David Needham

Abstract TITLE Tumor Ablation and Immune Microenvironment Modulation Following Laser Interstitial Thermal Therapy in a Murine Glioblastoma Model AUTHORS Ashley Puentes, Prazwal Athukuri, MD, Karina V. Moreno, Yuhui Yang, MS, Malcolm F. MacDonald, Anantha Marisetty, PhD, Sungho Lee, MD, Khatri Latha, PhD, David Needham, PhD, DSc, Ganesh Rao, MD ABSTRACT Glioblastoma (GBM) is an aggressive brain tumor with limited treatment options, particularly when surgical resection is not feasible. Laser interstitial thermal therapy (LITT) is a minimally invasive technique that delivers focused thermal ablation to tumor tissue. We evaluated the efficacy and immunological effects of LITT using a murine GBM model. C57BL/6 mice were orthotopically implanted with GL261-luciferase cells in the right frontal cortex. Five days post-implantation, mice underwent LITT or Sham treatment (laser fiber placement without activation). LITT was administered using a 1064-nm laser probe with thermal regulation maintained at 46°C. Tumor progression was monitored via bioluminescent imaging (BLI), revealing significant signal reductions at days 3, 7, and 14 in LITT-treated mice, whereas Sham controls exhibited progressive tumor growth. H&E staining confirmed well-demarcated ablation zones with central necrosis. Immunofluorescence analyses showed increased activation of Iba1+ microglia/macrophages and infiltration of CD3+, CD4+, and CD8+ T cells following LITT, especially at early time points. LITT also disrupted the blood-brain barrier, as evidenced by Evans blue dye extravasation. Survival analysis demonstrated a significant benefit for LITT-treated mice compared to Sham controls (p = 0.0287). These findings establish LITT as an effective approach for reducing tumor burden and promoting immune cell infiltration in a preclinical GBM model. The observed microglial activation and T cell recruitment suggest that LITT may enhance responsiveness to immunotherapy, particularly checkpoint inhibitors. This combinatorial strategy may offer new therapeutic opportunities for GBM, especially in cases where surgical resection is not feasible.

Objective: To manufacture and characterize a modified niclosamide stearate (NS) prodrug therapeutic (mNSPT) for use in a small clinical trial in a metastatic canine osteosarcoma (OS) model. Animals: 10 dogs that presented for treatment of nondetectable metastatic OS underwent resection of primary tumors prior to systemic therapy. Four cycles of IV carboplatin (300 mg/m2, IV, q 3 wk) followed by 4 cycles of the experimental mNSPT (10 mg/kg, IV, weekly). Posttreatment surveillance included physical examination and thoracic radiographs every 3 months for 2 years. Samples for pharmacokinetic analysis were taken at the end of the 0.5- to 1-hour IV infusion of the mNSPTs and followed for 2 hours. Clinical Presentation: The NS average concentration at the end of infusion was 134.88 ± 13.32 μg/mL; the average area under the curve was 211.62 ± 27.89 h·µg/mL. The niclosamide concentration at the end of infusion was 23.11 μg/mL ± 3.77 μg/mL, and the area under the curve was 27.52 ± 5.92 h·µg/mL, well above the NSPT 0.75 μg/mL (1.27 μM) cell-effective concentrations for OS cells in culture. The average terminal half-life was 4.57 hours for NS and 3.23 hours for niclosamide. Three dogs developed metastatic disease on carboplatin and did not receive mNSPT. The median time to tumor progression and OS of the 7 treated dogs was 510 and 632 days, respectively, with 3 dogs living > 4 years. Results: The results support further translational investigation of this novel therapeutic approach to OS treatment in a randomized, prospective phase III study in dogs and, if successful, ultimately in OS patients. Clinical Relevance: These results suggest that niclosamide-when transformed into the highly bioavailable NSPT-may have potential as a novel therapeutic agent for treating OS.

As a helicopter descends towards a bed of sand, a high velocity particle laden cloud can form around the helicopter body, a phenomenon known as “brownout”, and a consequence of which can potentially be a significant deterioration in visibility for the helicopter pilot. Here we consider a recently developed physically based rational mathematical model for the generation of wind-driven particle flow fields from otherwise static particle beds, one application of which is the scenario considered here. We introduce a directional opacity measure, defined for each observation angle from the helicopter cockpit, and show how visibility may vary in the model as certain parameters are varied. In particular, we demonstrate a counterintuitive result suggesting that, with specific yet potentially realistic parameter choices, pilot visibility may be improved in some viewing directions if the helicopter were hovering at a lower altitude.

