Toxicity assessment after dose-escalation study in pigs.

<p>A dose-escalation study comparing CED of carboplatin NP with aCSF (co-infused with 0.2% Gd) was conducted in a porcine model. (A) Widespread putamenal distribution was observed using real-time MR imaging (B) Fluorescence immunohistochemistry demonstrates intact neuronal (E & L) and glial (D & K) networks at the site of infusion. Repeat dosing of carboplatin NP was not associated with increased macrophage activation compared to control (G & N). Minimal toxicity was also confirmed by maintenance of cellular architecture (H & O) (Scale bar; 250μm in magnified images, 100μm in images showing entire section (B&I)</p

Repurposing the anti-epileptic drug sodium valproate as an adjuvant treatment for diffuse intrinsic pontine glioma

<div><p>Targeting epigenetic changes in diffuse intrinsic pontine glioma (DIPG) may provide a novel treatment option for patients. This report demonstrates that sodium valproate, a histone deacetylase inhibitor (HDACi), can increase the cytotoxicity of carboplatin in an additive and synergistic manner in DIPG cells <i>in vitro</i>. Sodium valproate causes a dose-dependent decrease in DIPG cell viability in three independent <i>ex vivo</i> cell lines. Furthermore, sodium valproate caused an increase in acetylation of histone H3. Changes in cell viability were consistent with an induction of apoptosis in DIPG cells <i>in vitro</i>, determined by flow cytometric analysis of Annexin V staining and assessment of apoptotic markers by western blotting. Subsequently, immunofluorescent staining of neuronal and glial markers was used to determine toxicity in normal rat hippocampal cells. Pre-treatment of cells with sodium valproate enhanced the cytotoxic effects of carboplatin, in three DIPG cell lines tested. These results demonstrate that sodium valproate causes increased histone H3 acetylation indicative of HDAC inhibition, which is inversely correlated with a reduction in cell viability. Cell viability is reduced through an induction of apoptosis in DIPG cells. Sodium valproate potentiates carboplatin cytotoxicity and prompts further work to define the mechanism responsible for the synergy between these two drugs and determine <i>in vivo</i> efficacy. These findings support the use of sodium valproate as an adjuvant treatment for DIPG.</p></div

<i>In vitro</i> cytotoxicity and uptake of carboplatin NP.

<p>(A) Carboplatin NP showed increased cytotoxicity over 48 hours. The cytotoxic effect of the carboplatin nanoparticles (Carboplatin NP) were assessed by MTT assay in UPAB and SNB19 human glioblastoma multiforme (GBM) cell lines. For UPAB, 0.18mg/ml and for SNB19, 0.03mg/ml carboplatin was used as these concentrations represent the IC50 after 72 hours. Paired <i>t-test</i> statistical analysis comparing cytotoxicity revealed significant differences for SNB19 at 24hours (p = 0.001) and 48 hours (p = 0.004), indicated by asterix. (B) Uptake of fluorescein-labelled carboplatin NP (green) occurs within 24 hours of dosing. Cells were dosed and fixed after defined periods of culture. Cells were stained with phalloidin (red), to visualise actin cytoskeleton and DAPI (blue) for cell nuclei. Uptake into cells is indicated by white arrows.</p

CED of carboplatin NP into the striatum of rat brains.

<p>Immediately after CED, nanoparticles distribute throughout the striatum. (A) CED of aCSF alone (5μl volume) or fluorescein-labelled carboplatin NP into the striatum of rat brain was conducted (1mg/ml; 5μl volume). These were analysed by sectioning around the needle track and counterstaining with DAPI (Scale bar; 250μm). (B) Carboplatin NP show increased tissue retention after 24 and 48 hours. Following CED of carboplatin NP (1mg/ml; 5μl volume), punches of the brain tissue obtained around the site of infusion were analysed by ICP-MS. Paired <i>t-test</i> analysis revealed a significant increase in the tissue half-life of carboplatin NP at 24 hours (indicated by asterix).</p

<i>In vitro</i> neurotoxicity in primary rat hippocampal cultures.

<p>Carboplatin NP are less toxic to neurones compared to the free drug. Primary rat brain hippocampal cultures were dosed with either carboplatin (0.03mg/ml) or carboplatin NP (1mg/ml) and assayed after 72 hours of culture. (A) MTT analysis shows significant increase in cell viability with carboplatin NP (p<0.001). (B) Carboplatin alone causes deregulation of the neurones and loss of the glial cells, whilst carboplatin NP retained neuronal connections and glial cells. Immunofluorescent analysis of the neurones (B3tubulin; green) and glial cells (GFAP; red). Cells were counterstained with DAPI to visualise cell nuclei (blue) (Scale bar; 100μm).</p

Toxicity analysis after CED into the striatum of rat brains.

<p>CED of aCSF (negative control), carboplatin (0.72mg/ml; 5μl volume) (or carboplatin NP (1mg/ml; 5μl volume) was conducted and toxicity in rat striatum assessed. Dual IHC analysis of neurons (NeuN) and Glial cell (GFAP) demonstrated minimal toxicity localised to the needle track. No glial or neuronal cell loss was observed elsewhere. Scale bar: 100μm.</p

<i>In vitro</i> primary neuronal and glial cell toxicity analysis.

<p>Primary rat hippocampal cells were dosed with valproate for 72 hours and assessed by immunofluorescent staining of neuronal (B3 tubulin) and glial (GFAP) markers. Hippocampal cultures show intact neuronal networks at 5 mM valproate and glial cells with normal morphology indicative of no significant toxicity.</p

Characterisation of carboplatin NP.

<p>(A) Carboplatin NP show a consistent spherical shape, less than 200nm in size. Morphological analysis of the nanoparticles was conducted using TEM scaled at 200nm. (B) Approximately 100% of carboplatin is released within 24 hours. Release profile of carboplatin from nanoparticles was conducted by analysing platinum content by ICP-MS at defined timepoints.</p

Valproate induced apoptosis in <i>ex vivo</i> DIPG cells <i>in vitro</i>.

<p>Cells were seeded in 12-well tissue culture plates and treated with sodium valproate for 72 hours prior to analysis by flow cytometry for Annexin V and 7-AAD. (A) Dose responses from at least 3 independent experiments were assessed and demonstrate an increase in Annexin V binding occurs from 1–10 mM valproate, with a significant induction of apoptosis occurring at doses of ≥5 mM valproate (p = <0.05). (B) Representative images of apoptosis analysis, all experiments were compensated based on single dyes alone. (C) To confirm the induction of apoptosis western blotting was carried out on all three cell lines treated with ≤5 mM valproate for 72 hours. * denotes statistical significance p = <0.05.</p