Quantitative MRI to Characterize Hypoxic Tumors in Comparison to FMISO PET/CT for Radiotherapy in Oropharynx Cancers.

Intratumoral hypoxia is associated with a poor prognosis and poor response to treatment in head and neck cancers. Its identification would allow for increasing the radiation dose to hypoxic tumor subvolumes. 18F-FMISO PET imaging is the gold standard; however, quantitative multiparametric MRI could show the presence of intratumoral hypoxia. Thus, 16 patients were prospectively included and underwent 18F-FDG PET/CT, 18F-FMISO PET/CT, and multiparametric quantitative MRI (DCE, diffusion and relaxometry T1 and T2 techniques) in the same position before treatment. PET and MRI sub-volumes were segmented and classified as hypoxic or non-hypoxic volumes to compare quantitative MRI parameters between normoxic and hypoxic volumes. In total, 13 patients had hypoxic lesions. The Dice, Jaccard, and overlap fraction similarity indices were 0.43, 0.28, and 0.71, respectively, between the FDG PET and MRI-measured lesion volumes, showing that the FDG PET tumor volume is partially contained within the MRI tumor volume. The results showed significant differences in the parameters of SUV in FDG and FMISO PET between patients with and without measurable hypoxic lesions. The quantitative MRI parameters of ADC, T1 max mapping and T2 max mapping were different between hypoxic and normoxic subvolumes. Quantitative MRI, based on free water diffusion and T1 and T2 mapping, seems to be able to identify intra-tumoral hypoxic sub-volumes for additional radiotherapy doses.

Advances in PET and MRI imaging of tumor hypoxia.

Tumor hypoxia is a complex and evolving phenomenon both in time and space. Molecular imaging allows to approach these variations, but the tracers used have their own limitations. PET imaging has the disadvantage of low resolution and must take into account molecular biodistribution, but has the advantage of high targeting accuracy. The relationship between the signal in MRI imaging and oxygen is complex but hopefully it would lead to the detection of truly oxygen-depleted tissue. Different ways of imaging hypoxia are discussed in this review, with nuclear medicine tracers such as [18F]-FMISO, [18F]-FAZA, or [64Cu]-ATSM but also with MRI techniques such as perfusion imaging, diffusion MRI or oxygen-enhanced MRI. Hypoxia is a pejorative factor regarding aggressiveness, tumor dissemination and resistance to treatments. Therefore, having accurate tools is particularly important.

First Comparison between [18f]-FMISO and [18f]-Faza for Preoperative Pet Imaging of Hypoxia in Lung Cancer.

Hypoxic areas are typically resistant to treatment. However, the fluorine-18-fluoroazomycin-arabinoside (FAZA) and fluorine 18 misonidazole (FMISO) tracers have never been compared in non small cell lung cancer (NSCLC). This study compares the capability of 18F-FAZA PET/CT with that of 18F-FMISO PET/CT for detecting hypoxic tumour regions in early and locally advanced NSCLC patients. We prospectively evaluated patients who underwent preoperative PET scans before surgery for localised NSCLC (i.e., fluorodeoxyglucose (FDG)-PET, FMISO-PET, and FAZA-PET). The PET data of the three tracers were compared with each other and then compared to immunohistochemical analysis (GLUT-1, CAIX, LDH-5, and HIF1-Alpha) after tumour resection. Overall, 19 patients with a mean age of 68.2 ± 8 years were included. There were 18 lesions with significant uptake (i.e., SUVmax >1.4) for the F-MISO and 17 for FAZA. The mean SUVmax was 3 (±1.4) with a mean volume of 25.8 cc (±25.8) for FMISO and 2.2 (±0.7) with a mean volume of 13.06 cc (±13.76) for FAZA. The SUVmax of F-MISO was greater than that of FAZA (p = 0.0003). The SUVmax of F-MISO shows a good correlation with that of FAZA at 0.86 (0.66-0.94). Immunohistochemical results are not correlated to hypoxia PET regardless of the staining. The two tracers show a good correlation with hypoxia, with FMISO being superior to FAZA. FMISO, therefore, remains the reference tracer for defining hypoxic volumes.

Targeting the Urotensin II/UT G Protein-Coupled Receptor to Counteract Angiogenesis and Mesenchymal Hypoxia/Necrosis in Glioblastoma.