• LDLR-OTNs achieved selective receptor-mediated uptake in breast cancer cells. • Nanoparticles prepared were ∼ 30 nm with > 99% peptide conjugation efficiency. • LDLR-OTNs showed rapid uptake and lowered IC 50 values significantly. • Uptake kinetics revealed rapid, exponential uptake of LDLR-OTNs. • Receptor mediated uptake confirmed through suramin-blocking studies. Many cancers overexpress low-density lipoprotein receptors (LDLR), facilitating cholesterol metabolism for tumour growth. Targeting LDLR offers a promising strategy for selective drug delivery. Orlistat, a fatty acid synthase (FAS) inhibitor, has shown anti-cancer potential, particularly in tumours with high FAS expression. This study introduces an LDLR-Orlistat Targeted Nanoparticles (LDLR-OTNs) to enhance cancer cell uptake via LDLR-mediated endocytosis. The objectives include synthesizing lipid-based orlistat nanoparticles, functionalizing them with an 11-mer LDLR-binding peptide, assessing uptake and cytotoxicity in three LDLR- and FAS-expressing breast cancer cell lines (BT-474, MDA MB 453, MCF-7), and comparing uptake kinetics with non-targeted nanoparticles. Orlistat nanoparticles (ONs) were synthesised via rapid solvent exchange, producing uncoated ONs, POPC-coated ONs (POPC-ONs), and LDLR-targeted ONs (LDLR-OTNs). Targeting was achieved by conjugating an 11-mer binding peptide (RLTRKRGLKLA) to DSPE-PEG5000 maleimide via click chemistry, confirmed by Ellman’s test. Nanoparticles were characterised using DLS and TEM. Cellular uptake over 24 hours was assessed using fluorescence-labelled POPC-ONs and LDLR-OTNs, and uptake kinetics were analysed. Suramin-blocking studies were used to confirm LDLR-mediated uptake. A 48-hour cytotoxicity assay quantified IC 50 values in the aforementioned cell lines. TEM data showed that LDLR-OTNs (33 nm) were smaller than untargeted POPC-ONs (58 nm) and uncoated ONs (67 nm). Ellman’s test confirmed > 99.2% peptide conjugation. Cellular uptake of LDLR-OTNs was rapid, with significant fluorescence by 1 hour and a kinetic plateau at 24–48 hours, with data fitting to a modified exponential model, while that of untargeted POPC-ONs had lower initial uptake, following a logistic model. Suramin blocking reduced LDLR-OTN uptake, confirming receptor-mediated entry. Cytotoxicity assays yielded IC 50 values of 23.8 µM (BT-474), 25.8 µM (MDA MB 453), and 8.2 µM (MCF-7), with maximal inhibition at 48 h. LDLR-OTNs demonstrated receptor-mediated uptake and potent cytotoxicity in LDLR- and FAS- overexpressing breast cancer cells. These findings support LDLR-targeted nanoparticles as a promising approach for delivering FAS inhibitors to LDLR-rich tumours, meriting further investigation in targeted cancer therapy development.

