Enzyme Replacement Therapy Therapy Research Articles

Complement activation and cellular inflammation in Fabry disease patients despite enzyme replacement therapy.

Defective α-galactosidase A (AGAL/GLA) due to missense or nonsense mutations in the GLA gene results in accumulation of the glycosphingolipids globotriaosylceramide (Gb3) and its deacylated derivate globotriaosylsphingosine (lyso-Gb3) in cells and body fluids. The aberrant glycosphingolipid metabolism leads to a progressive lysosomal storage disorder, i. e. Fabry disease (FD), characterized by chronic inflammation leading to multiorgan damage. Enzyme replacement therapy (ERT) with agalsidase-alfa or -beta is one of the main treatment options facilitating cellular Gb3 clearance. Proteome studies have shown changes in complement proteins during ERT. However, the direct activation of the complement system during FD has not been explored. Here, we demonstrate strong activation of the complement system in 17 classical male FD patients with either missense or nonsense mutations before and after ERT as evidenced by high C3a and C5a serum levels. In contrast to the strong reduction of lyso-Gb3 under ERT, C3a and C5a markedly increased in FD patients with nonsense mutations, most of whom developed anti-drug antibodies (ADA), whereas FD patients with missense mutations, which were ADA-negative, showed heterogenous C3a and C5a serum levels under treatment. In addition to the complement activation, we found increased IL-6, IL-10 and TGF-ß1 serum levels in FD patients. This increase was most prominent in patients with missense mutations under ERT, most of whom developed mild nephropathy with decreased estimated glomerular filtration rate. Together, our findings demonstrate strong complement activation in FD independent of ERT therapy, especially in males with nonsense mutations and the development of ADAs. In addition, our data suggest kidney cell-associated production of cytokines, which have a strong potential to drive renal damage. Thus, chronic inflammation as a driver of organ damage in FD seems to proceed despite ERT and may prove useful as a target to cope with progressive organ damage.

Unusual Evolution of Hypertrophic Cardiomyopathy in Non-Compaction Myocardium in a Pompe Disease Patient

Classic infantile Pompe disease is characterized by a severe phenotype with cardiomyopathy and hypotonia. Cardiomyopathy is generally hypertrophic and rapidly regresses after enzyme replacement therapy. In this report, for the first time, we describe a patient with infantile Pompe disease and hypertrophic cardiomyopathy that evolved into non-compaction myocardium after treatment. The male newborn had suffered since birth with hypertrophic cardiomyopathy and heart failure. He was treated with standard enzyme replacement therapy (ERT) (alglucosidase alfa) and several immunomodulation cycles due to the development of anti-ERT antibodies, without resolution of the hypertrophic cardiomyopathy. At the age of 2.5 years, he was treated with a new combination of ERT therapy (cipaglucosidase alfa) and a chaperone (miglustat) for compassionate use. After 1 year, the cardiac hypertrophy was resolved, but it evolved into non-compaction myocardium. Non-compaction cardiomyopathy is often considered to be a congenital, primitive cardiomyopathy, due to an arrest of compaction of the myocardium wall during the embryonal development. Several genetic causes have been identified. We first describe cardiac remodeling from hypertrophic cardiomyopathy to a non-compaction form in a patient with infantile Pompe disease treated with a new ERT. This has important implications both for the monitoring of Pompe disease patients and for the understanding of the pathophysiological basis of non-compaction myocardium.

P0068APABETALONE, A CLINICAL STAGE BET INHIBITOR, REDUCES THE PRO-INFLAMMATORY RESPONSE IN PBMCS FROM FABRY DISEASE PATIENTS ON ERT THERAPY