Glioblastomas (GBMs) are the most common primary brain tumors characterized by strong invasiveness and angiogenesis. GBM cells and microenvironment secrete angiogenic factors and also express chemoattractant G protein-coupled receptors (GPCRs) to their advantage. We investigated the role of the vasoactive peptide urotensin II (UII) and its receptor UT on GBM angiogenesis and tested potential ligand/therapeutic options based on this system. On glioma patient samples, the expression of UII and UT increased with the grade with marked expression in the vascular and peri-necrotic mesenchymal hypoxic areas being correlated with vascular density. In vitro human UII stimulated human endothelial HUV-EC-C and hCMEC/D3 cell motility and tubulogenesis. In mouse-transplanted Matrigel sponges, mouse (mUII) and human UII markedly stimulated invasion by macrophages, endothelial, and smooth muscle cells. In U87 GBM xenografts expressing UII and UT in the glial and vascular compartments, UII accelerated tumor development, favored hypoxia and necrosis associated with increased proliferation (Ki67), and induced metalloproteinase (MMP)-2 and -9 expression in Nude mice. UII also promoted a "tortuous" vascular collagen-IV expressing network and integrin expression mainly in the vascular compartment. GBM angiogenesis and integrin αvß3 were confirmed by in vivo 99mTc-RGD tracer imaging and tumoral capture in the non-necrotic area of U87 xenografts in Nude mice. Peptide analogs of UII and UT antagonist were also tested as potential tumor repressor. Urotensin II-related peptide URP inhibited angiogenesis in vitro and failed to attract vascular and inflammatory components in Matrigel in vivo. Interestingly, the UT antagonist/biased ligand urantide and the non-peptide UT antagonist palosuran prevented UII-induced tubulogenesis in vitro and significantly delayed tumor growth in vivo. Urantide drastically prevented endogenous and UII-induced GBM angiogenesis, MMP, and integrin activations, associated with GBM tumoral growth. These findings show that UII induces GBM aggressiveness with necrosis and angiogenesis through integrin activation, a mesenchymal behavior that can be targeted by UT biased ligands/antagonists.

The added value of clinical radiopharmacists in nuclear medicine: The example of glomerular filtration rate assessment in kidney donors.

WHAT IS KNOWN AND OBJECTIVES: In Europe, the pharmaceutical supply of chromium-51 has been stopped. However, this isotope is necessary for the evaluation of glomerular filtration rates. It is possible to replace it with technetium-99m, but the validation of this change in the measurement method must be carried out. METHODS: A retrospective analysis of chromium-51 data from January 2018 to January 2019 was performed, followed by a study from January 2019 to January 2020 using the technetium tracer. The patients were different in the both study groups, and none had an eGFR below 50 mL min-1 . A cost analysis was performed. Patient exposure to ionizing radiation was studied for both methods. RESULTS AND DISCUSSION: Seventy-eight patients were included in the study. In total, 42 EDTA-51 Cr and 36 DTPA-99m Tc examinations were conducted and compared. There were no significant differences between the methods used to assess renal function (P = .351). The results of cost analysis and patient radiation exposure were in favour of DTPA-99m Tc examinations. WHAT IS NEW AND CONCLUSION: Within the limitations of a retrospective study of two patient cohorts, there was no significant difference between the results obtained with chromium-51 and technetium-99m tracers. In addition, with the use of DTPA-99m Tc, operating costs and patient exposure to ionizing radiation were reduced, and clinical activity was maintained for the patients' benefit. Radiopharmacists are able to react quickly to supply contingencies, reduce operating costs and maintain the quality of medical examinations.

Development of Novel 111-In-Labelled DOTA Urotensin II Analogues for Targeting the UT Receptor Overexpressed in Solid Tumours.

