Convection enhanced delivery of panobinostat (LBH589)-loaded pluronic nano-micelles prolongs survival in the F98 rat glioma model.

BACKGROUND: The pan-histone deacetylase inhibitor panobinostat is a potential therapy for malignant glioma, but it is water insoluble and does not cross the blood-brain barrier when administered systemically. In this article, we describe the in vitro and in vivo efficacy of a novel water-soluble nano-micellar formulation of panobinostat designed for administration by convection enhanced delivery (CED). MATERIALS AND METHODS: The in vitro efficacy of panobinostat-loaded nano-micelles against rat F98, human U87-MG and M059K glioma cells and against patient-derived glioma stem cells was measured using a cell viability assay. Nano-micelle distribution in rat brain was analyzed following acute CED using rhodamine-labeled nano-micelles, and toxicity was assayed using immunofluorescent microscopy and synaptophysin enzyme-linked immunosorbent assay. We compared the survival of the bioluminescent syngenic F98/Fischer344 rat glioblastoma model treated by acute CED of panobinostat-loaded nano-micelles with that of untreated and vehicle-only-treated controls. RESULTS: Nano-micellar panobinostat is cytotoxic to rat and human glioma cells in vitro in a dose-dependent manner following short-time exposure to drug. Fluorescent rhodamine-labelled nano-micelles distribute with a volume of infusion/volume of distribution (Vi/Vd) ratio of four and five respectively after administration by CED. Administration was not associated with any toxicity when compared to controls. CED of panobinostat-loaded nano-micelles was associated with significantly improved survival when compared to controls (n=8 per group; log-rank test, P<0.001). One hundred percent of treated animals survived the 60-day experimental period and had tumour response on post-mortem histological examination. CONCLUSION: CED of nano-micellar panobinostat represents a potential novel therapeutic option for malignant glioma and warrants translation into the clinic.

Triggering apoptosis in cancer cells with an analogue of cribrostatin 6 that elevates intracellular ROS.

Elevation of reactive oxygen species (ROS) is both a consequence and driver of the upregulated metabolism and proliferation of transformed cells. The resulting increase in oxidative stress is postulated to saturate the cellular antioxidant machinery, leaving cancer cells susceptible to agents that further elevate their intracellular oxidative stress. Several small molecules, including the marine natural product cribrostatin 6, have been demonstrated to trigger apoptosis in cancer cells by increasing intracellular ROS. Here, we report the modular synthesis of a series of cribrostatin 6 derivatives, and assessment of their activity in a number of cell lines. We establish that placing a phenyl ring on carbon 8 of cribrostatin 6 leads to increased potency, and observe a window of selectivity towards cancer cells. The mechanism of activity of this more potent analogue is assessed and demonstrated to induce apoptosis in cancer cells by increasing ROS. Our results demonstrate the potential for targeting tumors with molecules that enhance intracellular oxidative stress.

Convection-enhanced delivery of MANF--volume of distribution analysis in porcine putamen and substantia nigra.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a 20kDa human protein which has both neuroprotective and neurorestorative activity on dopaminergic neurons and therefore may have application for the treatment of Parkinson's Disease. The aims of this study were to determine the translational potential of convection-enhanced delivery (CED) of MANF for the treatment of PD by studying its distribution in porcine putamen and substantia nigra and to correlate histological distribution with co-infused gadolinium-DTPA using real-time magnetic resonance imaging. We describe the distribution of MANF in porcine putamen and substantia nigra using an implantable CED catheter system using co-infused gadolinium-DTPA to allow real-time MRI tracking of infusate distribution. The distribution of gadolinium-DTPA on MRI correlated well with immunohistochemical analysis of MANF distribution. Volumetric analysis of MANF IHC staining indicated a volume of infusion (Vi) to volume of distribution (Vd) ratio of 3 in putamen and 2 in substantia nigra. This study confirms the translational potential of CED of MANF as a novel treatment strategy in PD and also supports the co-infusion of gadolinium as a proxy measure of MANF distribution in future clinical studies. Further study is required to determine the optimum infusion regime, flow rate and frequency of infusions in human trials.

Convection-enhanced delivery of AAV2 in white matter--a novel method for gene delivery to cerebral cortex.