Abstract Background and Aims Fabry disease (FD) is a rare X-linked recessive genetic disorder caused by mutations in the α-galactosidase A gene. Absence or defects of this lysosomal enzyme lead to globotriaosylceramide (Gb3) accumulation. Gb3 disrupts basic cellular metabolic processes, inducing oxidative stress and promoting inflammation. Systemically, Gb3 accumulation leads to organ damage throughout the body with an increase in renal, cardiac, cerebrovascular and skin complications. Enzyme replacement therapy (ERT) is current standard of care. This therapy may reduce Gb3 deposits but the impact on cardiac and kidney function is unclear based on the limited studies. Further, ERT is less effective in the late phases of FD, partially due to the development of uncontrolled inflammation and fibrosis. Apabetalone is a clinical stage orally administered bromo and extra terminal domain inhibitor (BETi) that has beneficial effects on vascular inflammation and vascular calcification through an epigenetic pathway. Based on its mechanism of action and impact on multiple disease drivers in both preclinical and clinical studies, apabetalone has therapeutic potential to modulate inflammation in FD patients on ERT therapy. Method PBMCs and neutrophils were isolated from fresh blood of FD patients ± ERT therapy using density gradient centrifugation. The baseline immune status of PBMCs was compared in naïve FD patients (without ERT, n=3) and ERT treated patients (n=8) via flow cytometry. PBMCs or neutrophils from ERT patients were treated ex vivo with 5µM or 20µM apabetalone ± LPS stimulation. Expression of key pro-inflammatory mediators was analyzed with real time quantitative PCR. The production of reactive oxygen species (ROS) in neutrophils was assessed by flow cytometry. Results The PBMC inflammatory profile in baseline showed monocytes from ERT patients produced lower amounts of pro-inflammatory mediators (TNFα and IL6) than naïve patients (∼60%, trending p≤0.06 and p≤0.08 respectively). However, surface abundance of CCR2, a chemokine receptor for the MCP1 chemokine that promotes monocyte recruitment to local inflamed tissue during inflammatory process, was ∼2-fold greater on monocytes from ERT subjects than the naïve controls (trending p≤0.06). Gene expression of MCP1 was approximately 5-fold higher in PBMC from ERT patients versus naïve controls (p=0.01). In response to ex vivo LPS stimulation, PBMCs from FD patients on ERT showed a robust induction of pro-inflammatory responses with increases in gene expression of MCP1, IL12B, TNFA and IL6. Ex vivo treatment with apabetalone (5µM) blocked the LPS-induction of MCP1 gene expression (∼90%, p<0.0001), and suppressed the upregulation of IL12B, TNFA and IL6 by 85%, 27% and 17%, respectively (p<0.0001,p=0.04,p=0.004). In response to LPS stimulation, neutrophils induced reactive oxygen species (ROS), an indicator of oxidative damage caused by intracellular Gb3 deposit in FD patients, by ∼6 fold. Apabetalone treatment reduced this induction by 32% and 62% at 5µM and 20 µM, respectively (p=0.01, p=0.0007). Conclusion Monocytes from FD patients on ERT therapy display an enhanced expression of the CCR2-MCP-1 axis as compared to naïve controls, indicating the potential for immune cell tissue infiltration and local inflammation. Apabetalone counters this increase by preventing MCP1 gene transcription. Apabetalone also reduces PBMC mediated pro-inflammatory gene transcription (TNFA, IL12B, IL6) and neutrophil mediated ROS production in response to LPS stimulation. Therefore, apabetalone treatment may reduce pathological inflammation in FD patients and thus complement ERT to optimize patient outcomes, which will be tested further with warranted clinical studies.

Evaluation of the efficacy and safety of the biosimilar drug Glurazyme (imiglucerase) in patients with Gaucher disease type 1