Overexpression of G protein-coupled receptors (GPCRs) in tumours is widely used to develop GPCR-targeting radioligands for solid tumour imaging in the context of diagnosis and even treatment. The human vasoactive neuropeptide urotensin II (hUII), which shares structural analogies with somatostatin, interacts with a single high affinity GPCR named UT. High expression of UT has been reported in several types of human solid tumours from lung, gut, prostate, or breast, suggesting that UT is a valuable novel target to design radiolabelled hUII analogues for cancer diagnosis. In this study, two original urotensinergic analogues were first conjugated to a DOTA chelator via an aminohexanoic acid (Ahx) hydrocarbon linker and then -hUII and DOTA-urantide, complexed to the radioactive metal indium isotope to successfully lead to radiolabelled DOTA-Ahx-hUII and DOTA-Ahx-urantide. The 111In-DOTA-hUII in human plasma revealed that only 30% of the radioligand was degraded after a 3-h period. DOTA-hUII and DOTA-urantide exhibited similar binding affinities as native peptides and relayed calcium mobilization in HEK293 cells expressing recombinant human UT. DOTA-hUII, not DOTA-urantide, was able to promote UT internalization in UT-expressing HEK293 cells, thus indicating that radiolabelled 111In-DOTA-hUII would allow sufficient retention of radioactivity within tumour cells or radiolabelled DOTA-urantide may lead to a persistent binding on UT at the plasma membrane. The potential of these radioligands as candidates to target UT was investigated in adenocarcinoma. We showed that hUII stimulated the migration and proliferation of both human lung A549 and colorectal DLD-1 adenocarcinoma cell lines endogenously expressing UT. In vivo intravenous injection of 111In-DOTA-hUII in C57BL/6 mice revealed modest organ signals, with important retention in kidney. 111In-DOTA-hUII or 111In-DOTA-urantide were also injected in nude mice bearing heterotopic xenografts of lung A549 cells or colorectal DLD-1 cells both expressing UT. The observed significant renal uptake and low tumour/muscle ratio (around 2.5) suggest fast tracer clearance from the organism. Together, DOTA-hUII and DOTA-urantide were successfully radiolabelled with 111Indium, the first one functioning as a UT agonist and the second one as a UT-biased ligand/antagonist. To allow tumour-specific targeting and prolong body distribution in preclinical models bearing some solid tumours, these radiolabelled urotensinergic analogues should be optimized for being used as potential molecular tools for diagnosis imaging or even treatment tools.

Immunotherapy by Immune Checkpoint Inhibitors and Nuclear Medicine Imaging: Current and Future Applications.

Immunotherapy by using immune checkpoint inhibitors is a revolutionary development in oncology. Medical imaging is also impacted by this new therapy, particularly nuclear medicine imaging (also called radionuclide imaging), which uses radioactive tracers to visualize metabolic functions. Our aim was to review the current applications of nuclear medicine imaging in immunotherapy, along with their limitations, and the perspectives offered by this imaging modality. Method: Articles describing the use of radionuclide imaging in immunotherapy were researched using PubMed by April 2019 and analyzed. Results: More than 5000 articles were analyzed, and nearly 100 of them were retained. Radionuclide imaging, notably 18F-FDG PET/CT, already has a major role in many cancers for pre-therapeutic and therapeutic evaluation, diagnoses of adverse effects, called immune-related adverse events (IrAE), and end-of-treatment evaluations. However, these current applications can be hindered by immunotherapy, notably due to atypical response patterns such as pseudoprogression, which is defined as an increase in the size of lesions, or the visualization of new lesions, followed by a response, and hyperprogression, which is an accelerated tumor growth rate after starting treatment. To overcome these difficulties, new opportunities are offered, particularly therapeutic evaluation criteria adapted to immunotherapy and immuno-PET allowing us to predict responses to immunotherapy. Moreover, some new technological solutions are also promising, such as radiomic analyses and body composition on associated anatomical images. However, more research has to be done, notably for the diagnosis of hyperprogression and pseudoprogression. Conclusion: Immunotherapy, by its major impact on cancer and by the new patterns generated on images, is revolutionary in the field of medical images. Nuclear medicine imaging is already established and will be able to help meet new challenges through its plasticity.

Radiotherapy boost in patients with hypoxic lesions identified by 18F-FMISO PET/CT in non-small-cell lung carcinoma: can we expect a better survival outcome without toxicity? [RTEP5 long-term follow-up].

