Martin Hrebicek

Novel oral treatment of Gaucher's disease with N-butyldeoxynojirimycin (OGT 918) to decrease substrate biosynthesis

BACKGROUND Current treatment for Gauchers disease involves administration of intravenous glucocerebrosidase to degrade glucocerebroside stored in lysosomes. Lowering the rate of biosynthesis of glucocerebroside should decrease accumulation of this substrate. We investigated the safety and efficacy of OGT 918 (N-butyldeoxynojirimycin), an inhibitor of glucosyltransferase, as a novel oral treatment for non-neuronopathic Gauchers disease. METHODS We recruited, into a 1-year open-label study, 28 adults (seven with previous splenectomies) from four national Gauchers referral clinics, who were unable or unwilling to receive enzyme treatment. We measured liver and spleen volume by computed tomography or magnetic resonance imaging at baseline and at months 6 and 12, and biochemical and haematological variables monthly, including chitotriosidase activity (a sensitive marker of Gauchers disease activity). Patients were started on 100 mg oral OGT 918 three times daily. FINDINGS Baseline liver volumes were 1.1-2.7 times normal and spleen volumes 5.1-24.8 times normal. At 12 months, mean liver and spleen volumes were significantly lowered by 12% (95% CI 7.8-16.4) and 19% (14.3-23.7), respectively (each p<0.001). Haematological variables improved slightly. Mean organ volume and blood counts improved continually between 6 months and 12 months of treatment. Mean chitotriosidase concentrations fell by 16.4% over 12 months (p<.0001). Six patients withdrew because of gastrointestinal complaints (two), personal reasons (two), or severe pre-existing disease (two). The most frequent adverse effect was diarrhoea, which occurred in 79% of patients shortly after the start of treatment. INTERPRETATION Decrease of substrate formation by OGT 918 improves key clinical features of non-neuronopathic Gauchers disease. The strategy justifies further trials in this and other glycosphingolipid storage disorders.

Sustained therapeutic effects of oral miglustat (Zavesca, N-butyldeoxynojirimycin, OGT 918) in type I Gaucher disease

Summary: It has been shown that treatment with miglustat (Zavesca, N-butyldeoxynojirimycin, OGT 918) improves key clinical features of type I Gaucher disease after 1 year of treatment. This study reports longer-term efficacy and safety data. Patients who had completed 12 months of treatment with open-label miglustat (100-300 mg three times daily) were enrolled to continue with therapy in an extension study. Data are presented up to month 36. Liver and spleen volumes measured by CT or MRI were scheduled every 6 months. Biochemical and haematological parameters, including chitotriosidase activity (a sensitive marker of Gaucher disease activity) were monitored every 3 months. Safety data were also collected every 3 months. Eighteen of 22 eligible patients at four centres entered the extension phase and 14 of these completed 36 months of treatment with miglustat. After 36 months, there were statistically significant improvements in all major efficacy endpoints. Liver and spleen organ volumes were reduced by 18% and 30%, respectively. In patients whose haemoglobin value had been below 11.5 g/dl at baseline, mean haemoglobin increased progressively from baseline by 0.55 g/dl at month 12 (NS), 1.28 g/dl at month 24 (p=0.007), and 1.30 g/dl at month 36 (p=0.013). The mean platelet count at month 36 increased from baseline by 22×109/L. No new cases of peripheral neuropathy occurred since previously reported. Diarrhoea and weight loss, which were frequently reported during the initial 12-month study, decreased in magnitude and prevalence during the second and third years. Patients treated with miglustat for 3 years show significant improvements in organ volumes and haematological parameters. In conclusion, miglustat was increasingly effective over time and showed acceptable tolerability in patients who continued with treatment for 3 years.

