Focused Ultrasound-Induced Suppression of Auditory Evoked Potentials in Vivo.

The goal of this study was to determine the feasibility of focused ultrasound-based neuromodulation affecting auditory evoked potentials (AEPs) in animals. Focused ultrasound-induced suppression of AEPs was performed in 22 rats and 5 pigs: Repetitive sounds were produced, and the induced AEPs were recorded before and repeatedly after FUS treatment of the auditory pathway. All treated animals exhibited a decrease in AEP amplitude post-treatment in contrast to animals undergoing the sham treatment. Suppression was weaker for rats treated at 2.3 W/cm2 (amplitudes decreased to 59.8 ± 3.3% of baseline) than rats treated at 4.6 W/cm2 (36.9 ± 7.5%, p <0.001). Amplitudes of the treated pigs decreased to 27.7 ± 5.9% of baseline. This effect lasted between 30 min and 1 mo in most treated animals. No evidence of heating during treatment or later brain damage/edema was observed. These results demonstrate the feasibility of inducing significant neuromodulation with non-thermal, non-invasive, reversible focused ultrasound. The long recovery times may have clinical implications.

Liposomal temozolomide drug delivery using convection enhanced delivery.

Even though some progress in diagnosis and treatment has been made over the years, there is still no definitive treatment available for Glioblastoma multiforme (GBM). Convection-enhanced delivery (CED), a continuous infusion-mediated pressure gradient via intracranial catheters, studied in clinical trials, enables in situ drug concentrations several orders of magnitude greater than those achieved by systemic administration. We hypothesized that the currently limited efficacy of CED could be enhanced by a liposomal formulation, thus achieving enhanced drug localization to the tumor site with minimal toxicity. We hereby describe a novel approach for treating GBM by CED of liposomes containing the known chemotherapeutic agent, temozolomide (TMZ). A new technique for encapsulating TMZ in hydrophilic (PEGylated) liposomes, characterized by nano-size (121nm), low polydispersity index (<0.13) and with near-neutral charge (-ʒ,0.2mV), has been developed. Co-infusion of PEGylated Gd-DTPA liposomes and TMZ-liposomes by CED in GBM bearing rats, resulted in enhanced tumor detection with longer residence time than free Gd-DTPA. Treatment of GBM-bearing rats with either TMZ solution or TMZ-liposomes resulted in greater tumor inhibition and significantly higher survival. However, the longer survival and smaller tumor volumes exhibited by TMZ liposomal treatment in comparison to TMZ in solution were insignificant (p<0.053); and only significantly lower edema volumes were observed. Thus, there are no clear-cut advantages to use a liposomal delivery system of TMZ via CED over a drug solution.

Combined local blood-brain barrier opening and systemic methotrexate for the treatment of brain tumors.

Despite aggressive therapy, existing treatments offer poor prognosis for glioblastoma multiforme patients, in part due to poor penetration of most drugs across the blood-brain barrier (BBB). We propose a minimal-invasive combined treatment approach consisting of local BBB disruption in the tumor in parallel to systemic drug administration. Local BBB disruption is obtained by convection-enhanced delivery of a novel BBB disruption agent, enabling efficient/targeted delivery of the systemically administered drug by the tumors own vasculature. Various human serum albumin (HSA) analogs were synthesized and screened for BBB disruption efficacy in custom in vitro systems. The candidate analogs were then delivered into naïve rat brains by convection-enhanced delivery and screened for maximal BBB disruption and minimal brain toxicity. These studies found a noncationized/neutralized analog, ethylamine (EA)-HSA, to be the optimal BBB-opening agent. Immunocytochemical studies suggested that BBB disruption by EA-HSA may be explained by alterations in occludin expression. Finally, an efficacy study in rats bearing intracranial gliomas was performed. The rats were treated by convection-enhanced delivery of EA-HSA in parallel to systemic administration of Methotrexate, showing significant antineoplastic effects of the combined approached reflected in suppressed tumor growth and significantly (~x3) prolonged survival.

