Preclinical investigation of artesunate as a therapeutic agent for hepatocellular carcinoma via impairment of glucosylceramidase-mediated autophagic degradation.

Artesunate (ART) has been indicated as a candidate drug for hepatocellular carcinoma (HCC). Glucosylceramidase (GBA) is required for autophagic degradation. Whether ART regulates autophagic flux by targeting GBA in HCC remains to be defined. Herein, our data demonstrated that the dramatic overexpression of GBA was significantly associated with aggressive progression and short overall survival times in HCC. Subsequent experiments revealed an association between autophagic activity and GBA expression in clinical HCC samples, tumor tissues from a rat model of inflammation-induced HCC and an orthotopic mouse model, and human HCC cell lines. Interestingly, probe labeling identified GBA as an ART target, which was further verified by both a glutathione-S-transferase pulldown assay and surface plasmon resonance analysis. The elevated protein expression of LC3B, the increased numbers of GFP-LC3B puncta and double-membrane vacuoles, and the enhanced expression of SQSTM1/p62 indicated that the degradation of autophagosomes in HCC cells was inhibited by ART treatment. Both the in vitro and in vivo data revealed that autophagosome accumulation through targeting of GBA was responsible for the anti-HCC effects of ART. In summary, this preclinical study identified GBA as one of the direct targets of ART, which may have promising potential to inhibit lysosomal autophagy for HCC therapy.

Improvement of life quality measured by Lansky Score after enzymatic replacement therapy in children with Gaucher disease type 1.

BACKGROUND: Gaucher disease type 1 (GD1, OMIM# 230800), is a condition with high impact in patient's quality of life (QoL). We report the improvement in QoL of children with GD1 measured by Lansky play-performance scale (LS) after enzymatic replacement therapy (ERT) and to describe our experience in the treatment of children with GD1. METHODS: Five children with diagnosis of GD1 received imiglucerase 60 mg/kg every two weeks. LS, hepatomegaly, splenomegaly, hemoglobin, platelets, and growth rate were measured every 6 months after beginning ERT for 30 months. RESULTS: After ERT, LS increased significantly from 28 ± 16.48 points before ERT to 70 ± 10 (P = 0.0046) and 95 ± 10 (P = 0.0022) points after 6 and 30 months of ERT, respectively; hemoglobin and platelets changed significantly from 9.28 ± 0.61 to 12.40 ± 0.85 (P = 0.0198) and from 71.50 ± 14.89 to 205.00 ± 65.34 (P = 0.0428) after 30 months of ERT, respectively. All patients demonstrated decreased hepatic and splenic size with mean reductions of 66% and 80% at 30 months of treatment and the USG longitudinal axis was reduced in both liver and spleen after ERT. CONCLUSION: The use of ERT with imiglucerase 60 mg/kg every two weeks has substantial benefits and significantly improves QoL, assessed with Lansky Score, of the five children with GD1 studied.

Antiviral activity of acid beta-glucosidase 1 on enterovirus 71, a causative agent of hand, foot and mouth disease.

Enterovirus 71 (EV71) is a causative agent of hand, foot and mouth disease (HFMD). EV71 causes fever, rash, diarrhoea and, in some cases, acute encephalopathy/encephalitis, which can be fatal. No specific treatment is currently available for EV71 infection. Here, we conducted a cDNA library screen and identified acid ß-glucosidase 1 (GBA1; also known as ß-glucocerebrosidase) as an EV71 resistance factor. The anti-EV71 function of GBA1 was verified by gene transduction and knockdown experiments. Cerezyme, a molecular drug used to treat Gaucher's disease and having recombinant human GBA1 as the active ingredient, protected against EV71 infection. The anti-EV71 activity of GBA1 was bimodal: endogenous GBA1 restricted cell surface expression levels of scavenger receptor class B, member 2 (SCARB2), also known as lysosomal integral membrane protein 2 (LIMP-2), and exogenous recombinant GBA1 interfered with EV71 to interact with SCARB2 outside the cell. Thus, our findings suggest that GBA1 may represent a novel molecular target for the treatment of EV71 infection.

Comorbidities and pharmacotherapies in patients with Gaucher disease type 1: The potential for drug-drug interactions.

