorders. JAMA. 1999, 281: 249-254.

Lenders M, Stypmann J, Duning T, Schmitz B, Brand SM, Brand E. Serum-mediated inhibition of enzyme replacement therapy in Fabry disease. J Am Soc Nephrol. 2016, 27:256-264

Lenders M, Schmitz B, Brand SM, Foell D, Brand E. Characterization of drug-neutralizing antibodies in patients with Fabry disease during infusion. J Allergy Clin Immunol. 2018, 141: 2289-2292.e7

Linthorst GE, Hollak CE, Donker-Koopman WE, Strijland A, Aerts JM. Enzyme therapy for Fabry disease: neutralizing antibodies towards agalsidase alpha and beta. Kidney Int. 2004, 66:1589-1595.

Rombach SM, Aerts JM, Poorthuis BJ, Groener JE, Donker-Koopman W, Hendriks E, Mirzaian M, Kuiper S, Wijburg FA, Hollak CE, Linthorst GE. Long-term effect of antibodies against infused alpha-galactosidase A in Fabry disease on plasma and urinary (lyso)Gb3 reduction and treatment outcome. PLoS One. 2012, 7:e47805.

Spada M, Pagliardini S, Yasuda M, Tukel T, Thiagarajan G, Sakuraba H, Ponzone A, Desnick RJ: High incidence of later-onset Fabry disease revealed by newborn screening. Am J Hum Genet. 2006;79: 31-40.

Zarate YA, Hopkin RJ. Fabry’s disease. Lancet. 2008; 372: 1427-1435.

VII. Funding

This project is funded by Sanofi-Aventis Deutschland GmbH.

I. Project title

German observational multicenter study of patients with Fabry disease under chaperone therapy with Migalastat-HCl

II. Principle investigators

Malte Lenders^a, Peter Nordbeck^b, Christine Kurschat^c, Maria Eveslage^d; Nesrin Karabul^e, Jessica Kaufeld^f, Julia B. Hennermann^g, Monica Patten^h, Markus Cybullaⁱ, Jonas Müntze^b, Nurcan Üçeyler^j, Dan Liu^b, Anibh M. Das^k, Claudia Sommer^j, Christian Pogoda^l, Stefanie Reiermann^a, Thomas Duning^m, Jens Gaedekeⁿ, Katharina von Cossel^o, Daniela Blaschke^p, Stefan-Martin Brand^q, W. Alexander Mann^e, Christoph Kampmann^g, Nicole Muschol^o, Sima Canaan-Kühlⁿ, Eva Brand^a

^aDepartment of Internal Medicine D, and Interdisciplinary Fabry Center (IFAZ), University Hospital Münster, Münster, ^bDepartment of Internal Medicine I, Comprehensive Heart Failure Center, and Fabry Center for Interdisciplinary Therapy (FAZIT), University of Würzburg, Würzburg, ^cDepartment II of Internal Medicine, Center for Molecular Medicine Cologne and Center for Rare Diseases, University of Cologne, Cologne, ^dInstitute of Biostatistics and Clinical Research (IBKF), University of Münster, Münster, ^eEndokrinologikum Frankfurt, Center of Hormonal and Metabolic Diseases, Rheumatology, Osteology and Neurology, Frankfurt, ^fDepartment of Nephrology and Hypertension, Hannover Medical School, Hannover, ^gVilla Metabolica, Department for Pediatric and Adolescent Medicine, University Medical Center Mainz, Mainz, ^hDepartment of general and interventional Cardiology, University Heart Center Hamburg, Hamburg, ⁱFGM, Center of Internal Medicine, Department of Nephrology and Rheumatology, Mühlheim, ^jDepartment of Neurology, University of Würzburg, Würzburg, ^kDepartment of Paediatrics, Hannover Medical School, Hannover, ^lDepartment of Cardiology I – Coronary and Peripheral Vascular Disease, Heart Failure, and Interdisciplinary Fabry Center (IFAZ), University Hospital Münster, Münster, ^mDepartment of Neurology, and Interdisciplinary Fabry Center (IFAZ), University Hospital Münster, Münster, ⁿMedizinische Klinik mit Schwerpunkt Nephrologie und Internistische Intensivmedizin, Fabry Zentrum, Zentrum für seltene Nierenerkrankungen (CeRKiD), Campus Charité Mitte, Charité - Universitätsmedizin Berlin, ^oDepartment of Pediatrics, University Medical Center Hamburg, Eppendorf, Hamburg, ^pDepartment of Medicine, Division of Cardiology, Charité, Campus Virchow-Klinikum, Berlin, ^qInstitute of Sports Medicine, Molecular Genetics of Cardiovascular Disease, and Interdisciplinary Fabry Center (IFAZ), University Hospital Münster, Münster, Germany