Background. Gaucher disease (GD) is a rare hereditary condition, which represents the most common form of lysosomal storage diseases. Enzyme replacement therapy (ERT) with recombinant glucocerebrosidase is used for the treatment of type 1 and type 3 GD. Imiglucerase (recombinant glucocerebrosidase) is the first biotechnological ERT medication possessing confirmed clinical efficacy and safety.Aim. To compare the efficacy and safety of the biosimilar drug Glurazyme and the reference drug Cerezyme during ERT therapy in patients with type 1 GD.Materials and methods. Thirty patients aged from 19 to 63 years (33 ± 9.7 years) with a stable course of type 1 GD were enrolled in the study. The patients were randomly divided into two equal treatment groups, one of which received Glurazyme and the other received the reference drug Cerezyme. In both groups, the drugs were used in doses of 15–40 U/kg intravenously once every 14 days for 52 weeks.Results. At the end of the study, at week 52 (primary endpoint of efficacy), both groups demonstrated a similar increase in hemoglobin concentration compared to the baseline data. In both groups, the average increase in hemoglobin concentration was 4.8 g/l, with this parameter varying between the groups by 0.1 g/l. At week 52, the calculated 95% confidence interval for the difference in hemoglobin concentration did not cross the non-inferiority margin of 18 g/l, which had been specified as the lower level of drug efficacy. This led to the conclusion that the studied drug Glurazyme is not inferior to Cerezyme in terms of efficacy. A comparison of the treatment groups by secondary endpoints of efficacy revealed: no decrease in hemoglobin concentration by more than 20 % in both groups; no differences between the groups by the number of patients with a change in platelet concentration relative to the baseline values; no increase in the spleen and liver volume by more than 20 and 10 %, respectively, in both groups. The doses of the drug under study and the reference drug remained unchanged during the research. The concentration of chemokine CCL-18 decreased slightly in 10 patients (64.3 %) and 6 patients (40 %) in the Glurazyme group and the Cerezyme group, respectively. The proportion of patients with improved physical and mental health components was 57.1 and 57.1 %, respectively, in the Glurazyme group compared to 73.3 and 60 %, respectively, in the Cerezyme group. Changes in the state of the bone tissue and the reserves of accumulated metabolites in the bone marrow showed a similar decrease in the severity of bone marrow infiltration in both groups.Conclusions. Glurazyme and Cerezyme showed comparable parameters of efficacy and safety in patients with GD type 1 during long-term enzyme replacement therapy.

Pharmacologic properties of high-dose ambroxol in four patients with Gaucher disease and myoclonic epilepsy

BackgroundAmbroxol (ABX) has been suggested as an augmentative pharmacological agent for neuronopathic Gaucher disease (nGD). This study assessed the long-term safety and efficacy of combined therapy with high-dose ABX and...

Long-Term Safety and Efficacy of Combined Therapy with High-Dose Ambroxol and Recombinant Enzyme in Neuronopathic Gaucher Disease

Background: Ambroxol (ABX) has been suggested as an augmentative pharmacological agent for neuronopathic Gaucher disease (nGD). This study assessed the long-term safety and efficacy of combined therapy with high-dose ABX and enzyme replacement therapy (ERT) in nGD. Methods: Four patients with nGD were enrolled. ABX ERT therapy was administered for 4·5 years. Ambroxol was initiated at a dose of 1·5 mg/kg/day, and the dose was escalated up to 27 mg/kg/day. The changes in glucocerebrosidase (GBA) activity, biochemical, safety, and neurocognitive findings were assessed. Findings: Enhanced residual GBA activity was observed in all patients, as evidenced in both in vitro and in vivo studies. Mean seizure frequencies markedly decreased from the baseline, and the neurocognitive function was maintained. Lyso-Gb1, a biomarker for the severity and progression of GD, gradually decreased in all patients. High-dose ABX was well-tolerated with no severe adverse events. Interpretation: Long-term treatment with high-dose ABX ERT was safe and shows promise for arresting the progression of the neurological manifestations in GD. Clinical Trial Number: The study protocol was registered at the Clinical Research Information Service, Korea Centers for Disease Control and Prevention, Ministry of Health and Welfare (Republic of Korea) (no. KCT0003218). Funding Statement: This research was supported in part by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (NRF- 2015K1A4A3046807, NRF-2016M3A9B4915706 and NRF-2018M3A9H1078335) and by ISU ABXIS, Gyeonggi-do, Korea. Declaration of Interests: None declared. Ethics Approval Statement: The study protocol was approved by the Institutional Review Board and the Ministry of Food and Drug Safety, Korea (no. 30449).

