Clostridium novyi-NT in cancer therapy

AbstractThe attenuated anaerobic bacterium Clostridium novyi-NT (C. novyi-NT) is known for its ability to precisely germinate in and eradicate treatment-resistant hypoxic tumors in various experimental animal models and spontaneously occurring canine sarcomas. In this article, we review the therapeutic and toxicologic aspects of C. novyi-NT therapy, key challenges and limitations, and promising strategies to optimize its performance via recombinant DNA technology and immunotherapeutic approaches, to establish C. novyi-NT as an essential tool in cancer therapy

Feasibility of using NF1-GRD and AAV for gene replacement therapy in NF1-associated tumors

Neurofibromatosis type 1, including the highly aggressive malignant peripheral nerve sheath tumors (MPNSTs), is featured by the loss of functional neurofibromin 1 (NF1) protein resulting from genetic alterations. A major function of NF1 is suppressing Ras activities, which is conveyed by an intrinsic GTPase-activating protein-related domain (GRD). In this study, we explored the feasibility of restoring Ras GTPase via exogenous expression of various GRD constructs, via gene delivery using a panel of adeno-associated virus (AAV) vectors in MPNST and human Schwann cells (HSCs). We demonstrated that several AAV serotypes achieved favorable transduction efficacies in those cells and a membrane-targeting GRD fused with an H-Ras C-terminal motif (C10) dramatically inhibited the Ras pathway and MPNST cells in a NF1-specific manner. Our results opened up a venue of gene replacement therapy in NF1-related tumors

Exploring transcriptional regulators Ref-1 and STAT3 as therapeutic targets in malignant peripheral nerve sheath tumours

Background MPNST is a rare soft-tissue sarcoma that can arise from patients with NF1. Existing chemotherapeutic and targeted agents have been unsuccessful in MPNST treatment, and recent findings implicate STAT3 and HIF1-α in driving MPNST. The DNA-binding and transcriptional activity of both STAT3 and HIF1-α is regulated by Redox factor-1 (Ref-1) redox function. A first-generation Ref-1 inhibitor, APX3330, is being tested in cancer clinical trials and could be applied to MPNST. Methods We characterised Ref-1 and p-STAT3 expression in various MPNST models. Tumour growth, as well as biomarkers of apoptosis and signalling pathways, were measured by qPCR and western blot following treatment with inhibitors of Ref-1 or STAT3. Results MPNSTs from Nf1-Arfflox/floxPostnCre mice exhibit significantly increased positivity of p-STAT3 and Ref-1 expression when malignant transformation occurs. Inhibition of Ref-1 or STAT3 impairs MPNST growth in vitro and in vivo and induces apoptosis. Genes highly expressed in MPNST patients are downregulated following inhibition of Ref-1 or STAT3. Several biomarkers downstream of Ref-1 or STAT3 were also downregulated following Ref-1 or STAT3 inhibition. Conclusions Our findings implicate a unique therapeutic approach to target important MPNST signalling nodes in sarcomas using new first-in-class small molecules for potential translation to the clinic

Erythrocytes as drug-carrier for insoluble substances using Amphotericin B as a model