PURPOSE: Chemoradiotherapy is the reference curative-intent treatment for nonresectable locally advanced non-small-cell lung carcinoma (NSCLC), with unsatisfactory survival, partially due to radiation resistance in hypoxic tissues. The objective was to update survival and toxicity at 3 years following radiotherapy boost to hypoxic tumours in NSCLC patients treated with curative-intent chemoradiotherapy. METHODS: This was an open-label, nonrandomized, multicentre, phase II clinical trial. 18F-Fluoromisonidazole (18F-FMISO) PET/CT was used to determine the hypoxic profile of the patients. 18F-FMISO-positive patients and those without organ-at-risk constraints received a radiotherapy boost (70-84 Gy); the others received standard radiotherapy (66 Gy). Overall survival (OS), progression-free survival (PFS) and safety were assessed. RESULTS: A total of 54 patients were evaluated. OS and PFS rates at 3 years were 48.5% and 28.8%, respectively. The median OS in the 18F-FMISO-positive patients was 25.8 months and was not reached in the 18F-FMISO-negative patients (p = 0.01). A difference between the groups was also observed for PFS (12 months vs. 26.2 months, p = 0.048). In 18F-FMISO-positive patients, no difference was observed in OS in relation to dose, probably because of the small sample size (p = 0.30). However, the median OS seemed to be in favour of patients who received the radiotherapy boost (26.5 vs. 15.3 months, p = 0.71). In patients who received the radiotherapy boost, no significant late toxicities were observed. CONCLUSION: 18F-FMISO uptake in NSCLC patients is strongly associated with features indicating a poor prognosis. In 18F-FMISO-positive patients, the radiotherapy boost seemed to improve the OS by 11.2 months. A further clinical trial is needed to investigate the efficacy of a radiotherapy boost in patients with hypoxic tumours.

Real-time molecular optical micro-imaging of EGFR mutations using a fluorescent erlotinib based tracer.

BACKGROUND: EGFR mutations are routinely explored in lung adenocarcinoma by sequencing tumoral DNA. The aim of this study was to evaluate a fluorescent-labelled erlotinib based theranostic agent for the molecular imaging of mutated EGFR tumours in vitro and ex vivo using a mice xenograft model and fibred confocal fluorescence microscopy (FCFM). METHODS: The fluorescent tracer was synthesized in our laboratory by addition of fluorescein to an erlotinib molecule. Three human adenocarcinoma cell lines with mutated EGFR (HCC827, H1975 and H1650) and one with wild-type EGFR (A549) were xenografted on 35 Nude mice. MTT viability assay was performed after exposure to our tracer. In vitro imaging was performed at 1 µM tracer solution, and ex vivo imaging was performed on fresh tumours excised from mice and exposed to a 1 µM tracer solution in PBS for 1 h. Real-time molecular imaging was performed using FCFM and median fluorescence intensity (MFI) was recorded for each experiment. RESULTS: MTT viability assay confirmed that addition of fluorescein to erlotinib did not suppress the cytotoxic of erlotinib on tumoral cells. In vitro FCFM imaging showed that our tracer was able to distinguish cell lines with mutated EGFR from those lines with wild-type EGFR (p < 0.001). Ex vivo FCFM imaging of xenografts with mutated EGFR had a significantly higher MFI than wild-type (p < 0.001). At a cut-off value of 354 Arbitrary Units, MFI of our tracer had a sensitivity of 100% and a specificity of 96.3% for identifying mutated EGFR tumours. CONCLUSION: Real time molecular imaging using fluorescent erlotinib is able to identify ex vivo tumours with EGFR mutations.

FDG and FMISO PET-guided dose escalation with intensity-modulated radiotherapy in lung cancer.