382. Correction of Autophagic Dysregulation in Pompe Disease Following Gene Therapy

Pompe Disease (PD) is a fatal metabolic disorder caused by mutations in the GAA gene leading to a deficiency in acid alpha-glucosidase (GAA) and affects 1 in 40,000 births. Clinically, this lysosomal storage disorder presents with cardiomegaly and skeletal muscle weakness, leading to cardiorespiratory failure. GAA is responsible for the breakdown of glycogen in the lysosome, an important energy source for striated muscle and neurons. Currently, the only approved treatment for PD is enzyme replacement therapy (ERT). While ERT has increased patient survival, there are several limitations including treatment cost and the inability of the enzyme to cross the blood-brain barrier and breakdown lysosomal glycogen deposition within the central nervous system. ERT therapy is dependent upon receptor-mediated endocytosis of the exogenous enzyme; much of which remains in amphisomes resulting in insufficiently trafficking of GAA to the lysosome. These issues illustrate the need for an alternative treatment. We propose that endogenous production of GAA enzyme mediated by adeno-associated virus (AAV)-delivery of the GAA gene will improve targeting of GAA to the lysosome and reduce the overall dysregulation of vesicular systems. In this study, we performed intravenous delivery of AAV9-DES-coGAA to 12-week old Gaa−/− animals at three doses (1×1011 vg/kg, 1×1013 vg/kg, and 1×1014vg/kg) and compared to AAV9-CMV-GFP (1×1013 vg/kg) and vehicle-control groups. Previous research has revealed that cellular dysregulation due to lysosomal storage of glycogen is amassed by twelve-weeks of age prior to onset of the physiological phenotype of PD in the Gaa−/− murine model. One month after AAV or sham injection, cardiac and skeletal muscles were harvested for biochemical and histological analyses. GAA activity assays demonstrate that therapeutic levels of enzyme activity are not attained with doses 1×1011 vg/kg and 1×1013 vg/kg, but were when a dose of 1×1014 vg/kg is administered. Western Blot analysis of autophagy-associated proteins, such as Beclin, LAMP1, LC3-I, and LC3-II show a decrease in protein levels following gene therapy indicating an improvement in autophagic regulation. Hematoxalin & Eosin staining of tibialis anterior and gastrocnemius muscle sections show an inverse relationship between level of vacuolarization of muscle fibers and dose of AAV9-DES-coGAA vector. Additionally, preliminary data suggest increased endocytosis of AAVs without reaching the therapeutic threshold may activate autophagy. Further analysis by RT-qPCR and immunofluorescence will elucidate the mechanism by which treatment modifies autophagic flux.

Comparative impact of AAV and enzyme replacement therapy on respiratory and cardiac function in adult Pompe mice

Pompe disease is an autosomal recessive genetic disorder characterized by a deficiency of the enzyme responsible for degradation of lysosomal glycogen (acid α-glucosidase (GAA)). Cardiac dysfunction and respiratory muscle weakness are primary features of this disorder. To attenuate the progressive and rapid accumulation of glycogen resulting in cardiorespiratory dysfunction, adult Gaa–/– mice were administered a single systemic injection of rAAV2/9-DES-hGAA (AAV9-DES) or bimonthly injections of recombinant human GAA (enzyme replacement therapy (ERT)). Assessment of cardiac function and morphology was measured 1 and 3 months after initiation of treatment while whole-body plethysmography and diaphragmatic contractile function was evaluated at 3 months post-treatment in all groups. Gaa–/– animals receiving either AAV9-DES or ERT demonstrated a significant improvement in cardiac function and diaphragmatic contractile function as compared to control animals. AAV9-DES treatment resulted in a significant reduction in cardiac dimension (end diastolic left ventricular mass/gram wet weight; EDMc) at 3 months postinjection. Neither AAV nor ERT therapy altered minute ventilation during quiet breathing (eupnea). However, breathing frequency and expiratory time were significantly improved in AAV9-DES animals. These results indicate systemic delivery of either strategy improves cardiac function but AAV9-DES alone improves respiratory parameters at 3 months post-treatment in a murine model of Pompe disease.