Follow-up study of subunit c of mitochondrial ATP synthase (SCMAS) in Batten disease and in unrelated lysosomal disorders

Abstract Immunohistochemical and biochemical studies of subunit c of mitochondrial ATP synthase (SCMAS) storage were carried out in neuronal ceroid lipofuscinosis (NCL) and in a series of unrelated inherited and acquired lysosomal disorders. In the NCL group, represented by the late infantile, early juvenile and juvenile types, SCMAS storage was generalized neurovisceral, with considerable difference in the visceral storage pattern between the types. In late infantile NCL the SCMAS storage was intensive and corresponded to the generalized, autofluorescent, uniformly curvilinear material, irrespective of the cell type affected. In both early juvenile and juvenile NCLs the SCMAS storage was strong and almost uniform in brain neurons, but did not correlate entirely with the visceral autofluorescent storage pool, being undetectable in autofluorescent storage deposits in a constant set of tissues. In the adult (Kufs) type, the brain neurons were stained with various intensity. In infantile NCL, SCMAS storage was restricted to some of the persisting neurons. In a series of inherited lysosomal enzymopathies and acquired lysosomal disorders, excessive SCMAS accumulation was found only in secondary neuronal lipopigments. It occurred as an early and more uniform phenomenon in mucopolysaccharidosis types I, II, IIIA and in polysulphatase deficiency, or as a delayed varied phenomeno in protracted variants of mucolipidosis I, Niemann-Pick types A and C, and GM2 and GM1 gangliosidoses. Neuronal ageing led to an irregular increase in immunodetectable SCMAS epitope in some neuronal lipofuscin granules. There was no evidence of significant SCMAS lysosomal accumulation in non-neural cells in the whole group, regardless of whether lipofuscin or ceroid accumulation occurred or not. The neuronal SCMAS storage is thus nosologically a common unspecific phenomenon, which is especially amplified in NCL. The specificity of the NCL storage process is shown by the fact that even lysosomes of non-neuronal cells in NCL accumulate SCMAS.

Relationship between X-inactivation and clinical involvement in Fabry heterozygotes. Eleven novel mutations in the α-galactosidase A gene in the Czech and Slovak population

We have identified 21 different α-galactosidase A gene (GLA) mutations in 22 unrelated Czech and Slovak families with Fabry disease. Eleven of these mutations were novel (point mutations D93N, A135V, D155H, G171R, Q280K, G360S, Q330X, splicing errors c.194ins14, c.801ins36 and deletions c.674_732del59, g.3405_6021del2617). Genotyping of family members for family-specific mutations revealed 55 heterozygotes that manifested clinical symptoms of different severity. To examine the contribution of X-inactivation skewing to disease manifestation in Fabry heterozygotes, we have adopted the Mainz severity scoring scheme and compared the score values with the X-inactivation status in 39 carriers in an age-dependent manner. The age-score trendline of Fabry females who had a predominantly inactivated X-chromosome bearing a wild-type GLA allele (10 of 38 females) was markedly steeper than in the rest of the cohort. One female carrier with an inactivated mutated allele had a low score value when compared to the other heterozygotes of the same age. These data suggest that X-inactivation is indeed a major factor determining the severity of clinical involvement in Fabry heterozygotes. There was a statistically significant difference between the severity score values of heterozygotes with random and non-random X-chromosome inactivation at the 5% level of significance. Further studies will show if the degree of the wildtype allele inactivation will be useful as a predictive marker of severity of phenotype in Fabry heterozygotes. Although the correlation between X-inactivation skewing and presentation of the disease in Fabry heterozygotes has previously been suggested in the literature, this report is among the first attempts to examine this relationship systematically.

Inhibition of substrate synthesis as a strategy for glycolipid lysosomal storage disease therapy.