In den letzten Jahrzehnten hat die Häufigkeit invasiver Pilzinfektionen - vor allem verursacht durch C. albicans - dramatisch zugenommen und ist insbesondere bei immungeschwächten Patienten mit einer erheblichen Mortalität verbunden. Dabei spielen die phagozytierenden Zellen des Immunsystems, Mononzyten (MNC) und Neutrophile (PMN), eine entscheidende Rolle in der Infektionsbegrenzung und Dissemination. Das Polyenantibiotikum Amphotericin B (AmB) gehört zum therapeutischen Goldstandard; der klinische Einsatz ist jedoch durch ein fulminantes Nebenwirkungsprofil stark eingeschränkt. Anstrengungen die Toxizität mit der Synthese von AmB Analoga und Lipid- Formulierungen zu mindern, führten nicht zum Erfolg und machen die Entwicklung neuer Strategien dringend erforderlich. Daher hypothesierten wir, dass die Rekruitierung von Leukozyten zum AmB Transport einen idealen Ansatz zur gezielten Verstärkung der Pilzabwehr und Erhöhung der Wirkstoffkonzentration am Infektionsherd bildet. Um das Risiko einer AmB induzierten Zytotoxizität auf die Leukozyten zu reduzieren, wurden Erythrozyten (RBCs) aufgrund ihrer exzellenten Beladungseigenschaften als primäre Medikamententransporter genutzt. Die AmB-Beladung der RBCs erfolgte mit einer AmB Nanosuspension (AmB- NS, Partikelgröße 65 nm) via hypotoner Hämolyse und führte zu Beladungskonzentrationen von 3.81 pg/RBC, entsprechend einer Einschlusseffizienz von 15 – 19% sowie einem prozentualen molekularen AmB Anteil von 41 mol%, der damit deutlich höher lag als bei anderen Lipid- assoziierten Formulierungen. Durch die zusätzliche Inkorporation von Magnetit konnten AmB-NS-RBCs im MRT visualisiert werden und offerieren somit die Möglichkeit des therapeutischen Monitorings. Die leukzytäre Erkennung und Aufnahme der AmB-NS-RBCs war PS vermittelt und sehr effzient. Innerhalb von 4 hr internalisierten > 98% der Leukozyten 4 - 5 AmB-NS-RBCs pro Zelle, das einer AmB-Endkonzentrationen von 14 - 16 pg pro PMN bzw. MNC entsprach. Dies führte bei Inkubation mit C. albicans zu einer sofortigen und dauerhaften Hemmung der intra-und extrazellulären Pilz-Aktivität. Die durch Diffusion freigesetzte AmB-Menge lag dabei über den gesamten Beobachtungszeitraum weit oberhalb der MHK90, jedoch ohne offensichtliche Zytotoxizität für die Immunzellen. Lediglich eine transiente, AmB unabhängige Reduktion der Phagozytosekapazität um ~30% und der ROS Bildung um ~50% konnte identifiziert werden, die sich nach 24 hr wieder normalisierten. Daher muss geschlussfolgert werden, dass das beschriebene DDS eine exzellente Alternative zu den bisher entwickelten AmB-Formulierungen darstellt, angesichts der überragenden intra- und extrazellulären Pilzabtötung unter Erhaltung der leukozytären Funktionalität.The efficacy of antifungal treatment has been limited by the poor biodistribution of amphotericin B (AmB) due to its pharmacological profile, as well as its severe side effects. Efforts to improve the biodistribution and lower the toxicity with the synthesis of AmB analogues such as AmB esters and lipid-based formulations, have not led to the anticipated success. Therefore, new drug delivery strategies for AmB are urgently needed in order to improve antifungal treatment and enhance host survival. Based on these issues we have developed a cellular drug-delivery system, which incorporates human erythrocytes (RBCs) loaded with an AmB nanosuspension (AmB-NS) thereby allowing subsequent targeting to the leucocytes, the main effector arm of the antifungal immune response. AmB-NS encapsulation in RBCs is achieved by hypotonic hemolysis, leading to intracellular AmB concentrations of 3.8 pg per RBC and an entrapment efficacy of approximately 15%. Furthermore, AmB-NS-RBCs were loaded with magnetite thereby achieving MRI visualization of the drug delivery system and offering additional targeting opportunities. AmB-NS-RBCs were quickly recognized and ingested by the leucocytes. On average more than 98% of the leucocytes ingested 4-5 AmB-NS-RBCs which correlated with an AmB loading capacity of 14-16 pg per leucocyte. Upon phagocytosis, leukocytes showed a slow AmB release over ten days that led to a significant improvement of the intra- and extracellular killing activity while the leucocytes remained functional with no permanent alterations in their cell viability, phagocytic capacity and oxidative burst production. Thus, this drug-delivery method is effective for the transport of water-insoluble substances, such as AmB, and warrants consideration for further testing

Dynamic contrast-enhanced CEST MRI using a low molecular weight dextran

Natural and synthetic sugars have great potential for developing highly biocompatible and translatable chemical exchange saturation transfer (CEST) MRI contrast agents. In this study, we aimed to develop the smallest clinically available form of dextran, Dex1 (molecular weight, MW ~ 1 kDa), as a new CEST agent. We first characterized the CEST properties of Dex1 in vitro at 11.7 T and showed that the Dex1 had a detectable CEST signal at ~1.2 ppm, attributed to hydroxyl protons. In vivo CEST MRI studies were then carried out on C57BL6 mice bearing orthotopic GL261 brain tumors (n = 5) using a Bruker BioSpec 11.7 T MRI scanner. Both steady-state full Z-spectral images and single offset (1.2 ppm) dynamic dextran-enhanced (DDE) images were acquired before and after the intravenous injection of Dex1 (2 g/kg). The steady-state Z-spectral analysis showed a significantly higher CEST contrast enhancement in the tumor than in contralateral brain (∆MTRasym 1.2 ppm = 0.010 ± 0.006 versus 0.002 ± 0.008, P = 0.0069) at 20 min after the injection of Dex1. Pharmacokinetic analyses of DDE were performed using the area under the curve (AUC) in the first 10 min after Dex1 injection, revealing a significantly higher uptake of Dex1 in the tumor than in brain tissue for tumor-bearing mice (AUC[0-10 min] = 21.9 ± 4.2 versus 5.3 ± 6.4%·min, P = 0.0294). In contrast, no Dex1 uptake was foundling in the brains of non-tumor-bearing mice (AUC[0-10 min] = -1.59 ± 2.43%·min). Importantly, the CEST MRI findings were consistent with the measurements obtained using DCE MRI and fluorescence microscopy, demonstrating the potential of Dex1 as a highly translatable CEST MRI contrast agent for assessing tumor hemodynamics

Target Product Profile for Cutaneous Neurofibromas: Clinical Trials to Prevent, Arrest, or Regress Cutaneous Neurofibromas.

Cutaneous neurofibromas (cNFs) are benign tumors of the skin that affect >95% of adults with neurofibromatosis type 1. Despite their benign histology, cNFs can significantly impact QOL due to disfigurement, pain, and pruritus. There are no approved therapies for cNFs. Existing treatments are limited to surgery or laser-based treatments that have had mixed success and cannot be readily applied to a large number of tumors. We review cNF treatment options that are currently available and under investigation, discuss the regulatory considerations specific to cNFs, and propose strategies to improve cNF clinical trial design and standardize clinical trial endpoints

Podocalyxin-like protein Is expressed in glioblastoma multiforme stem-like cells and is associated with poor outcome

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