PURPOSE: Clinical care for patients with rare diseases may be complicated by comorbidities. Administration of medications to treat comorbidities may elicit potentially harmful drug-drug interactions (DDIs). Genetic background may also influence DDI occurrence. We investigated the range of comorbid conditions in patients with Gaucher disease type I (GD1), the pharmacotherapies prescribed and the potential for DDI with enzyme replacement and substrate reduction therapies and additional medications, specifically cytochrome P450 (CYP) metabolizing medications. METHODS: A literature review examined comorbid conditions and pharmacotherapies reported in GD1. Analysis of two national databases reported real-world prescription practices in patients with GD1 (Germany, N=87; US, N=374). Prescribed drugs were assessed for known interactions with isoenzymes from the hepatic CYP enzyme family. RESULTS: The literature reported GD1 symptomatology and comorbid conditions in broad agreement with the known clinical picture. German patients received 86 different medications whereas US patients received 329 different medications. An average of 3.2 medications (Germany) and 7 medications (US) per patient were prescribed. Moderate/strong inhibitors of CYP isoenzymes were prescribed to 20% and 57% of patients in the US and Germany, respectively. CONCLUSION: This study describes the extensive number of comorbid conditions and drugs prescribed to patients with GD1, and the importance of determining CYP isoenzyme interaction to reduce DDI risk.

Role of LIMP-2 in the intracellular trafficking of ß-glucosidase in different human cellular models.

Acid ß-glucosidase (GCase), the enzyme deficient in Gaucher disease (GD), is transported to lysosomes by the lysosomal integral membrane protein (LIMP)-2. In humans, LIMP-2 deficiency leads to action myoclonus-renal failure (AMRF) syndrome. GD and AMRF syndrome share some clinical features. However, they are different from clinical and biochemical points of view, suggesting that the role of LIMP-2 in the targeting of GCase would be different in different tissues. Besides, the role of LIMP-2 in the uptake and trafficking of the human recombinant (hr)GCase used in the treatment of GD is unknown. Thus, we compared GCase activity and intracellular localization in immortalized lymphocytes, fibroblasts, and a neuronal model derived from multipotent adult stem cells, from a patient with AMRF syndrome, patients with GD, and control subjects. In fibroblasts and neuronlike cells, GCase targeting to the lysosomes is completely dependent on LIMP-2, whereas in blood cells, GCase is partially targeted to lysosomes by a LIMP-2-independent mechanism. Although hrGCase cellular uptake is independent of LIMP-2, its trafficking to the lysosomes is mediated by this receptor. These data provide new insights into the mechanisms involved in the intracellular trafficking of GCase and in the pathogeneses of GD and AMRF syndrome.

Management of bone disease in Gaucher disease type 1: clinical practice.

Gaucher disease is a rare autosomal recessive disorder of glycosphingolipid metabolism resulting from deficient activity of the lysosomal enzyme beta-glucocerebrosidase that causes accumulation of glucosylceramide in tissue macrophage with damage to hematological, visceral, and skeletal organ systems. Severity and progression may vary independently among these domains, necessitating individualized therapy. Skeletal involvement is highly prevalent and often associated with intense pain, impaired mobility, and reduced quality of life. Enzyme replacement therapy improves parameters in all affected domains, but skeletal involvement requires longer treatment and higher dosages to obtain significant results. Despite numerous papers on bone complications in patients with Gaucher disease, there are no specific indications on how to assess properly bone involvement in such condition, the frequency of assessment, the use of markers for osteoblast and osteoclast activity, or the administration of bisphosphonates or other symptomatic drugs in adult and pediatric patients. Starting from a re-evaluation of cases with bone involvement, we have identified some common errors in the diagnostic approach and management. The aim of this paper was to propose a methodological and critical approach to the diagnosis, follow-up and treatment of bone disease in patients with Gaucher disease type 1.

Impact of velaglucerase alfa on bone marrow burden score in adult patients with type 1 Gaucher disease: 7-year follow-up.

