Improving the anti-tumour efficacy of Albendazole

Albendazole (ABZ) is a benzamidazole derivative that binds to β-tubulin and inhibits the polymerisation of microtubules. ABZ has a remarkable activity against a variety of tumour cell lines including colorectal, liver, and ovarian cancer cells. In pre-clinical models, ABZ has been shown to have both anti-tumour and anti-angiogenic activities. Nevertheless, poor aqueous solubility of ABZ limits its absorption, and as a result, its efficacy. Therefore, the first aim of this project was to improve the aqueous solubility of ABZ. Using a combination of ionisation with acid and complexation with hydroxypropyl-β-cyclodextrin (HPβCD), a relatively high concentration of ABZ in solution was achieved. Comparison of pharmacokinetic profiles of ABZ/HPβCD with a conventional formulation of ABZ in hydroxypropyl methylcellulose (ABZ/HPMC) in nude mice revealed that complexation with HPβCD results in a significantly higher peak plasma concentration (Cmax) and area under the curve (AUC) of ABZ and its metabolites. Moreover, in mice-bearing human colorectal cancer cells, HCT-116, ABZ/HPβCD treatment led to a significant delay in tumour growth with increase in survival of animals as compared with vehicle and ABZ/HPMC treatments.The second aim of this research was to improve the efficacy of ABZ using combination therapy. To this end, the interaction between ABZ and different chemotherapeutic agents were assessed using the Sulforhodamine B assay (SRB) and quantified by median effect analysis method. Among the tested agents, a synergistic anti-proliferative effect was observed with the combination of ABZ and 2-methoxyestradiol (2ME) in HCT-116 and DU145. Of interest, 2ME, a microtubule targeting agent binds to similar colchicine-binding site of β-tubulin as ABZ and inhibits microtubules polymerisation. Synergistic interaction of ABZ and 2ME was accompanied with the activation of extrinsic pathway of apoptosis.In vivo, the combination of low concentration of ABZ with 2ME resulted in an increase in the survival rate of mice-bearing HCT-116 tumours. This effect was accompanied by a decrease in plasma and tumour vascular endothelial growth factor (VEGF) as well as a reduction in microvessel density. In addition, combination therapy led to a significant decrease in proliferation rate of the tumour and an increase in apoptosis. Noticeably, high concentration of ABZ, in combination with 2ME, resulted in an antagonistic effect on tumour growth and survival of the animals. Taken together, the solubility and anti-tumour efficacy of ABZ was highly increased by complexation with HPβCD, leading to the conclusion that the formulation may be suitable for parenteral administration. Moreover, combination of ABZ and 2ME has shown promising results in pre-clinical model. Additionally, the finding that the combination of two microtubule-binding agents that share the same binding site can act synergistically in vitro and in vivo may lead to the development of new therapeutic strategies in cancer treatment

RARE-08. POTENTIAL NEW THERAPIES FOR DIFFUSE INTRINSIC PONTINE GLIOMAS IDENTIFIED THROUGH HIGH THROUGHPUT DRUG SCREENING

Diffuse Intrinsic Pontine Gliomas (DIPGs) are the most devastating of all brain tumors. There are no effective treatments, hence almost all children will die of their tumor within 12-months. There is an urgent need for novel effective therapies for this aggressive tumor. We performed a high-throughput drug screen with over 3,570 biologically active, clinically approved compounds against a panel of neurosphere-forming DIPG cells. We identified 7 compounds - auranofin, fenretinide, ivermectin, lanatoside, parthenolide, SAHA and mefloquine - that were confirmed to have potent anti-tumor activity against a panel of DIPG-neurospheres, with minimal effect on normal cells. Using cytotoxicity and clonogenic assays, we found that these drugs were able to inhibit DIPG-neurosphere proliferation and colony formation in vitro. To determine whether the in vitro efficacy could be replicated in vivo, we tested the activity of each of these compounds in an orthotopic DIPG model. Of the agents tested, fenretinide, auranofin and SAHA were the most active anti-tumor agents, significantly enhancing the survival of tumor bearing animals. Mechanistic studies showed fenretinide enhancing apoptotic cell death of DIPG cells via inhibition of PDGFRa transcription and downregulation of the PI3K/AKT/MTOR pathway. We therefore examined the therapeutic efficacy of fenretinide using a second orthotopic model with PDGFRa amplification. We used two different fenretinide formulations which were found to enhance survival. Fenretinide is clinically available with safety data in children. Validation of the activity of Fenretinide in PDGFRa-amplified or overexpressed DIPGs will lead to the development of a clinical trial, allowing the advancement of fenretinide as potentially the first active therapy for DIPG

DIPG-07. HIGH THROUGHPUT DRUG SCREENING IDENTIFIES POTENTIAL NEW THERAPIES FOR DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPGs)