The glycosphingolipid (GSL) lysosomal storage diseases are caused by mutations in the genes encoding the glycohydrolases that catabolize GSLs within lysosomes. In these diseases the substrate for the defective enzyme accumulates in the lysosome and the stored GSL leads to cellular dysfunction and disease. The diseases frequently have a progressive neurodegenerative course. The therapeutic options for treating these diseases are relatively limited, and for the majority there are no effective therapies. The problem is further compounded by difficulties in delivering therapeutic agents to the brain. Most research effort to date has focused on strategies for augmenting enzyme levels to compensate for the underlying defect. These include bone marrow transplantation (BMT), enzyme replacement and gene therapy. An alternative strategy that we have been exploring is substrate deprivation. This approach aims to balance the rate of GSL synthesis with the impaired rate of GSL breakdown. The imino sugar N-butyldeoxynojirimycin (NB-DNJ) inhibits the first step in GSL biosynthesis and has been used to evaluate this approach. Studies in an asymptomatic mouse model of Tay–Sachs disease have shown that substrate deprivation prevents GSL storage in the CNS. In a severe neurodegenerative mouse model of Sandhoff disease, substrate deprivation delayed the onset of symptoms and disease progression and significantly increased life expectancy. Combining NB-DNJ and BMT was found to be synergistic in the Sandhoff mouse model. A clinical trial in type I Gaucher disease has been undertaken and has shown beneficial effects. Efficacy was demonstrated on the basis of significant decreases in liver and spleen volumes, gradual but significant improvement in haematological parameters and disease activity markers, together with diminished GSL biosynthesis and storage as determined by independent biochemical assays. Further trials in type I Gaucher disease are in progress; studies are planned in patients with GSL storage in the CNS.

Force majeure: therapeutic measures in response to restricted supply of imiglucerase (Cerezyme) for patients with Gaucher disease.

Gaucher disease is the first lysosomal disorder for which clinically effective enzyme replacement therapy has been introduced. Lifelong treatment with imiglucerase, the recombinant glucocerebrosidase manufactured by the Genzyme Corporation (MA, USA), is administered intravenously - usually at biweekly intervals. An acute shortage of imiglucerase (to 20% of prior global supply) has occurred as a result of viral contamination of the production facility; production was halted, and a full supply of imiglucerase is not anticipated until January 2010. An urgent meeting of physicians, researchers, and patients was convened through the agency of the European Working Group for Gaucher Disease; this was instigated by patients internationally represented by the European Gaucher Alliance. Here we present a position statement based on the findings of the group, with key recommendations about identification and monitoring of at-risk patients threatened by the abrupt withdrawal of treatment, the equitable distribution of residual imiglucerase - and access to alternative treatments including those that have completed phase III clinical trials but have not yet been licensed.

Neuroinflammation, mitochondrial defects and neurodegeneration in mucopolysaccharidosis III type C mouse model.

Severe progressive neurological paediatric disease mucopolysaccharidosis III type C is caused by mutations in the HGSNAT gene leading to deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase involved in the lysosomal catabolism of heparan sulphate. To understand the pathophysiology of the disease we generated a mouse model of mucopolysaccharidosis III type C by germline inactivation of the Hgsnat gene. At 6-8 months mice showed hyperactivity, and reduced anxiety. Cognitive memory decline was detected at 10 months and at 12-13 months mice showed signs of unbalanced hesitant walk and urinary retention. Lysosomal accumulation of heparan sulphate was observed in hepatocytes, splenic sinus endothelium, cerebral microglia, liver Kupffer cells, fibroblasts and pericytes. Starting from 5 months, brain neurons showed enlarged, structurally abnormal mitochondria, impaired mitochondrial energy metabolism, and storage of densely packed autofluorescent material, gangliosides, lysozyme, phosphorylated tau, and amyloid-β. Taken together, our data demonstrate for the first time that deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase causes lysosomal accumulation of heparan sulphate in microglial cells followed by their activation and cytokine release. They also show mitochondrial dysfunction in the neurons and neuronal loss explaining why mucopolysaccharidosis III type C manifests primarily as a neurodegenerative disease.

Observational, retrospective study of a large cohort of patients with Niemann-Pick disease type C in the Czech Republic: a surprisingly stable diagnostic rate spanning almost 40 years