BACKGROUND: Bone marrow infiltration by substrate-engorged "Gaucher" cells manifests early in Gaucher disease (GD). The impact of velaglucerase alfa on bone marrow burden (BMB) was evaluated as an exploratory assessment. METHODS: BMB scores were assessed using T1- and T2-weighted magnetic resonance images of the lumbar spine (LS) and femora among symptomatic GD patients who participated in the 9-month Phase I/II trial and long-term extension study for velaglucerase alfa. A post-hoc assessment of marrow involvement was performed. BMB scores per site are 0-8 (0/1=normal; 8=severe infiltration). RESULTS: The median LS-BMB score at baseline was 6 (n=12; range 3-8); at 9 months, compared with baseline, there was a median change of -2 (n=11; two-sided p-value=0.0078). LS-BMB scores continued to decrease through 5 years (n=8; median change from baseline -5 [p=0.0078], median score 1 [range 1-4]) and were subsequently sustained through 7 years (n=8). LS-BMB decreases of ≥2 points occurred in 6/11 patients at 9 months, and in all assessable patients (8/8) by 5 years. Long-term femoral BMB (F-BMB) assessment was possible for three patients; all experienced reductions of ≥2 points at 5 years with a total score (LS-BMB+F-BMB) decrease ≥4. CONCLUSIONS: This post hoc analysis suggests improvement in BMB scores through 5 years that was sustained through 7 years, despite dose reduction from 15 months. Prospective studies in a large cohort are needed to validate these findings.

Evoked potentials and neurocognitive functions in pediatric Egyptian Gaucher patients on enzyme replacement therapy: a single center experience.

BACKGROUND: Effectiveness of enzyme replacement therapy (ERT) in reverting hematologic, skeletal, and visceral symptoms in Gaucher disease (GD) has been demonstrated, although, its efficacy in neurologic involvement is still debated. AIM: We evaluated the extent of neuro-cognitive dysfunction using brain stem evoked potential in GD3 patients, age-matched controls, and GD1 patients without neurological manifestations served as disease control group. METHODS: Study included 56 GD (36 had type 1, 20 had type 3) under ERT. Investigations included complete blood count, beta glucosidase assay in peripheral leucocytes, plasma chitotriosidase and bone marrow examination, electroencephalography, brain stem auditory (AEP), somatosensory (SSEP) and visual evoked potentials (VEP) as well as IQ testing. RESULTS: Both types of GD showed significantly higher mean latency at 75 on left eye, lower PP amplitude ratio, higher latency at 75, 100, 145, lower amplitude, and higher Lat Diff LT-RT ms and Lt-Rt % compared to controls (p < 0.05) with no difference between both groups in other values of VEP. Both groups showed significantly prolonged latency of N 13-19 compared to controls (p < 0.05) with positive correlation between age and duration of therapy with parameters of SSEP (p < 0.01). Both groups of GD had significantly prolonged latency of the mean waves of AEP compared to controls (p < 0.05) with no significant difference between both groups. There was a negative correlation between age and waves II, III, I-III, I-V and threshold values of AEP. IQ level was positively correlated with AEP values. Severity scoring tool was positively correlated with AEP and SSEP values. CONCLUSIONS: Electrophysiological abnormalities were present in both types of GD and have been correlated to cognitive function and disease characteristics.

Enzyme replacement therapy with velaglucerase alfa in Gaucher disease: Results from a randomized, double-blind, multinational, Phase 3 study.

Type 1 Gaucher disease (GD1), resulting from glucocerebrosidase deficiency, leads to splenomegaly, hepatomegaly, anemia, thrombocytopenia, and bone involvement. Current standard treatment is enzyme replacement therapy. Velaglucerase alfa is an enzyme replacement product for GD1, with the same amino acid sequence as naturally occurring human glucocerebrosidase. This multinational, Phase 3 trial evaluated the efficacy and safety of two doses of velaglucerase alfa in 25 treatment-naïve, anemic patients with GD1 (4-62 years of age), randomized to intravenous velaglucerase alfa 60 U/kg (n=12) or 45 U/kg body weight (n=13) every other week for 12 months. The primary endpoint was change from baseline in hemoglobin concentration in the 60 U/kg arm. At 12 months, mean hemoglobin concentrations increased from baseline [60 U/kg: +23.3%; +2.43 g/dL (P<0.001); 45 U/kg: +23.8%; +2.44 g/dL (P<0.001)], as did mean platelet counts [60 U/kg: +65.9%; +50.9 × 10(9) /L (P=0.002); 45 U/kg: +66.4%; +40.9 × 10(9) /L(P=0.01)]. Mean splenic volume decreased from baseline [60 U/kg: -50.4%, from 14.0 to 5.8 multiples of normal (MN) (P=0.003); 45 U/kg: -39.9%, from 14.5 to 9.5 MN (P=0.009)]. No drug-related serious adverse events or withdrawals were observed. One patient developed antibodies. Velaglucerase alfa was generally well tolerated and effective for adults and children with GD1 in this study. All disease-specific parameters measured demonstrated clinically meaningful improvements after 12 months.