BACKGROUND: Convection-enhanced delivery (CED) is currently under investigation for delivering therapeutic agents to subcortical targets in the brain. Direct delivery of therapies to the cerebral cortex, however, remains a significant challenge. NEW METHOD: We describe a novel method of targeting adeno-associated viral vector (AAV) mediated gene therapies to specific cerebral cortical regions by performing high volume, high flow rate infusions into underlying white matter in a large animal (porcine) model. RESULTS: Infusion volumes of up to 700 µl at flow rates as high as 10 µl/min were successfully performed in white matter without adverse neurological sequelae. Co-infusion of AAV2/5-GFP with 0.2% Gadolinium in artificial CSF confirmed transgene expression in the deep layers of cerebral cortex overlying the infused areas of white matter. COMPARISON WITH EXISTING METHODS: AAV-mediated gene therapies have been previously targeted to the cerebral cortex by performing intrathalamic CED and exploiting axonal transport. The novel method described in this study facilitates delivery of gene therapies to specific regions of the cerebral cortex without targeting deep brain structures. CONCLUSIONS: AAV-mediated gene therapies can be targeted to specific cortical regions by performing CED into underlying white matter. This technique could be applied to the treatment of neurological disorders characterised by cerebral cortical degeneration.

In vitro and in vivo testing of a novel recessed-step catheter for reflux-free convection-enhanced drug delivery to the brain.

INTRODUCTION: The optimisation of convection-enhanced drug delivery (CED) to the brain is fundamentally reliant on minimising drug reflux. The aim of this study was to evaluate the performance of a novel reflux-resistant CED catheter incorporating a recessed-step and to compare its performance to previously described stepped catheters. METHODS: The in vitro performance of the recessed-step catheter was compared to a conventional "one-step" catheter with a single transition in outer diameter (OD) at the catheter tip, and a "two-step" design comprising two distal transitions in OD. The volumes of distribution and reflux were compared by performing infusions of Trypan blue into agarose gels. The in vivo performance of the recessed-step catheter was then analysed in a large animal model by performing infusions of 0.2% Gadolinium-DTPA in Large White/Landrace pigs. RESULTS: The recessed-step catheter demonstrated significantly higher volumes of distribution than the one-step and two-step catheters (p=0.0001, one-way ANOVA). No reflux was detected until more than 100 ul had been delivered via the recessed-step catheter, whilst reflux was detected after infusion of only 25 ul via the 2 non-recessed catheters. The recessed-step design also showed superior reflux resistance to a conventational one-step catheter in vivo. Reflux-free infusions were achieved in the thalamus, putamen and white matter at a maximum infusion rate of 5 ul/min using the recessed-step design. CONCLUSION: The novel recessed-step catheter described in this study shows significant potential for the achievement of predictable high volume, high flow rate infusions whilst minimising the risk of reflux.

Intermittent convection-enhanced delivery to the brain through a novel transcutaneous bone-anchored port.

Convection-enhanced delivery (CED) describes a novel method of drug delivery to the brain through intraparenchymal microcatheters. One of the barriers to effective translation of CED to clinical trials is the requirement for intermittent delivery over prolonged periods. This is particularly relevant for delivery of neurotrophins for the treatment of Parkinson's disease where chronic infusion of glial cell-line derived neurotrophic factor (GDNF) with subcutaneously implanted pumps has been associated with poor distribution and local toxicity due to point source accumulation. We have previously described the development of an implantable catheter for CED which facilitates repeated drug administrations at intervals of up to one month. The aim of this study was to determine the feasibility of implanting a transcutaneous bone-anchored port (TBAP) which facilitates chronic intermittent drug delivery to the brain. We describe the design and development of a titanium port which was implanted in Large White and NIH miniature pigs for periods of up to three months. By intermittently accessing the port with a needle administration set it was possible to repeatedly perform CED infusions at one month intervals. This study confirms the safety and feasibility of performing intermittent CED through a transcutaneous bone-anchored port. The use of a transcutaneous port has the potential to facilitate clinical translation of CED of therapeutics requiring intermittent delivery to achieve optimum efficacy whilst negating the need for subcutaneously implanted pumps.

Elemental imaging of MRI contrast agents: benchmarking of LA-ICP-MS to MRI.

