Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Salinomycin inhibits HepG2 and Huh7 autophagic flux.

<p>HepG2 and Huh7 cells were exposed to different concentrations of Salinomycin (0.5, 2 and 8 µM) for different time points and analyzed for activity of autophagy. (<b>A</b>) Immunoblot analysis of LC3-I and LC3-II-isoforms (up) with densitometry quantitative analysis (down) in HepG2 cells revealed Sal-induced LC3-II-accumulation due to inhibition of autophagic flux as demonstrated by reduced LC3-II-accumulation after addition of ACH. (<b>B</b>) Basic and PP242-activated autophagic flux in Huh7 cells (black bars). Treatment with autophagy inhibitors 3MA (2 and 10 mM), ACH (5 and 20 mM) or CQ (5, 20 µM) for 24 h counteracts PP242 activation of autophagic flux. (<b>C</b>) Inhibition of autophagic flux in Huh7 cells after shRNA-mediated knockdown of ATG7. (<b>D+E</b>) Decreased accumulation of autophagic compartments after the blockage of autophagolysosomal degradation by ACH indicates reduced autophagic flux in HepG2 (<b>D</b>) and Huh7 (<b>E</b>) cells treated with Sal for 24 h. Next to the bar graphs representative histograms are depicted. All experiments are presented as mean ± SD of n = 3 independent experiments.*p<0.05; **p<0.01, ***p<0.001.</p

Impact of Salinomycin-treatment on PHH synthesis function.

<p>Functionality of PHH after treatment with Salinomycin was analyzed by urea formation and albumin synthesis. (<b>A</b>) 24 hours of Salinomycin exposure at varying concentrations was not accompanied by impaired urea formation at all. (<b>B</b>) In contrast, treatment for 48 hours resulted in a dose-dependent decrease of urea formation at days 1, 3 and 5 without reaching statistical significance. (<b>C+D</b>) Albumin synthesis was markedly impaired at day 1 after stimulation with Salinomycin for 24 and 48 hours, respectively. Further incubation led to continuous recovery of albumin synthesis in the groups with 24 hours of drug exposure. In contrast, treatment for 48 hours resulted only in moderate recovery of albumin synthesis (n = 3). * p<0.05, ** p<0.005.</p

Salinomycin-mediated inhibition of autophagic flux relates to accumulation of dysfunctional mitochondria and increased ROS-production.

<p>(<b>A</b>) Inhibition of basic and HBSS-activated autophagic flux in HepG2 cells stably expressing LC3-GFP treated with 3 MA (2 and 10 mM), LY (2 and 10 mM), nocodazole (2 and 10 µM) or ACH (0.8 and 4 mM) for 7 h. (<b>B</b>) Inhibition of autophagic flux in HepG2 cells treated with Sal (0.5, 2 and 8 µM) for 24 h (left) with representative histograms (right). (<b>C</b>) Flow-cytometric analysis of total mitochondrial mass using MitoTracker Green (MTR green) reveals accumulation of dysfunctional mitochondria. (<b>D</b>) Evidence of increased ROS-production using CM-H2DCFDA (left) with representative histograms (right). All experiments are presented as mean ± SD of n = 3 independent experiments. *p<0.05, **p<0.01.</p

Impact of Salinomycin-treatment on <i>in vitro</i> morphology of PHH.

<p>After one day of incubation cultured PHH were exposed to increasing concentrations of Salinomycin (1, 2, 5 and 10 µM) at varying time of exposure (24 and 48 hours, respectively). (<b>A</b>) Impairment of <i>in vitro</i> morphology without subsequent recovery were only detectable after treatment with high concentrations of Salinomycin (10 mM) whereas treatment with up to 5 µM Salinomycin resulted in morphological recovery after lapse of the agent. (<b>B+C</b>) Cell viability was assessed by MTS-assay. Salinomycin treatment for 24 (<b>B</b>) and 48 (<b>C</b>) hours led to significantly impaired cell viability at day 1 and 3. Upon further incubation recovery of the cells was observed as indicated by increasing production of the coloured formazaan-product (n = 6). (<b>D+E</b>) Cell damage of PHH as represented by AST release was only detectable immediately after drug exposure (day 1) to 10 µM Salinomycin for 24 (<b>D</b>) and 48 (<b>E</b>) hours, respectively. This difference did not reach statistical significance (n = 3). Ongoing incubation was accompanied by barely measurable AST release. * p<0.05, ** p<0.005.</p

Salinomycin impairs HCC cell survival.

<p>HepG2 and Huh7 cells were exposed to increasing concentrations of Salinomycin (1, 2, 5 and 10 µM) for 24 hours. (<b>A</b>) Cell viability was assessed by MTS-assay; high concentrations of Salinomycin led to significantly decreased viability of both cell lines (n = 5). (<b>B</b>) HCC cells revealed significantly reduced proliferation after exposure to Salinomycin as demonstrated by decreased ³H-Thymidine uptake. (<b>C</b>) Low concentrations of Salinomycin (left panel, solid line) led to weak increase of apoptotic cells compared to untreated cells (dotted line in overlay). High concentrations markedly induced apoptosis (right panel, solid line). Results are shown as representative scatter-grams of Annexin-V⁺-cells or summarized as mean ± SD of n = 4 independent experiments (<b>D</b>). *p<0.05, **p<0.01.</p

Assessment of apoptosis-induction in PHH by Salinomycin.

<p>Cultured PHH were exposed to increasing concentrations of Salinomycin (1, 2, 5 and 10 µM) for 24 hours. Apoptosis was detected by M30 Cytodeath kit. Treatment with Fas Ligand (FasL) served as a positive control. Staining of PHH with DAPI (blue) and M30 Cytodeath kit (red) revealed no evidence for induction of apoptosis in PHH even after treatment with high concentrations of Salinomycin. In contrast, FasL clearly induced apoptosis in PHH.</p

50 години Катедра „Социална медицина и организация на здравеопазването`

22 Май 201