Velaglucerase alfa enzyme replacement therapy compared with imiglucerase in patients with Gaucher disease.

Enzyme replacement therapy for Gaucher disease (GD) has been available since 1991. This study compared the efficacy and safety of velaglucerase alfa with imiglucerase, the previous standard of care. A 9-month, global, randomized, double-blind, non-inferiority study compared velaglucerase alfa with imiglucerase (60 U/kg every other week) in treatment-naïve patients aged 3-73 years with anemia and either thrombocytopenia or organomegaly. The primary endpoint was the difference between groups in mean change from baseline to 9 months in hemoglobin concentration. 35 patients were randomized: 34 received study drug (intent-to-treat: 17 per arm), 20 were splenectomized. Baseline characteristics were similar in the two groups. The per-protocol population included 15 patients per arm. The mean treatment difference for hemoglobin concentration from baseline to 9 months (velaglucerase alfa minus imiglucerase) was 0.14 and 0.16 g/dL in the intent-to-treat and per-protocol populations, respectively. The lower bound of the 97.5% one-sided confidence interval in both populations lay within the pre-defined non-inferiority margin of -1.0 g/dL, confirming that velaglucerase alfa is non-inferior to imiglucerase. There were no statistically significant differences in the secondary endpoints. Most adverse events were mild to moderate. No patient receiving velaglucerase alfa developed antibodies to either drug, whereas four patients (23.5%) receiving imiglucerase developed IgG antibodies to imiglucerase, which were cross-reactive with velaglucerase alfa in one patient. This study demonstrates the efficacy and safety of velaglucerase alfa compared with imiglucerase in adult and pediatric patients with GD clinically characterized as Type 1. Differences in immunogenicity were also observed.

Safety and efficacy of velaglucerase alfa in Gaucher disease type 1 patients previously treated with imiglucerase.

Velaglucerase alfa is a glucocerebrosidase produced by gene activation technology in a human fibroblast cell line (HT-1080), and it is indicated as an enzyme replacement therapy (ERT) for the treatment of Gaucher disease type 1 (GD1). This multicenter, open-label, 12-month study examined the safety and efficacy of velaglucerase alfa in patients with GD1 previously receiving imiglucerase. Eligible patients, ≥2 years old and clinically stable on imiglucerase therapy, were switched to velaglucerase alfa at a dose equal to their prior imiglucerase dose. Infusion durations were 1 hr every other week. Forty patients received velaglucerase alfa (18 male, 22 female; four previously splenectomized; age range 9-71 years). Velaglucerase alfa was generally well tolerated with most adverse events (AEs) of mild or moderate severity. The three most frequently reported AEs were headache (12 of 40 patients), arthralgia (9 of 40 patients), and nasopharyngitis (8 of 40 patients). No patients developed antibodies to velaglucerase alfa. There was one serious AE considered treatment-related: a grade 2 anaphylactoid reaction within 30 min of the first infusion. The patient withdrew; this was the only AE-related withdrawal. Hemoglobin concentrations, platelet counts, and spleen and liver volumes remained stable through 12 months. In conclusion, adult and pediatric patients with GD1, previously treated with imiglucerase, successfully transitioned to velaglucerase alfa, which was generally well tolerated and demonstrated efficacy over 12 months' treatment consistent with that observed in the velaglucerase alfa phase 3 clinical trial program.

Minimal disease activity in Gaucher disease: criteria for definition.