Delayed contrast extravasation MRI: a new paradigm in neuro-oncology.

BACKGROUND: Conventional magnetic resonance imaging (MRI) is unable to differentiate tumor/nontumor enhancing tissues. We have applied delayed-contrast MRI for calculating high resolution treatment response assessment maps (TRAMs) clearly differentiating tumor/nontumor tissues in brain tumor patients. METHODS: One hundred and fifty patients with primary/metastatic tumors were recruited and scanned by delayed-contrast MRI and perfusion MRI. Of those, 47 patients underwent resection during their participation in the study. Region of interest/threshold analysis was performed on the TRAMs and on relative cerebral blood volume maps, and correlation with histology was studied. Relative cerebral blood volume was also assessed by the study neuroradiologist. RESULTS: Histological validation confirmed that regions of contrast agent clearance in the TRAMs >1 h post contrast injection represent active tumor, while regions of contrast accumulation represent nontumor tissues with 100% sensitivity and 92% positive predictive value to active tumor. Significant correlation was found between tumor burden in the TRAMs and histology in a subgroup of lesions resected en bloc (r(2) = 0.90, P < .0001). Relative cerebral blood volume yielded sensitivity/positive predictive values of 51%/96% and there was no correlation with tumor burden. The feasibility of applying the TRAMs for differentiating progression from treatment effects, depicting tumor within hemorrhages, and detecting residual tumor postsurgery is demonstrated. CONCLUSIONS: The TRAMs present a novel model-independent approach providing efficient separation between tumor/nontumor tissues by adding a short MRI scan >1 h post contrast injection. The methodology uses robust acquisition sequences, providing high resolution and easy to interpret maps with minimal sensitivity to susceptibility artifacts. The presented results provide histological validation of the TRAMs and demonstrate their potential contribution to the management of brain tumor patients.

Dynamic effects of point source electroporation on the rat brain tissue.

In spite of aggressive therapy, existing treatments offer poor prognosis for glioblastoma multiforme due to tumor infiltration into the surrounding brain as well as poor blood-brain barrier penetration of most therapeutic agents. In this paper we present a novel approach for a minimally invasive treatment and a non-invasive response assessment methodology consisting of applying intracranial point-source electroporation and assessing treatment effect volumes using magnetic resonance imaging. Using a unique setup of a single intracranial electrode and an external surface electrode we treated rats' brains with various electroporation protocols and applied magnetic resonance imaging to study the dependence of the physiological effects on electroporation treatment parameters. The extent of blood-brain barrier disruption and later volumes of permanent brain tissue damage were found to correlate significantly with the treatment voltages (r(2)=0.99, p<0.001) and the number of treatment pulses (r(2)=0.94, p<0.002). Blood-brain barrier disruption depicted 3.2±0.3 times larger volumes than the final permanent damage volumes (p<0.0001). These results indicate that it may be beneficial to use more than one modality of electroporation when planning a treatment for brain tumors.

Using MRI for the assessment of paraoxon-induced brain damage and efficacy of antidotal treatment.