III. Background

Fabry disease (FD; OMIM #301500) is an X-linked (Xq22.1) inborn error of glycosphingolipid catabolism resulting from deficient alpha-galactosidase A activity (GLA/AGAL; 300644) leading to a progressive lysosomal accumulation of glycosphingolipids (mainly globotriaosylceramide [Gb₃]) within the vascular endothelium, as well as renal, cardiac, and neuronal cells. These accumulations result in a multisystemic disease with early myocardial failure and stroke, end-stage renal disease and severely decreased life expectancy. The enzymatic deficiency is based on GLA gene mutations (n>900) (http://www.hgmd.cf.ac.uk/ac/gene.php?gene=GLA). Identified mutations can be classified in nonsense and missense mutations. While nonsense mutations mostly result in a complete loss of function, missense mutations might result only in a partial loss of enzymatic activity due to single amino acid substitutions. Enzyme replacement therapy (ERT) is based on infusions of biotechnologically produced AGAL enzyme (Agalsidase-alfa, Replagal, 0.2 mg/kg BW every other week, Takeda/Shire, and Agalsidase-beta, Fabrazyme, 1 mg/kg BW every other week, Sanofi Genzyme), to compensate for the loss of endogenous enzyme. In contrast, the orally administered pharmacological chaperone Migalastat-HCl (Galafold, 1 capsule [123 mg] every other day, Amicus Therapeutics) enables the correct folding of the endogenous AGAL enzyme in patients with amenable GLA mutations. Several GLA missense mutations code for AGAL enzyme variants that are delivered to the proteasome for degeneration due to folding mistakes and failed quality control within the endoplasmic reticulum (ER). Many of these GLA variants might not only pass the quality control, but also meet up their function within the lysosomes, if a proper folding would be achieved. Since Migalastat-HCl serves as an AGAL folding template, the folding of mutated enzymes may be achieved and GLA enzyme function will be stabilized. By a proper tertiary protein structure, the AGAL enzyme passes the quality control within the ER and will be subsequently translocated to the Golgi apparatus and lysosomes. The pH shift (from neutral to acidic) within the lysosomes leads to a release of Migalastat-HCl and Gb3 as natural substrate can interact with the catalytic center of AGAL. The advantages of the pharmacological chaperone therapy over ERT are: 1) orally administered drug therapy, 2) encompassing the blood-brain barrier that cannot be encompassed by ERT (probably reduced effectiveness of ERT within the brain) and 3) no immunogenicity, which protects against an antibody-mediated ERT inhibition and loss of effectiveness in affected patients. The rationale of the current project is that disease progression of patients with FD with amenable GLA mutations for chaperones can be stabilized comparable to patients under ERT, leading to a validation of the clinical phase 3-studies and a transfer of these previous outcomes to a nationwide “real world” designed study in Germany. The published one year data on 60 patients with FD demonstrated that therapy with migalastat was generally safe and resulted in an amelioration of left ventricular mass (Lenders et al. 2020). In terms of impaired renal function, blood pressure control seems to be an unattended important goal.

IV. Aims

The amendment of this study aimed to analyze treatment effects in patients with genetic variants of unknown significance (GVUS) within the GLA gene.

V. Results

In detail, 15 (9 females) patients carrying p.A143T or p.D313Y were recruited and treated for 24 months with migalastat. 10 patients carried p.A143T (6 females) and 5 patients carried p.D313Y (3 females). Eight patients (4 females) were previously treated with ERT for a mean duration of 2.7±2.6 years. No differences for baseline characteristics between females and males were observed. Baseline characteristics demonstrated no FD-related renal or cardiac manifestations (including normal estimated glomerular filtration rate [eGFR], left ventricular septum thickness in diastole [lVSd] and left ventricular mass index [LVMi] values) independent of sex. However, females and males presented with relevant neurologic manifestations including the presence of FD-related pain and cerebrovascular events (transient ischemic attack [TIA]/stroke). Treatment with migalastat was well tolerated and safe and no adverse reactions were reported. During treatment, a total of 4 clinical events were assessed. In detail, one female suffered from 2 arrhythmia, one male suffered from one arrhythmia and one male from a TIA, resulting in an event rate of 133 events per 1,000 patient-years. Females and males demonstrated stable health conditions (including eGFR, lVSd and LVMi values) over 2 years. 

VI. Conclusion

15 patients carrying a GVUS within the GLA gene were treated for 24 months with migalastat. None of the treated individuals suffered from adverse reactions, demonstrating the safety and tolerability of this drug. Individuals presented with stable health conditions including normal renal function and cardiac parameters. Whether patients with GVUS (p.A143T, p.D313Y) and Fabry-like manifestations benefit from migalastat therapy is unclear because a comparison group of individuals without migalastat therapy is lacking. However, since individuals presented with stable health conditions over time, it can be concluded that migalastat was not harmful in this very cohort.

VII. References

Lenders M, Nordbeck P, Kurschat C, Karabul N, Kaufeld J, Hennermann JB, Patten M, Cybulla M, Müntze J, Üçeyler N, Liu D, Das AM, Sommer C, Pogoda C, Reiermann S, Duning T, Gaedeke J, Stumpfe K, Blaschke D, Brand SM, Mann WA, Kampmann C, Muschol N, Canaan-Kühl S, Brand E. Treatment of Fabry's disease with migalastat: Outcome from a prospective observational multicenter study (FAMOUS). Clin Pharmacol Ther. 2020,108:326-337.

VIII. Funding

This project was funded by Amicus Therapeutics.