Abstract Motivation The low solubility, weak acid drug, niclosamide is a host cell modulator with broad-spectrum anti-viral cell-activity against many viruses, including stopping the SARS-CoV-2 virus from infecting cells in cell culture. As a result, a simple universal nasal spray preventative was proposed and investigated in earlier work regarding the dissolution of niclosamide into simple buffers. However, starting with pharmaceutical grade, niclosamide represents a new 505(b)(2) application. The motivation for this second paper in the series was therefore to explore if and to what extent niclosamide could be extracted from commercially available and regulatory-approved niclosamide oral tablets that could serve as a preventative nasal spray and an early treatment oral/throat spray, with possibly more expeditious testing and regulatory approval. Experimental Measurements of supernatant niclosamide concentrations were made by calibrated UV-Vis for the dissolution of niclosamide from commercially available Yomesan crushed into a powder for dissolution into Tris Buffer (TB) solutions. Parameters tested were as follows: time (0–2 days), concentration (300 µM to -1 mM), pH (7.41 to 9.35), and anhydrous/hydrated state. Optical microscopy was used to view the morphologies of the initial crushed powder, and the dissolving and equilibrating undissolved excess particles to detect morphologic changes that might occur. Results Concentration dependence : Niclosamide was readily extracted from powdered Yomesan at pH 9.34 TB at starting Yomesan niclosamide equivalents concentrations of 300 µM, 600 µM, and 1 mM. Peak dissolved niclosamide supernatant concentrations of 264 µM, 216 µM, and 172 µM were achieved in 1 h, 1 h, and 3 h respectively. These peaks though were followed by a reduction in supernatant concentration to an average of 112.3 µM ± 28.4 µM after overnight stir on day 2. pH dependence : For nominal pHs of 7.41, 8.35, 8.85, and 9.35, peak niclosamide concentrations were 4 µM, 22.4 µM, 96.2 µM, and 215.8 µM, respectively. Similarly, the day 2 values all reduced to 3 µM, 12.9 µM, 35.1 µM, and 112.3 µM. A heat-treatment to 200 °C dehydrated the niclosamide and showed a high 3 h concentration (262 µM) and the least day-2 reduction (to 229 µM). This indicated that the presence, or formation during exposure to buffer, of lower solubility polymorphs was responsible for the reductions in total solubilities. These morphologic changes were confirmed by optical microscopy that showed initially featureless particulate-aggregates of niclosamide could grow multiple needle-shaped crystals and form needle masses, especially in the presence of Tris-buffered sodium chloride, where new red needles were rapidly made. Scale up : A scaled-up 1 L solution of niclosamide was made achieving 165 µM supernatant niclosamide in 3 h by dissolution of just one fifth (100 mg niclosamide) of a Yomesan tablet. Conclusion These comprehensive results provide a guide as to how to utilize commercially available and approved tablets of niclosamide to generate aqueous niclosamide solutions from a simple dissolution protocol. As shown here, just one 4-tablet pack of Yomesan could readily make 165 L of a 20 µM niclosamide solution giving 16,500 10 mL bottles. One million bottles, from just 60 packs of Yomesan, would provide 100 million single spray doses for distribution to mitigate a host of respiratory infections as a universal preventative-nasal and early treatment oral/throat sprays throughout the world. Graphical Abstract pH dependence of niclosamide extraction from crushed Yomesan tablet material into Tris buffer (yellow-green in vial) and Tris-buffered saline solution (orange-red in vial). Initial anhydrous dissolution concentration is reduced by overnight stirring to likely monohydrate niclosamide; and is even lower if in TBSS forming new niclosamide sodium needle crystals grown from the original particles.

The gamma-interferon (IFNγ)-inducible guanylate-binding proteins (GBPs) promote host defense against gram-negative cytosolic bacteria in part through the induction of an inflammatory cell death pathway called pyroptosis. To activate pyroptosis, GBPs facilitate sensing of the gram-negative bacterial outer membrane component lipopolysaccharide (LPS) by the noncanonical caspase-4 inflammasome. There are seven human GBP paralogs, and it is unclear how each GBP contributes to LPS sensing and pyroptosis induction. GBP1 forms a multimeric microcapsule on the surface of cytosolic bacteria through direct interactions with LPS. The GBP1 microcapsule recruits caspase-4 to bacteria, a process deemed essential for caspase-4 activation. In contrast to GBP1, closely related paralog GBP2 is unable to bind bacteria on its own but requires GBP1 for direct bacterial binding. Unexpectedly, we find that GBP2 overexpression can restore gram-negative-induced pyroptosis in GBP1 KO cells, without GBP2 binding to the bacterial surface. A mutant of GBP1 that lacks the triple arginine motif required for microcapsule formation also rescues pyroptosis in GBP1 KO cells, showing that binding to bacteria is dispensable for GBPs to promote pyroptosis. Instead, we find that GBP2, like GBP1, directly binds and aggregates “free” LPS through protein polymerization. We demonstrate that supplementation of either recombinant polymerized GBP1 or GBP2 to an in vitro reaction is sufficient to enhance LPS-induced caspase-4 activation. This provides a revised mechanistic framework for noncanonical inflammasome activation where GBP1 or GBP2 assembles cytosol-contaminating LPS into a protein-LPS interface for caspase-4 activation as part of a coordinated host response to gram-negative bacterial infections.

