Alain Le Pape

ROS implication in a new antitumor strategy based on non-thermal plasma

Non‐thermal plasma (NTP) is generated by ionizing neutral gas molecules/atoms leading to a highly reactive gas at ambient temperature containing excited molecules, reactive species and generating transient electric fields. Given its potential to interact with tissue or cells without a significant temperature increase, NTP appears as a promising approach for the treatment of various diseases including cancer. The aim of our study was to evaluate the interest of NTP both in vitro and in vivo. To this end, we evaluated the antitumor activity of NTP in vitro on two human cancer cell lines (glioblastoma U87MG and colorectal carcinoma HCT‐116). Our data showed that NTP generated a large amount of reactive oxygen species (ROS), leading to the formation of DNA damages. This resulted in a multiphase cell cycle arrest and a subsequent apoptosis induction. In addition, in vivo experiments on U87MG bearing mice showed that NTP induced a reduction of bioluminescence and tumor volume as compared to nontreated mice. An induction of apoptosis was also observed together with an accumulation of cells in S phase of the cell cycle suggesting an arrest of tumor proliferation. In conclusion, we demonstrated here that the potential of NTP to generate ROS renders this strategy particularly promising in the context of tumor treatment.

Effects of a Non Thermal Plasma Treatment Alone or in Combination with Gemcitabine in a MIA PaCa2-luc Orthotopic Pancreatic Carcinoma Model

Pancreatic tumors are the gastrointestinal cancer with the worst prognosis in humans and with a survival rate of 5% at 5 years. Nowadays, no chemotherapy has demonstrated efficacy in terms of survival for this cancer. Previous study focused on the development of a new therapy by non thermal plasma showed significant effects on tumor growth for colorectal carcinoma and glioblastoma. To allow targeted treatment, a fibered plasma (Plasma Gun) was developed and its evaluation was performed on an orthotopic mouse model of human pancreatic carcinoma using a MIA PaCa2-luc bioluminescent cell line. The aim of this study was to characterize this pancreatic carcinoma model and to determine the effects of Plasma Gun alone or in combination with gemcitabine. During a 36 days period, quantitative BLI could be used to follow the tumor progression and we demonstrated that plasma gun induced an inhibition of MIA PaCa2-luc cells proliferation in vitro and in vivo and that this effect could be improved by association with gemcitabine possibly thanks to its radiosensitizing properties.

Tumor burden influences exposure and response to rituximab: pharmacokinetic-pharmacodynamic modeling using a syngeneic bioluminescent murine model expressing human CD20

Clinical studies have shown a large interindividual variability in rituximab exposure and its significant influence on clinical response in patients receiving similar doses of antibody. The aim of this study was to evaluate the influence of tumor burden on dose-concentration-response relationships of rituximab. Murine lymphoma cells (EL4, 8 x 10(3)), transduced with human CD20 cDNA and transfected with luciferase plasmid (EL4-huCD20-Luc), were intravenously injected into C57BL/6J mice. Tumor burden detection, dissemination, and progression were evaluated quantitatively by in vivo bioluminescence imaging. Different doses of rituximab (6, 12, 20, or 40 mg/kg) were infused 13 days after lymphoma cell inoculation, and rituximab serum concentrations were measured by enzyme-linked immunosorbent assay. Without rituximab, all mice developed disseminated lymphoma and died within 30 days, whereas a significant dose-response relationship was observed in mice receiving rituximab. The 20-mg/kg dose was adequate to study interindividual variability in response because 23% of mice were cured, 59% had partial response, and 18% had disease progression. Rituximab concentrations were inversely correlated with tumor burden; mice with low tumor burden had high rituximab concentrations. Furthermore, rituximab exposure influenced response and survival. Finally, using a pharmacokinetic-pharmacodynamic model, we demonstrated that tumor burden significantly influenced rituximab efficacy.

Hybrid gadolinium oxide nanoparticles combining imaging and therapy

The cytotoxicity of luminescent paramagnetic gadolinium oxide nanoparticles activated by harmless thermal neutron irradiation was evaluated on a luciferase coding gene transfected lymphome cells (EL4-Luc). Cellular uptake of nanoparticles was determined by fluorescence and magnetic resonance imaging and by ICP-MS analyses while the metabolic activity of irradiated EL4-Luc cells was monitored by bioluminescence. The alteration of the irradiated cells depends both on neutron irradiation dose and on gadolinium content within cells. In addition to their dual modality imaging ability, the application of these multifunctional particles for neutron capture therapy can be envisaged.

Preclinical activity of F14512, designed to target tumors expressing an active polyamine transport system

SummaryWe have exploited the polyamine transport system (PTS) to deliver selectively a spermine-drug conjugate, F14512 to cancer cells. This study was aimed to define F14512 anticancer efficacy against tumor models and to investigate whether fluorophor-labeled polyamine probes could be used to identify tumors expressing a highly active PTS and that might be sensitive to F14512 treatments. Eighteen tumor models were used to assess F14512 antitumor activity. Cellular uptake of spermine-based fluorescent probes was measured by flow cytometry in cells sampled from tumor xenografts by needle biopsy. The accumulation of the fluorescent probe within B16 tumors in vivo was assessed using infrared fluorescence imaging. This study has provided evidence of a major antitumor activity for F14512. Significant responses were obtained in 67% of the tumor models evaluated, with a high level of activity recorded in 33% of the responsive models. Complete tumor regressions were observed after i.v., i.p. or oral administrations of F14512 and its antitumor activity was demonstrated over a range of 2–5 dose levels, providing evidence of its good tolerance. The level of cellular fluorescence emitted by the fluorescent probes was higher in cells sampled from tumors sensitive to F14512 treatments than from F14512-refractory tumors. We suggest that these probes could be used to identify tumors expressing a highly active PTS and guide the selection of patients that might be treated with F14512. These results emphasize the preclinical interest of this novel molecule and support its further clinical development.