BACKGROUND: Concomitant chemo-radiotherapy is the reference treatment for non-resectable locally-advanced Non-Small Cell Lung Cancer (NSCLC). Increasing radiotherapy total dose in the whole tumour volume has been shown to be deleterious. Functional imaging with positron emission tomography (PET/CT) offers the potential to identify smaller and biologically meaningful target volumes that could be irradiated with larger doses without compromising Organs At Risk (OAR) tolerance. This study investigated four scenarios, based on 18FDG and 18F-miso PET/CT, to delineate the target volumes and derive radiotherapy plans delivering up to 74Gy. METHOD: Twenty-one NSCLC patients, selected from a prospective phase II trial, had 18FDG- and 18F-miso PET/CT before the start of radiotherapy and 18FDG PET/CT during the radiotherapy (42Gy). The plans were based planned on a standard plan delivering 66 Gy (plan 1) and on three different boost strategies to deliver 74Gy total dose in pre-treatment 18FDG hotspot (70% of SUVmax) (plan 2), pre-treatment 18F-miso target (SUVmax > 1.4) (plan 3) and per-treatment 18FDG residual (40% of SUVmax). (plan 4). RESULTS: The mean target volumes were 4.8 cc (± 1.1) for 18FDG hotspot, 38.9 cc (± 14.5) for 18F-miso and 36.0 cc (± 10.1) for per-treatment 18FDG. In standard plan (66 Gy), the mean dose covering 95% of the PTV (D95%) were 66.5 (± 0.33), 66.1 (± 0.32) and 66.1 (± 0.32) Gy for 18FDG hotspot, 18F-miso and per-treatment 18FDG. In scenario 2, the mean D95% was 72.5 (± 0.25) Gy in 18FDG hotspot versus 67.9 (± 0.49) and 67.9 Gy (± 0.52) in 18F-miso and per-treatment 18FDG, respectively. In scenario 3, the mean D95% was 72.2 (± 0.27) Gy to 18F-miso versus 70.4 (± 0.74) and 69.5Gy (± 0.74) for 18FDG hotspot and per-treatment 18FDG, respectively. In scenario 4, the mean D95% was 73.1 (± 0.3) Gy to 18FDG per-treatment versus 71.9 (± 0.61) and 69.8 (± 0.61) Gy for 18FDG hotspot and 18F-miso, respectively. The dose/volume constraints to OARs were matched in all scenarios. CONCLUSION: Escalated doses can be selectively planned in NSCLC target volumes delineated on 18FDG and 18F-miso PET/CT functional images. The most relevant strategy should be investigated in clinical trials. TRIAL REGISTRATION: (RTEP5, NCT01576796 , registered 15 june 2012).

Physicochemical stability of oxycodone-ketamine solutions in polypropylene syringe and polyvinyl chloride bag for patient-controlled analgesia use.

OBJECTIVES: The study evaluated the stability of three combinations of oxycodone and ketamine diluted in normal saline (NS) after storage for 7 days at 23°C and exposed to light. METHODS: The stability of three mixtures of oxycodone and ketamine (oxycodone 0.4 mg/mL+ketamine 40 mg/mL, oxycodone 10 mg/mL+ketamine 0.1 mg/mL and oxycodone 10 mg/mL+ketamine 40 mg/mL) in NS stored in a polypropylene syringe and a polyvinyl chloride (PVC) bag was studied. The physical characteristics, including pH, colour and precipitation, were evaluated. The samples were analysed in triplicate by a stability-indicating high-performance liquid chromatography method. RESULTS: There was no significant change in the pH values of any solution. No precipitation or change in colour was observed. All formulations maintained more than 95% of the initial concentration of each drug on day 7. No trace of degradation products was detected. CONCLUSIONS: Ketamine (0.1-40 mg/mL) combined with oxycodone (0.4-10 mg/mL) is physically compatible and chemically stable for 7 days when diluted with NS, packaged in polypropylene syringe or PVC bag and stored at 23°C.

In- and ex-vivo molecular imaging of apoptosis to assess sensitivity of non-small cell lung cancer to EGFR inhibitors using probe-based confocal laser endomicroscopy.

BACKGROUND: Prediction of treatment outcome of non-small cell lung cancer (NSCLC) with EGFR inhibitors on the basis of the genetic analysis of the tumor can be incorrect in case of rare or complex mutations, bypass molecular activation pathways, or pharmacodynamic variations. The aim of this study was to develop an ex vivo and in vivo real-time quantitative imaging test for EGFR inhibitors sensitivity assessment. METHODS: Erlotinib resistant (A549, H460, H1975), insensitive (H1650) and hypersensitive (HCC827) cell lines were injected subcutaneously in Nude mice. Tumor xenografts from mice treated with Erlotinib were imaged ex vivo and in vivo using probe-based confocal laser endomicroscopy (pCLE) and NucView 488 Caspase 3 substrate, a fluorescent probe specific for the activated caspase 3. RESULTS: Assessment of apoptosis at 24h post treatment, both ex vivo in explanted tumor xenografts and in vivo, showed a significant difference between resistant cell lines (A549, H460 and H1975) and insensitive (H1650) or hypersensitive (HCC827) ones (p<0.05 for ex vivo imaging, p≤0.02 for in vivo imaging). There was also a significant difference between insensitive and hypersensitive cell lines, both ex vivo (p<0.05) and in vivo (p = 0.01). CONCLUSION: Real-time in vivo and ex vivo assessment of apoptosis using pCLE differentiates resistant from sensitive NSCLC xenografts to Erlotinib.