<div>Abstract<p>Therapeutic advances for osteosarcoma have stagnated over the past several decades, leading to an unmet clinical need for patients. The purpose of this study was to develop a novel therapy for osteosarcoma by reformulating and validating niclosamide, an established anthelminthic agent, as a niclosamide stearate prodrug therapeutic (NSPT). We sought to improve the low and inefficient clinical bioavailability of oral dosing, especially for the relatively hydrophobic classes of anticancer drugs. Nanoparticles were fabricated by rapid solvent shifting and verified using dynamic light scattering and UV-vis spectrophotometry. NSPT efficacy was then studied <i>in vitro</i> for cell viability, cell proliferation, and intracellular signaling by Western blot analysis; <i>ex vivo</i> pulmonary metastatic assay model; and <i>in vivo</i> pharmacokinetic and lung mouse metastatic model of osteosarcoma. NSPT formulation stabilizes niclosamide stearate against hydrolysis and delays enzymolysis; increases circulation <i>in vivo</i> with <i>t</i><sub>1/2</sub> approximately 5 hours; reduces cell viability and cell proliferation in human and canine osteosarcoma cells <i>in vitro</i> at 0.2–2 μmol/L IC<sub>50</sub>; inhibits recognized growth pathways and induces apoptosis at 20 μmol/L; eliminates metastatic lesions in the <i>ex vivo</i> lung metastatic model; and when injected intravenously at 50 mg/kg weekly, it prevents metastatic spread in the lungs in a mouse model of osteosarcoma over 30 days. In conclusion, niclosamide was optimized for preclinical drug delivery as a unique prodrug nanoparticle injected intravenously at 50 mg/kg (1.9 mmol/L). This increased bioavailability of niclosamide in the blood stream prevented metastatic disease in the mouse. This chemotherapeutic strategy is now ready for canine trials, and if successful, will be targeted for human trials in patients with osteosarcoma.</p></div>

<p>Supplementary Materials including Text and Data</p>

<div>Abstract<p>Therapeutic advances for osteosarcoma have stagnated over the past several decades, leading to an unmet clinical need for patients. The purpose of this study was to develop a novel therapy for osteosarcoma by reformulating and validating niclosamide, an established anthelminthic agent, as a niclosamide stearate prodrug therapeutic (NSPT). We sought to improve the low and inefficient clinical bioavailability of oral dosing, especially for the relatively hydrophobic classes of anticancer drugs. Nanoparticles were fabricated by rapid solvent shifting and verified using dynamic light scattering and UV-vis spectrophotometry. NSPT efficacy was then studied <i>in vitro</i> for cell viability, cell proliferation, and intracellular signaling by Western blot analysis; <i>ex vivo</i> pulmonary metastatic assay model; and <i>in vivo</i> pharmacokinetic and lung mouse metastatic model of osteosarcoma. NSPT formulation stabilizes niclosamide stearate against hydrolysis and delays enzymolysis; increases circulation <i>in vivo</i> with <i>t</i><sub>1/2</sub> approximately 5 hours; reduces cell viability and cell proliferation in human and canine osteosarcoma cells <i>in vitro</i> at 0.2–2 μmol/L IC<sub>50</sub>; inhibits recognized growth pathways and induces apoptosis at 20 μmol/L; eliminates metastatic lesions in the <i>ex vivo</i> lung metastatic model; and when injected intravenously at 50 mg/kg weekly, it prevents metastatic spread in the lungs in a mouse model of osteosarcoma over 30 days. In conclusion, niclosamide was optimized for preclinical drug delivery as a unique prodrug nanoparticle injected intravenously at 50 mg/kg (1.9 mmol/L). This increased bioavailability of niclosamide in the blood stream prevented metastatic disease in the mouse. This chemotherapeutic strategy is now ready for canine trials, and if successful, will be targeted for human trials in patients with osteosarcoma.</p></div>

<p>Supplementary Materials including Text and Data</p>

<p>AVI file - 34.5MB, Real-time intravascular release of doxorubicin from Dox-TSL and subsequent redistribution of drug in B16 melanoma tumor tissue. The video shows drug (red) delivered over the first 20min, at 20x magnification. The eNOS-GFPtg mouse model was used in this experiment, so the blood vessels are green. (Download before playing in IE8)</p>

<div>Abstract<p>Traditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumors. The enhanced permeability and retention effect can permit passive accumulation into tumor interstitium. However, suboptimal delivery is achieved with most nanoparticles because of heterogeneities of vascular permeability, which limits nanoparticle penetration. Furthermore, slow drug release limits bioavailability. We developed a fast drug-releasing liposome triggered by local heat that has already shown substantial antitumor efficacy and is in human trials. Here, we show that thermally sensitive liposomes (Dox-TSL) release doxorubicin inside the tumor vasculature. Real-time confocal imaging of doxorubicin delivery to murine tumors in window chambers and histologic analysis of flank tumors illustrates that intravascular drug release increases free drug in the interstitial space. This increases both the time that tumor cells are exposed to maximum drug levels and the drug penetration distance, compared with free drug or traditional pegylated liposomes. These improvements in drug bioavailability establish a new paradigm in drug delivery: rapidly triggered drug release in the tumor bloodstream. <i>Cancer Res; 72(21); 5566–75. ©2012 AACR</i>.</p></div>