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been used to map the spatial distribution of magnetic resonance imaging (MRI) contrast agents (Gd-based) in histological sections in order to explore synergies with in vivo MRI. Images from respective techniques are presented for two separate studies namely (1) convection enhanced delivery of a Gd nanocomplex (developmental therapeutic) into rat brain and (2) convection enhanced delivery, with co-infusion of Magnevist (commercial Gd contrast agent) and Carboplatin (chemotherapy drug), into pig brain. The LA technique was shown to be a powerful compliment to MRI not only in offering improved sensitivity, spatial resolution and signal quantitation but also in giving added value regarding the fate of administered agents (Gd and Pt agents). Furthermore simultaneous measurement of Fe enabled assignment of an anomalous contrast enhancement region in rat brain to haemorrhage at the infusion site.

The development of an implantable catheter system for chronic or intermittent convection-enhanced delivery.

Convection-enhanced delivery (CED) is a promising technique for the administration of therapeutic agents such as cytotoxics, neurotrophins and enzymes to the brain. In this study we describe the development of an implantable catheter system that is compatible with long-term intermittent CED. Catheters made from fused silica, PEEK or carbothane, and of various internal and external diameters were implanted in the striatum of rats and assessed for patency at 21 or 28 days. A high-rate of catheter blockage was observed with all fused silica and PEEK catheters. Carbothane catheters with an outer diameter of 0.6mm and an inner diameter of 0.35 mm had significantly lower rates of blockage (P≤0.01). Carbothane catheters were then implanted into 4 Large White/Landrace pigs and 4 NIH miniature pigs and infusions undertaken at monthly intervals to evaluate catheter patency and infusate distribution. Catheter patency was demonstrated for a maximum period of 163 days in one animal. Widespread and reproducible intraputamenal CED could be achieved with intermittent drug delivery at flow-rates as high as 5 µl/min. Problems were encountered using the pig model due to catheter distortion from rapid animal growth. In conclusion, it is possible to achieve intermittent high-flow CED with a chronic implanted carbothane catheter with a low rate of catheter blockage.

Evaluation and optimization of the administration of a selectively replicating herpes simplex viral vector to the brain by convection-enhanced delivery.

The direct intraparenchymal administration of oncolytic viral vectors by convection-enhanced delivery (CED) represents a promising new treatment strategy for malignant gliomas. However, there is no evidence to suggest that oncolytic viruses as large as herpes simplex virus-1 (HSV-1) can be administered by CED, as this has not been systematically examined in an animal model. In this study, the administration of a herpes simplex viral vector, HSV1, has been evaluated in detail in the gray and white matter of both rat and pig models, using high flow-rate infusions, co-infusing heparin or preinfusing the tissue with an isotonic albumin solution. Rat HSV-1 infusions at both slow (0.5 µl min(-1)) and high infusion rates (2.5 µl min(-1)) led to extensive tissue damage and negligible cell transduction. Co-infusion with heparin led to extensive hemorrhage. Preinfusion of tissue with an isotonic albumin solution facilitated widespread vector distribution and cell transduction in white matter only. Using this approach in pig brain led to widespread vector distribution with extensive transduction of astrocytes and activated microglia. In rat brain, enhanced green fluorescent protein expression peaked 48 h after vector administration and was associated with a vigorous immune response. These findings indicate that direct infusions of HSV-1-based viral vectors into the brain lead to minimal vector distribution, negligible cell transduction and extensive damage. Tissue preinfusion with an isotonic solution prior to vector administration represents an effective technique for achieving widespread HSV-1 distribution.

A robust MRI-compatible system to facilitate highly accurate stereotactic administration of therapeutic agents to targets within the brain of a large animal model.

Achieving accurate intracranial electrode or catheter placement is critical in clinical practice in order to maximise the efficacy of deep brain stimulation and drug delivery respectively as well as to minimise side-effects. We have developed a highly accurate and robust method for MRI-guided, stereotactic delivery of catheters and electrodes to deep target structures in the brain of pigs. This study outlines the development of this equipment and animal model. Specifically this system enables reliable head immobilisation, acquisition of high-resolution MR images, precise co-registration of MRI and stereotactic spaces and overall rigidity to facilitate accurate burr hole-generation and catheter implantation. To demonstrate the utility of this system, in this study a total of twelve catheters were implanted into the putamen of six Large White Landrace pigs. All implants were accurately placed into the putamen. Target accuracy had a mean Euclidean distance of 0.623 mm (standard deviation of 0.33 mm). This method has allowed us to accurately insert fine cannulae, suitable for the administration of therapeutic agents by convection-enhanced delivery (CED), into the brain of pigs. This study provides summary evidence of a robust system for catheter implantation into the brain of a large animal model. We are currently using this stereotactic system, implantation procedure and animal model to develop catheter-based drug delivery systems that will be translated into human clinical trials, as well as to model the distribution of therapeutic agents administered by CED over large volumes of brain.