Cytogenetic biomonitoring in mucopolyssacharosis I, II and IV patients treated with enzyme replacement therapy

Background and objectives: The aim of this study was to evaluate genotoxicity and mutagenicity in peripheral blood and buccal mucosal cells in mucopolysaccharidosis (MPS) I, II or VI patients.Methods: A total of 12 patients with MPS type I, II and VI attended at the Institute of Genetics and Inborn Errors of Metabolism treated with enzyme replacement therapy (ERT) and 10 healthy control volunteers were included in this study. Mechanically exfoliated cells from cheek mucosa (left and right side) were used to micronucleus test and single cell gel (comet) assay in peripheral blood cells.Results: The results of this study detected the presence of genetic damage in peripheral blood for all individuals with MPS treated with ERT, regardless of type of MPS as depicted by tail moment results. In addition, an increased number of micronucleated cells were found in buccal cells of MPS type II patients. It was also observed an increase of other nuclear alterations closely related to cytotoxicity as depicted by the frequency of pyknosis, karyolysis and karyorrhexis in buccal mucosa cells of MPS VI patients (p < 0.05).Conclusion: Taken together, such results demonstrate that metabolic alterations induced by the enzymatic deficiency characteristic of MPS associated with ERT therapy can induce genotoxicity and mutagenicity in peripheral blood and buccal mucosa cells, respectively. This effect appears to be more pronounced to MPS II.

Miglustat Therapy in Type 1 Gaucher Disease: Long-Term Treatment Experience From a Multicenter, Retrospective Cohort Study,

Abstract 3207▪▪This icon denotes a clinically relevant abstract Introduction:Miglustat (Zavesca®) was approved for the treatment of adults with mild-to-moderate type 1 Gaucher disease (GD1) for whom enzyme replacement therapy (ERT) is either unsuitable or not a therapeutic option in the EU in 2002 and in the USA in 2003. However, data from real-world clinical experience with miglustat remain limited. Methods:Medical chart data were collected from consecutive adult GD1 patients who initiated commercial miglustat therapy at centers in 9 EU countries or the US after 20th November 2002. Both ERT-naïve and ERT-pretreated patients were included. Follow-up data were collected from miglustat initiation to the end of the observation period (i.e. last information/visit before the end of the study on 31st December 2008, death or loss to follow up). Data on patient demographics, medical history and disease characteristics were collected. Outcome assessments included hematological and biochemical parameters, liver/spleen volumes, gastrointestinal signs/symptoms and neurological manifestations. Results:115 patients (55% female; mean±SD age at miglustat initiation 45±14 years) were enrolled, among whom 34 (30%) were ERT-naïve and 81 (70%) were ERT-pretreated. The median (range) miglustat exposure was 15 (1 – 53) months in ERT-naïve and 15 (0 – 62) months in ERT-pretreated patients. At the time of miglustat initiation the median (range) hemoglobin concentration in ERT-naïve patients (n=24) was 12.8 (10.2 – 16.4) g/dl, and in ERT-pretreated patients (n=65) it was 13.6 (7.3 – 17.4) g/dl; 5 patients in each group had anemia. The median (range) change in hemoglobin from miglustat initiation to last assessment in ERT-naïve patients was 0.3 (−2.5 – 3.6) g/dl, and in ERT-pretreated patients it was −0.3 (−4 – 4.6) g/dl. In the subgroup of patients with anemia the median (range) change was 1.3 (0.1 – 3.6) g/dl in the 5 ERT-naïve patients and 2.6 (−2.7 – 4.6) in the 5 ERT-pretreated patients. At the time of miglustat initiation the median (range) platelet count in ERT-naïve patients (n=25) was 101 (37 – 730) ×109/l, and in ERT-pretreated patients (n=65) it was 173 (43 – 382) ×109/l; 12 patients in the ERT-naïve and 9 in the ERT-pretreated group had thrombocytopenia. The median (range) change in platelet count was 8 (−77 – 145) ×109/l in ERT-naïve patients and −10 (−144 – 434) ×109/l in ERT-pretreated patients. In the subgroup of patients with thrombocytopenia, the median (range) change was 31 (−29 – 145) ×109/l in the 12 ERT-naïve patients and 14 (−32 – 434) ×109/l in the 9 ERT-pretreated patients. Plasma chitotriosidase was highly variable. Substantial median (range) reductions were seen in 20 evaluable ERT-naïve patients [−1365 (−13216 – 3477) nmol/ml/h] but not in the 43 evaluable ERT-pretreated patients [20 (−2700 – 12431) nmol/ml/h]. Few patients had spleen and liver organ volume data recorded. Forty-nine patients (43%) discontinued miglustat during the observation period. Tolerability issues, primarily gastrointestinal, were the most frequent reason for discontinuation, accounting for 32 (28%) of all 115 patients. Other reasons included: non-medical (n=7), insufficient efficacy (n=5), switch to ERT (n=4), and patient death (n=1). Tremor was observed in 3/115 (3%) patients before, and in 18/115 (16%) after miglustat initiation. Conclusions:Based on hematological parameters, we observed that miglustat can maintain disease stability in GD1 patients, irrespective of previous ERT therapy. Although based on a limited number of patients, benefits appeared more pronounced in analyses of anemic and thrombocytopenic patients. Gastrointestinal manifestations remain a concern, leading to discontinuation in around one-third of patients. The safety profile of miglustat was similar to that in previous clinical trials. Disclosures:Kuter:Actelion: Consultancy. Mehta:Actelion: Consultancy. Hollak:Actelion: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hughes:Actelion: Consultancy. Belmatoug:Actelion: Consultancy, Research Funding. Brand:Actelion: Employment. Muller:Actelion: Employment. Schaaf:Factum Gmbh: Consultancy. Giorgino:Actelion: Employment. Zimran:Actelion: Honoraria.