<p>AVI file - 34.5MB, Real-time intravascular release of doxorubicin from Dox-TSL and subsequent redistribution of drug in B16 melanoma tumor tissue. The video shows drug (red) delivered over the first 20min, at 20x magnification. The eNOS-GFPtg mouse model was used in this experiment, so the blood vessels are green. (Download before playing in IE8)</p>

<p>PDF file - 83K, Citation</p>

<p>AVI file - 41.7MB, Real-time drug delivery of free doxorubicin and Dox-TSL in heated FaDu tumor tissue. The blood vessels are highlighted in green and drug is red. The video shows drug delivered over the first 20min, at 20x magnification. Free drug quickly disperses throughout the tissue and then is reabsorbed into the vasculature, leaving a low drug concentration behind in the tumor cells. In the Dox-TSL treated tissue, drug is delivered uniformly across the tumor tissue, with visible uptake in tumor and endothelial cell nuclei. Drug in the Dox-TSL treated mouse continues to accumulate in the tumor tissue over the 20min period. (Download before playing in IE8)</p>

<p>AVI file - 41.7MB, Real-time drug delivery of free doxorubicin and Dox-TSL in heated FaDu tumor tissue. The blood vessels are highlighted in green and drug is red. The video shows drug delivered over the first 20min, at 20x magnification. Free drug quickly disperses throughout the tissue and then is reabsorbed into the vasculature, leaving a low drug concentration behind in the tumor cells. In the Dox-TSL treated tissue, drug is delivered uniformly across the tumor tissue, with visible uptake in tumor and endothelial cell nuclei. Drug in the Dox-TSL treated mouse continues to accumulate in the tumor tissue over the 20min period. (Download before playing in IE8)</p>

<p>PDF file - 83K, Citation</p>

<div>Abstract<p>Traditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumors. The enhanced permeability and retention effect can permit passive accumulation into tumor interstitium. However, suboptimal delivery is achieved with most nanoparticles because of heterogeneities of vascular permeability, which limits nanoparticle penetration. Furthermore, slow drug release limits bioavailability. We developed a fast drug-releasing liposome triggered by local heat that has already shown substantial antitumor efficacy and is in human trials. Here, we show that thermally sensitive liposomes (Dox-TSL) release doxorubicin inside the tumor vasculature. Real-time confocal imaging of doxorubicin delivery to murine tumors in window chambers and histologic analysis of flank tumors illustrates that intravascular drug release increases free drug in the interstitial space. This increases both the time that tumor cells are exposed to maximum drug levels and the drug penetration distance, compared with free drug or traditional pegylated liposomes. These improvements in drug bioavailability establish a new paradigm in drug delivery: rapidly triggered drug release in the tumor bloodstream. <i>Cancer Res; 72(21); 5566–75. ©2012 AACR</i>.</p></div>

Metabolic reprogramming, through increased uptake of cholesterol in the form of low-density lipoproteins (LDL), is one way by which cancer cells, including high grade gliomas (HGG), maintain their rapid growth. In this study, we determined LDL receptor (LDLR) expression in HGGs using immunohistochemistry on tissue microarrays from intra- and inter tumour regions of 36 adult and 133 paediatric patients to confirm LDLR as a therapeutic target. Additionally, we analysed expression levels in three representative cell line models to confirm their future utility to test LDLR-targeted nanoparticle uptake, retention, and cytotoxicity. Our data show widespread LDLR expression in adult and paediatric cohorts, but with significant intra-tumour variation observed between the core and either rim or invasive regions of adult HGG. Expression was independent of paediatric tumour grade or identified clinicopathological factors. LDLR-expressing tumour cells localized preferentially within perivascular niches, also with significant adult intra-tumour variation. We demonstrated variable levels of LDLR expression in all cell lines, confirming their suitability as models to test LDLR-targeted nanotherapy delivery. Overall, our study reveals the LDLR pathway as a ubiquitous metabolic vulnerability in high grade gliomas across all ages, amenable to future consideration of LDL-mediated nanoparticle/drug delivery to potentially circumvent tumour heterogeneity.