BackgroundNiemann-Pick disease type C (NPC) is a rare, fatal neurovisceral disorder with autosomal recessive inheritance, and featuring striking clinical variability dependent on the age at onset of neurological symptoms. We report data from a large cohort of 56 Czech patients with NPC diagnosed over a period of 37 years.MethodsAn observational, retrospective analysis of historic and current clinical and laboratory information was performed among all NPC patients originating from the area of the contemporary Czech Republic and diagnosed between 1975 and 2012. All patients with ≥1 positive diagnostic test and relevant clinical information were included. Data on diagnostic methods (histopathological and/or ultrastructural; biochemical; genetic), clinical status and general information on treatment were collated. Data were examined in accordance with international guidelines for the management of NPC.ResultsBetween 1975 and 1985 diagnoses were based exclusively on specific histopathological findings, often at autopsy. Bone marrow smear (BMS) analyses have proved to be a very specific indicator for NPC and have become an important part of our diagnostic algorithm. Filipin staining and cholesterol esterification assays became the definitive diagnostic tests after 1985 and were applied in 24 of our patients. Since 2005, more and more patients have been assessed using NPC1/NPC2 gene sequencing. Twelve patients were diagnosed with neonatal/early-infantile onset NPC, 13 with the late-infantile onset form, 20 with the juvenile onset form, and nine with the adolescent/adult onset form. Two diagnosed patients remained neurologically asymptomatic at study completion. Nineteen patients were siblings. Causal NPC1 mutations were determined in 38 patients; two identical NPC2 mutations were identified in one patient. In total, 30 different mutations were identified, 14 of which have been confirmed as novel. The frequency of individual mutated NPC1 alleles in our cohort differs compared with previous published data: the most frequent mutant NPC1 allele was p.R1186H (n = 13), followed by p.P1007A (n = 8), p.S954L (n = 8) and p.I1061T (n = 4).ConclusionsThese data demonstrate the evolution of the diagnostic process in NPC over the last four decades. We estimate the contemporary birth prevalence of NPC in the Czech Republic at 0.93 per 100,000.

Subclinical course of adult visceral Niemann-Pick type C1 disease. A rare or underdiagnosed disorder?

SummaryWe present the third case of Niemann–Pick disease type C without neurological symptoms. The patient was a 53-year-old woman without significant prior health problems who died of acute pulmonary embolism. Autopsy findings of hepatosplenomegaly, lymphadenopathy and ceroid-rich foam cells raised the suspicion of the visceral form of acid sphingomyelinase deficiency (Niemann–Pick disease type B; NPB) or a much rarer disorder, variant adult visceral form of Niemann–Pick disease type C (NPC). To verify the histopathological findings, SMPD1, NPC1 and NPC2 genes were analysed. Two novel sequence variants, c.1997G>A (S666N) and c.2882A>G (N961S) were detected in the NPC1 gene. No pathogenic sequence variants were found either in the SMPD1 gene mutated in NPB or in NPC2 gene. The pathogenicity of both NPC1 variants was supported by their location in regions important for the protein function. Both variations were not found in more than 300 control alleles. Identified sequence variations confirm the diagnosis of the extremely rare adult visceral form of Niemann–Pick disease type C, which is otherwise dominated by neurovisceral symptoms. Although only three patients have been reported, this (most probably underdiagnosed) form of NPC should be considered in differential diagnosis of isolated hepatosplenomegaly with foam cells in adulthood.

Riboflavin-responsive epilepsy in a patient with SER209 variant form of short-chain acyl-CoA dehydrogenase

Short-chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric mitochondrial flavoprotein that catalyses the first step in/3-oxidation of butyryl-CoA and hexanoylCoA. SCAD deficiency is associated with failure to thrive, metabolic acidosis, various degrees of neuromuscular dysfunction and abnormal urinary excretion of ethylmalonate, methylsuccinate and n-butyrylglycine (Amendt et al 1987; Coates et al 1988; Turnbull et al 1990). Recently, new variant SCAD alleles in patients with proven SCAD deficiency have been reported (Kristensen et al 1993). One of these alleles harbours adenine at position 625 (A625) of SCAD cDNA instead of guanine (G625), changing glycine at the position 209 of precursor SCAD to serine (Ser 209). Surprisingly, in 90 randomly selected controls we found that 4% were homozygous A625/A625 and 31% heterozygous A625/G625 (Kristensen et al 1994). As a result, this polymorphism is unlikely to be disease-causing itself. Significant overrepresentation of the A625 allele was found in a group of patients suspected of having a fatty oxidation defect (unpublished results). This implicates a susceptibility that may, in combination with other factors, provoke symptoms. Such a factor can, for example, be the effect of valproate and its metabolites on/3-oxidation, as presented here.