DIPGs are the most devastating of all brain tumors. There are no effective treatments, hence almost all children will die of their tumor within 12 months. There is an urgent need for novel effective therapies for this aggressive tumor. We performed a high-throughput drug screen with over 3,500 biologically active, clinically approved compounds against a panel of neurosphere-forming DIPG cells. We identified 7 compounds- auranofin, fenretinide, ivermectin, lanatoside, parthenolide, SAHA and mefloquine- that were confirmed to have potent anti-tumor activity against a panel of DIPG-neurospheres, with minimal effect on normal cells. Using cytotoxicity and clonogenic assays, we found that these drugs were able to inhibit DIPG-neurosphere proliferation and colony formation in-vitro. To determine whether the in-vitro efficacy could be replicated in-vivo, we tested the activity of each of these compounds in an orthotopic DIPG model. Of the agents tested, fenretinide and SAHA were the most active anti-tumor agents, significantly enhancing the survival of tumor bearing animals. Mechanistic studies showed fenretinide enhancing apoptotic cell death of DIPG cells via inhibition of PDGFRa transcription and downregulation of the PI3K/AKT/MTOR pathway. We therefore examined the therapeutic efficacy of fenretinide using a second orthotopic model with PDGFRa amplification. We used two different Fenretinide formulations (LYM-X-Sorb and NanoMicelle) which were found to enhance survival. Fenretinide is clinically available with safety data in children. Validation of the activity of Fenretinide in PDGFRa-amplified or overexpressed DIPGs will lead to the development of a clinical trial, allowing the advancement of fenretinide as potentially the first active therapy for DIPG

S100 protein, an early and prognostic serum marker for brain damage following cardiac surgery

S100 protein is a marker for brain damage easily analysed in blood. The serum level immediatly after termination of extracorporeal circulation (ECC) is increased and associated to age and the duration of perfusion. A prolonged residual concentration indicates cellular brain damage. The objective of this study was to ascertain if the prognosis of individual patients, with stroke following ECC, could be predicted by the S100 levels. 224 patients with an uneventful outcome constituted a reference population. Blood samples for the analysis of S100 were collected at termination of ECC (TO) and at 5, 15 and 48 hours thereafter. Eight patients with stroke following ECC were analysed. In 4, S100 levels continued to increase after ECC. The volume of brain infarctions were large and in 3 of the patients outcome was fatal. In 4 other patients with small volume brain injury S100 dropped initially, but after 5 hours the elimination slowed down, indicating a small but persistant release. In 3 of these patients recovery was complete or with minor residuals. The fourth patient never regained consciousness and died. The levels from serial S100 measurements early after cardiac surgery were related to the size of brain injury and a prognostic value is therefore suggested

No overall survival benefit with panobinostat treatments <i>in vivo</i>.

<p><b>(A)</b> Mice with tumors driven by PDGF-B and p53 loss harboring H3.3-K27M mutations (generated as described in Materials and Methods) were treated with 20 mg/kg panobinostat (n = 11) or vehicle (25% DMSO, 0.25x PBS, 5% glucose, n = 8) administered via intraperitoneal (i.p.) injection once per day twice a week beginning 21 days post-virus injection and continuing until mice reached humane endpoints. The mice were monitored daily and sacrificed upon moribund condition (lethargy, enlarged head circumference, ataxia, and/or > 25% weight loss) (p = 0.756, log rank test). <b>(B-D)</b> Effect of panobinostat treatment on the survival of mice-bearing H3.3-K27M HSJD-DIPG-007 orthotopic xenografts. NOD-SCID mice (7 weeks old) were orthotopically injected with HSJD-DIPG-007 cells (passage 39) into the brainstem via stereotactic coordinates. (<b>B</b>) Representative H&E (top panel) and IHC staining for Ki67 (middle panel) and Vimentin (bottom panel) of control mice (treated with vehicle and sacrificed immediately after the last dose, as described in the Materials and Methods). 400x magnification, scale bar = 50 μm. (<b>C-D</b>) Starting on day 28 post-implantation, mice were treated with panobinostat prepared in a vehicle containing 5% dextrose (<b>C</b>) or 2.5% DMSO, 5% PEG400 and 5% Tween80 in 0.9% saline (<b>D</b>) via intraperitoneal (i.p.) injection at 10 mg/kg, three times a week for four weeks (<i>p</i>>0.05, log-rank test). Shaded areas under the curves in <b>A, C-D</b> indicate treatment duration.</p

Short-term <i>in vivo</i> treatments with panobinostat reduces tumor cell proliferation and increases H3 acetylation without evidence of apoptosis.

<p>Neonatal Ntv-a;p53-fl/fl mice were injected with RCAS-PDGF-B, -H3.3-K27M, and–Cre viruses to induce tumor formation. Upon the first appearance of brain tumor symptoms (3–5 weeks post-injection), mice were treated with five doses of 20 mg/kg panobinostat (Drug) or vehicle (Veh, 25% DMSO, 0.25x PBS, 5% glucose, n = 6 in each group) once daily via i.p. injections and then sacrificed 1 hour after the final treatment. Shown is immunohistochemistry of brain tumor tissue for cell proliferation (phospho histone H3, pH3) <b>(A)</b>, cell apoptosis (cleaved caspase-3, cc3) (<b>B</b>), and H3 acetylation (AcH3) <b>(C</b>), 400x, scale bar = 50 μm, with quantification of total positive nuclear staining as a percentage of total nuclear area below in each panel. Statistical significance was determined using unpaired two-tailed t-test to compare groups. (ns: nonsignificant).</p