Hsp70 suppresses apoptosis in sympathetic neurones by preventing the activation of c-Jun.

The anti-apoptotic effects of heat-shock protein (Hsp70) were assessed in SCG neurones following nerve growth factor (NGF) withdrawal. The results showed that the virally mediated expression of Hsp70 mirrored the effects of the c-Jun-N-terminal kinase (JNK) binding domain (JBD) of JNK interacting protein (an inhibitor of JNK and c-Jun activation) and suppressed the phosphorylation of c-Jun. Preventing c-Jun transcriptional activity subsequently led to reduced cytochrome c release and prevented caspase activation as indicated by a decrease in poly (ADP-ribose) polymerase-1 (PARP) cleavage. Together, these results show that Hsp70 is a highly effective inhibitor of apoptosis in sympathetic neurones and that it mediates this effect primarily by suppressing c-Jun transcriptional signalling.

Disruption of the GluR2-NSF interaction protects primary hippocampal neurons from ischemic stress.

A specific interaction between the AMPA receptor subunits GluR2 and GluR3 and the fusion protein NSF has recently been identified. Disruption of this interaction by adenoviral-mediated expression of a peptide (pep2m) corresponding to the NSF-binding region of GluR2 results in a dramatic reduction in surface expression of AMPA receptors in primary hippocampal neurons. Here we report that expression of pep2m from a recently developed neuronal-specific adenoviral system gave significant neuroprotection to primary CA1-CA3 hippocampal neurons following stimulation with kainate (KA) and this was accompanied by a reduction in Ca(2+) influx. Protection was also observed following glucose deprivation and exposure to ischemic buffer in the absence of any NMDA receptor antagonists. These results provide strong evidence that AMPA receptors play a direct role in mediating postischemic neurotoxicity.

Inhibition of JNK by overexpression of the JNL binding domain of JIP-1 prevents apoptosis in sympathetic neurons.

Studies in non-neuronal cells show that c-Jun N-terminal kinases (JNK) play a key role in apoptotic cell death. In some neurons JNK is also thought to initiate cell death by the activation of c-Jun. JNK inhibition has been achieved pharmacologically by inhibiting upstream kinases, but there has been no direct demonstration that inhibition of JNK can prevent neuronal death. We have therefore examined whether the JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1, a scaffold protein and specific inhibitor of JNK) can inhibit c-Jun phosphorylation and support the survival of sympathetic neurons deprived of NGF. We show that expression of the JBD in >80% of neurons was sufficient to prevent the phosphorylation of c-Jun and its nuclear accumulation as well as abrogate neuronal cell death induced by NGF deprivation. JBD expression also preserved the capacity of mitochondria to reduce MTT. Interestingly, although the PTB domain of JIP was reported to interact with rhoGEF, expression of the JBD domain was sufficient to localize the protein to the membrane cortex and growth cones. Hence, JNK activation is a key event in apoptotic death induced by NGF withdrawal, where its point of action lies upstream of mitochondrial dysfunction.

Targeting of tetracycline-regulatable transgene expression specifically to neuronal and glial cell populations using adenoviral vectors.

Targeting regulatable transgene expression specifically to neuronal or glial cell populations would facilitate studies of CNS gene function. We have developed the tetracycline (Tet) regulatable adenoviral system by expressing the Tet-off transactivator (tTA) under the control of the neuronal-specific synapsin I promoter and the well characterized glial-specific glial fibrillary acidic protein (GFAP) promoter. Transfection of primary hippocampal cultures demonstrated that the respective promoters restricted reporter transgene expression exclusively to neuronal or glial populations. Delivery of the vectors into adult rat hippocampus resulted in a similar pattern of cell specific transgene expression. These novel vectors provide a highly effective means of directing regulated, cell-specific, transgene expression and as such are important tools for investigations of neuronal and glial cell function and advancing gene therapy studies.