Observational clinical study in juvenile-adult glycogenosis type 2 patients undergoing enzyme replacement therapy for up to 4 years

The objective of this study was to describe a large Italian cohort of patients with late-onset glycogen storage disease type 2 (GSDII) at various stages of disease progression and to evaluate the clinical effectiveness of alglucosidase alpha enzyme replacement therapy (ERT). Previous studies showed in late-onset patients ERT efficacy against placebo and variable response in uncontrolled studies. Seventy-four juvenile or adult GSDII patients were treated with ERT in a multicenter open label, non-randomized study, from 12 months up to 54 months. Recombinant human alpha glucosidase (rh-GAA) was injected by intravenous route at 20 mg/kg every second week. Patients were divided into three groups according to ERT duration: Group A received treatment for 12-23 months (n = 16), Group B for 24-35 months (n = 14), and Group C for more than 36 months (n = 44). Clinical assessment included a 6-min walk test (6MWT), forced vital capacity (FVC), the Walton and Gardner-Medwin score, the number of hours of ventilation, body mass index, echocardiography and blood creatine kinase (CK). Included in our cohort were 33 males and 41 females (M:F = 0.8:1), with a mean age at first symptoms of 28.3 years (range 2-55 years) and a mean age of 43 years at study entry (range 7-72 years). Seven wheelchair bound patients, as well as 27 patients requiring ventilation support, were included. After treatment we could observe an increase in distance walked on the 6MWT in the large majority of patients (48/58; 83%), with an overall mean increase of 63 m (from 320 ± 161 to 383 ± 178 m). After treatment in the majority of patients FVC was improved or unchanged (45/69; 65%). In ventilated patients we observed an improvement in average number of hours off the ventilator (from 15.6 to 12.1 h). Six patients stopped mechanical ventilation and two others started it. The effect of therapy was not related to ERT duration. Nine of 64 patients (13%) that underwent to echocardiography showed a variable degree of cardiac hypertrophy (left ventriculum or septum), and a positive effect was observed after 36 months of ERT in one adult case. Discontinuation of treatment occurred in four patients: one drop-off case, one patient died for a sepsis after 34 months of treatment and two patients stopped ERT for worsening of general clinical condition. Mild adverse effects were observed in four cases (5%). This study represents the largest cohort of late-onset GSDII patients treated with ERT, and confirm a positive effect of treatment. These results, obtained in a large case series on therapy, indicate a favourable effect of ERT therapy, even in more advanced stage of the disease.