Organophosphate intoxication induces neural toxicity as demonstrated in histological analysis of poisoned animals. Diffusion-weighted magnetic resonance imaging (DWMRI) enables early noninvasive characterization of biological tissues based on their water diffusion characteristics. Our objectives were to study the application of MRI for assessment of paraoxon-induced brain damage and the efficacy of antidotal treatments. Seventy-six rats were poisoned with paraoxon followed by treatment with atropine and obidoxime. The rats were then divided into five treatment groups consisting of midazolam after 1 or 30 min, scopolamine after 1 or 30 min and a no anticonvulsant treatment group. Five untreated rats served as controls. Animals underwent MRI on days 1, 8, 15, 29 and 50 post poisoning. Histological evaluation was performed on representative rat brains. Acute DWMRI effects, such as enhancement of temporal brain regions, and chronic effects such as ventricular enlargement and brain atrophy, depicted on T2-weighted MRI, were significantly more prominent in late anticonvulsant treatment groups. There was no significant difference between the neuroprotective effects of midazolam and scopolamine as shown by DWMRI. Early MRI abnormalities were found to correlate significantly with histological analysis of samples obtained 15 days post treatment. In conclusion, our results demonstrate the feasibility of using DWMRI for depiction of early cytotoxic response to paraoxon and T2-weighted MRI for later changes, thus enabling assessment of early/late brain damage as well as treatment efficacy in rats. The ability to depict these changes early and noninvasively may be applied clinically in the acute phase of organophosphate poisoning.

Convection-enhanced delivery of methotrexate-loaded maghemite nanoparticles.

Convection-enhanced delivery (CED) is a novel approach for delivering drugs directly into brain tumors by intracranial infusion, enabling the distribution of high drug concentrations over large tissue volumes. This study was designed to present a method for binding methotrexate (MTX) to unique crystalline, highly ordered and superparamagnetic maghemite nanoparticles via human serum albumin (HSA) coating, optimized for CED treatments of gliomas. Naked nanoparticles and HSA- or polyethylene glycol (PEG)-coated nanoparticles with/without MTX were studied. In vitro results showed no toxicity and a similar cell-kill efficacy of the MTX-loaded particles via HSA coating to that of free MTX, while MTX-loaded particles via PEG coating showed low efficacy. In vivo, the PEG-coated nanoparticles provided the largest distributions in normal rat brain and long clearance times, but due to their low efficacy in vitro, were not considered optimal. The naked nanoparticles provided the smallest distributions and shortest clearance times. The HSA-coated nanoparticles (with/without MTX) provided good distributions and long clearance times (nearly 50% of the distribution volume remained in the brain 3 weeks post treatment). No MTX-related toxicity was noted. These results suggest that the formulation in which HSA was bound to our nanoparticles via a unique precipitation method, and MTX was bound covalently to the HSA, could enable efficient and stable drug loading with no apparent toxicity. The cell-kill efficacy of the bound MTX remained similar to that of free MTX, and the nanoparticles presented efficient distribution volumes and slow clearance times in vivo, suggesting that these particles are optimal for CED.

Specific lipoplex-mediated antisense against Bcl-2 in breast cancer cells: a comparison between different formulations.

G3139 is an antisense oligonucleotide (ODN) that can down-regulate bcl-2, thus potentially acting as a potent anticancer drug. However, effective therapy requires efficient ODN delivery, which may be achieved by employing G3139 lipoplexes. Yet, lipofection is a complex, multifactorial process that is still poorly understood. In order to shed more light on this issue, we prepared 18 different G3139 lipoplex formulations and compared them in terms of their capability to transfect MCF-7 breast cancer cells. Each formulation was composed of a cationic lipid and sometimes a helper lipid. The cationic lipid was either DOTAP (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride), DC-CHOL (3ss[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol), or CCS (ceramide carbomoyl spermine). The helper lipid was either DOPC, DOPE, or cholesterol. Each lipid combination existed in two different structural forms--either large unilamellar vesicles (approximately 100 nm LUV) or unsized heterolamellar vesicles (UHV). Cell proliferation assays were used to evaluate the cytotoxicity of G3139 lipoplexes, control cationic lipid assemblies, and free G3139. Western blots were used to confirm the specific activity of G3139 as an anti-bcl-2 antisense agent. We determined that treatment of MCF-7 cells with G3139:CCS lipoplexes (UHV-derived) produced a maximal 50-fold improvement in antisense efficacy compared to treatment with free G3139. The other G3139 lipoplexes were not superior to free G3139. Thus, successful lipofection requires precise optimization of lipoplex lipid composition, structure, and concentration.