The First Generation of β-Galactosidase-Responsive Prodrugs Designed for the Selective Treatment of Solid Tumors in Prodrug Monotherapy†

Massive attack: Galactoside prodrugs have been designed that can be selectively activated by lysosomal β-galactosidase located inside cancer cells expressing a specific tumor-associated receptor. This efficient enzymatic process triggers a potent cytotoxic effect, releasing the potent antimitotic agent MMAE and allowing the destruction of both receptor-positive and surrounding receptor-negative tumor cells.

Aerosolized Gemcitabine in Patients with Carcinoma of the Lung: Feasibility and Safety Study

BACKGROUND We investigated the biodistribution, pharmacokinetics, safety profile, and feasibility of aerosolized gemcitabine (GCB) in patients with lung carcinoma. METHOD Eleven patients with carcinoma localized in the lungs were studied in a dose escalation study of aerosolized GCB administered 1 day/week for 9 consecutive weeks. Safety data, scintigraphic assessment of the delivered dose and pharmacokinetic monitoring were analyzed. Patients were treated with doses of between 1 mg/kg and 4 mg/kg (dose in the nebulizer), using a new inhaler device (Aeroneb Pro with an Idehaler Chamber). RESULTS AND CONCLUSIONS The total dose of GCB delivered to the patients lung was 42±16% of the initial amount of dose in the nebulizer. Safety data showed no hematologic toxicity, nephrotoxicity or neurotoxicity. At 4 mg/kg, one patient experienced grade 4 pulmonary toxicity (bronchospasm), which was the dose-limiting toxicity. Grade 2 and 3 toxic effects included fatigue, vomiting, dyspnea, and cough. Overall response: minor response in one patient, stable disease in four patients, progressive disease in four patients. Pharmacokinetic data showed very low plasma GCB levels. Maximal plasma concentration was observed at the end of nebulization. Aerosolized gemcitabin was safe, with minimal toxicity, for patients with lung carcinoma.

Modifications of glomerular basement membrane cross-links in experimental diabetic rats

Abstract Glomerular basement membranes were isolated from normal and streptozotocin-diabetic rats. after elimination of non collagenous components with 8M urea the extent of ketoamine-linked hexoses and cross-links in the preparation was determined using NaB 3 H 4 reduction and radiochromatography. The observed increase in the level of N-glycosylation appeared to be closely related to a lowered amount of intermolecular cross-links in diabetic rats as compared to normal animals. Such a glycosylation may affect the stability and physicochemical properties of G.B.M. and thereby contribute to the vascular sequelae of diabetes mellitus.

Radioisotopic Imaging Allows Optimization of Adenovirus Lung Deposition for Cystic Fibrosis Gene Therapy

Cystic fibrosis is a common, heriditary disease resulting from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Airway transfer of the CFTR gene is a potential strategy to treat or prevent the lung pathology that is the main cause of morbidity and mortality. Among the vectors used for gene therapy, adenoviruses have shown their ability to transfer the CFTR gene to respiratory epithelial cells, using either instillation or nebulization. Our objective was to characterize the lung deposition of aerosolized adenovirus by quantitative radioisotopic imaging, the only noninvasive technique allowing in vivo quantitation of inhaled drugs. We first labeled an adenovirus expressing human CFTR with the gamma-emitting radioisotope, technetium 99m (99mTc), and determined the best labeling conditions to allow preservation of virus bioactivity. We then administered the radioaerosol to baboons, determined lung regional deposition of 99mTc-labeled adenovirus, and compared the expression of CFTR transcripts 3 and 21 days after inhalation. The expression of vector-encoded mRNA ranged from 4 to 22% with respect to the endogenous CFTR mRNA depending on the lung segments. Moreover, we have developed a model using 99mTc-DTPA (diethylenetriamine pentaacetic acid), which can be used, as an alternative to adenovirus, to determine the profile of lung deposition of the vector. This study demonstrates that scintigraphy is a useful technique to achieve optimization of gene administration to the airways.

Influence of in vitro non-enzymatic glycosylation on the physicochemical parameters of type I collagen.

In vitro non-enzymatic condensation of glucose on acid soluble collagen is shown not to affect the molecular structure and stability as evaluated both by circular dichroism and differential spectrometry. Viscometric studies demonstrate a lowering in intermolecular interactions; whereas the hydrodynamic volume remains unaffected by nonenzymatic glycosylation. alpha 2(I) collagen chains exhibit a modified electrophoretic mobility previously found in collagen of diabetic rats; this peculiar behavior is discussed in terms of lowered hydrophobicity due to addition of hydrophilic groups from glucose. The lowered formation of hydrophobic areas, evaluated from the fluorescence emission of diphenylhexatriene, indicates that non-enzymatic glycosylation is able to influence the close packing of collagen fibers.