Gaucher disease type I is a metabolic disorder caused by a genetic deficiency of lysosomal ß-glucocerebrosidase that leads to accumulation of glucocerebroside in macrophages, thus causing damage in different organ systems. Enzyme replacement therapy with imiglucerase improves organ impairment and clinical manifestations, but patients differ in response to treatment. While clinical remission is the most desirable therapeutic outcome, a more realistic goal in patients with high disease burden is reasonably good clinical status despite persistence of residual biochemical or imaging abnormalities. Therefore, the concept of minimal disease activity--used in certain haematological or rheumatologic conditions--needs to be introduced in Gaucher disease, with a level of disease activity that patients and physicians consider a useful treatment target. In this paper, we propose specific parameters and criteria for defining minimal disease activity in Gaucher disease and its stability over time, based on three major systemic domains typically involved: haematological, visceral, and skeletal. Biomarker parameters were not included as criteria, because currently they do not adequately reflect disease evolution in individual patients. Neurological and respiratory domains were also excluded, as their involvement per se indicates severe disease unlikely to respond to enzyme replacement therapy and achieve minimal disease status. Our goal in defining minimal disease activity and stability is to identify a tool to facilitate treatment decisions in clinical practice.

Taliglucerase alfa for the treatment of Gaucher's disease.

Gaucher's disease is a rare inherited inborn error of metabolism caused by mutations in the gene encoding the lysosomal enzyme glucocerebrosidase, GBA. Glucocerebrosidase is involved in the metabolism of the lipid metabolism-derived substrate, glucocerebroside. Accumulation of glucocerebroside substrate in macrophages, as a result of loss of enzyme function, leads to the formation of Gaucher cells causing hypertrophy of the spleen and liver, hematological disorders, skeletal malformations and the neurological symptoms characteristic of Gaucher's disease. The disease is subdivided into three types that differ in their symptoms, severity and prognosis. Patients of any age can be affected but those of a younger age have a poor prognosis often dying in infancy. As a genetic disorder the incidence of the disease is variable on a global scale. Enzyme replacement therapy is the therapy of choice and has demonstrated good efficacy in treating the visceral and skeletal symptoms of Gaucher's disease. A cost-effective plant-cell-derived human recombinant glucocerebrosidase, taliglucerase alfa, has been developed that demonstrated a promising safety and efficacy profile in phase I clinical trials and is currently in phase III and IV trials for the treatment of pediatric and adult patients with Gaucher's disease.

Velaglucerase alfa: a new option for Gaucher disease treatment.

Type 1 Gaucher disease (GD) results from inherited ß-glucocerebrosidase gene mutations, leading to anemia, thrombocytopenia, splenomegaly, hepatomegaly and skeletal disease. Velaglucerase alfa is a ß-glucocerebrosidase produced by gene activation in a human cell line, and indicated for type 1 GD. A phase I/II clinical trial (TKT025; N = 12), its ongoing extension (TKT025EXT) and three phase III trials (total N = 82), showed that velaglucerase alfa is generally well tolerated in adult and pediatric patients. Many disease-related parameters improved significantly in two phase III trials in treatment-naïve patients, and were successfully maintained in imiglucerase-experienced patients in a phase II/III switch study. Ten adults in TKT025EXT sustained improvements through 5 years, including bone mineral density. Comparison with imiglucerase shows that velaglucerase alfa is an effective, generally well-tolerated alternative enzyme replacement therapy. In vitro data suggest velaglucerase alfa may be internalized into cells more efficiently and have a lower rate of seroconversion. However, these results do not necessarily correlate with clinical efficacy.

Significant and continuous improvement in bone mineral density among type 1 Gaucher disease patients treated with velaglucerase alfa: 69-month experience, including dose reduction.

Since bone pathology is a major concern in type 1 Gaucher disease (GD1), we evaluated bone mineral density (BMD) in adults receiving velaglucerase alfa in the seminal Phase I/II and extension trial. Ten treatment-naïve symptomatic patients with GD1 (four men, six women; median age 35years, range 18-62years) were included; of these, four patients were receiving bisphosphonates at enrollment. Using WHO criteria to classify the lumbar spine (LS) and femoral neck (FN) BMD T-scores, respectively, one (10%) and four (40%) patients had osteoporosis; eight (80%) and five (50%) had osteopenia; and one each (10%) was in the normal range, at baseline. By Month 69, two LS and one FN osteopenic patients normalized and one FN osteoporotic patient became osteopenic; change was seen only in patients not receiving bisphosphonates. Significant improvements in BMD Z-scores were seen at the LS by Month 24 and at the FN by Month 33 and were continuous thereafter. In linear mixed models, Z-scores were significantly lower than the reference population at baseline and improved significantly with treatment (LS and FN both P<0.01); analysis of the subgroup of patients not receiving bisphosphonates showed similar results. In conclusion, in this small cohort, velaglucerase alfa was associated with clinically meaningful and statistically significant LS and FN BMD improvements as early as Month 24 (LS) and 33 (FN), despite dose reduction and significant baseline skeletal pathology. These results suggest that velaglucerase alfa may hold promise in the management of skeletal pathology associated with GD1.

Surface localization of glucosylceramide during Cryptococcus neoformans infection allows targeting as a potential antifungal.

Cryptococcus neoformans (Cn) is a significant human pathogen that, despite current treatments, continues to have a high morbidity rate especially in sub-Saharan Africa. The need for more tolerable and specific therapies has been clearly shown. In the search for novel drug targets, the gene for glucosylceramide synthase (GCS1) was deleted in Cn, resulting in a strain (Δgcs1) that does not produce glucosylceramide (GlcCer) and is avirulent in mouse models of infection. To understand the biology behind the connection between virulence and GlcCer, the production and localization of GlcCer must be characterized in conditions that are prohibitive to the growth of Δgcs1 (neutral pH and high CO(2)). These prohibitive conditions are physiologically similar to those found in the extracellular spaces of the lung during infection. Here, using immunofluorescence, we have shown that GlcCer localization to the cell surface is significantly increased during growth in these conditions and during infection. We further seek to exploit this localization by treatment with Cerezyme (Cz), a recombinant enzyme that metabolizes GlcCer, as a potential treatment for Cn. Cz treatment was found to reduce the amount of GlcCer in vitro, in cultures, and in Cn cells inhabiting the mouse lung. Treatment with Cz induced a membrane integrity defect in wild type Cn cells similar to Δgcs1. Cz treatment also reduced the in vitro growth of Cn in a dose and condition dependent manner. Finally, Cz treatment was shown to have a protective effect on survival in mice infected with Cn. Taken together, these studies have established the legitimacy of targeting the GlcCer and other related sphingolipid systems in the development of novel therapeutics.

Comparative therapeutic effects of velaglucerase alfa and imiglucerase in a Gaucher disease mouse model.

Gaucher disease type 1 is caused by the defective activity of the lysosomal enzyme, acid beta-glucosidase (GCase). Regular infusions of purified recombinant GCase are the standard of care for reversing hematologic, hepatic, splenic, and bony manifestations. Here, similar in vitro enzymatic properties, and in vivo pharmacokinetics and pharmacodynamics (PK/PD) and therapeutic efficacy of GCase were found with two human GCases, recombinant GCase (CHO cell, imiglucerase, Imig) and gene-activated GCase (human fibrosarcoma cells, velaglucerase alfa, Vela), in a Gaucher mouse, D409V/null. About 80+% of either enzyme localized to the liver interstitial cells and <5% was recovered in spleens and lungs after bolus i.v. injections. Glucosylceramide (GC) levels and storage cell numbers were reduced in a dose (5, 15 or 60 U/kg/wk) dependent manner in livers (60-95%) and in spleens ( approximately 10-30%). Compared to Vela, Imig (60 U/kg/wk) had lesser effects at reducing hepatic GC (p = 0.0199) by 4 wks; this difference disappeared by 8 wks when nearly WT levels were achieved by Imig. Anti-GCase IgG was detected in GCase treated mice at 60 U/kg/wk, and IgE mediated acute hypersensitivity and death occurred after several injections of 60 U/kg/wk (21% with Vela and 34% with Imig). The responses of GC levels and storage cell numbers in Vela- and Imig-treated Gaucher mice at various doses provide a backdrop for clinical applications and decisions.