Donepezil-Based Central Acetylcholinesterase Inhibitors by Means of a "Bio-Oxidizable" Prodrug Strategy: Design, Synthesis, and in Vitro Biological Evaluation.

With the aim of reducing side effects of acetylcholinesterase inhibitors (AChEIs) during symptomatic treatment of Alzheimer's disease, we report herein a new class of donepezil-based "bio-oxidizable" prodrugs 1 designed to be converted into dual binding site AChEIs 2. While most of indanone-derived N-benzylpyridinium salts 2 revealed to be highly potent dual binding site hAChEIs (IC50 up to 3 nM), outperforming the standard drug donepezil (IC50 = 11 nM), most of the corresponding 1,4-dihydropyridines 1 were found to be inactive. Promisingly, whereas the selected prodrug 1r showed good permeability in the PAMPA-BBB model and high in vitro antioxidant activity, its conversion to AChEI 2r could be easily achieved under mild conditions when incubated in various oxidizing media. Lastly, both compounds 1r and 2r did not show genotoxicity in vitro and displayed high LD50 values in mice, making this prodrug 1r/drug 2r couple a good candidate for further in vivo biological experiments.

Phase II Study of a Radiotherapy Total Dose Increase in Hypoxic Lesions Identified by 18F-Misonidazole PET/CT in Patients with Non-Small Cell Lung Carcinoma (RTEP5 Study).

See an invited perspective on this article on page 1043.This multicenter phase II study investigated a selective radiotherapy dose increase to tumor areas with significant 18F-misonidazole (18F-FMISO) uptake in patients with non-small cell lung carcinoma (NSCLC). Methods: Eligible patients had locally advanced NSCLC and no contraindication to concomitant chemoradiotherapy. The 18F-FMISO uptake on PET/CT was assessed by trained experts. If there was no uptake, 66 Gy were delivered. In 18F-FMISO-positive patients, the contours of the hypoxic area were transferred to the radiation oncologist. It was necessary for the radiotherapy dose to be as high as possible while fulfilling dose-limiting constraints for the spinal cord and lungs. The primary endpoint was tumor response (complete response plus partial response) at 3 mo. The secondary endpoints were toxicity, disease-free survival (DFS), and overall survival at 1 y. The target sample size was set to demonstrate a response rate of 40% or more (bilateral α = 0.05, power 1-ß = 0.95). Results: Seventy-nine patients were preincluded, 54 were included, and 34 were 18F-FMISO-positive, 24 of whom received escalated doses of up to 86 Gy. The response rate at 3 mo was 31 of 54 (57%; 95% confidence interval [CI], 43%-71%) using RECIST 1.1 (17/34 responders in the 18F-FMISO-positive group). DFS and overall survival at 1 y were 0.86 (95% CI, 0.77-0.96) and 0.63 (95% CI, 0.49-0.74), respectively. DFS was longer in the 18F-FMISO-negative patients (P = 0.004). The radiotherapy dose was not associated with DFS when adjusting for the 18F-FMISO status. One toxic death (66 Gy) and 1 case of grade 4 pneumonitis (>66 Gy) were reported. Conclusion: Our approach results in a response rate of 40% or more, with acceptable toxicity. 18F-FMISO uptake in NSCLC patients is strongly associated with poor prognosis features that could not be reversed by radiotherapy doses up to 86 Gy.

SPECT-computed tomography in rats with TNBS-induced colitis: A first step toward functional imaging.

AIM: To assess the feasibility of SPECT-computed tomography (CT) in rats with trinitrobenzene sulfonic acid (TNBS)-induced acute colitis and confront it with model inflammatory characteristics. METHODS: Colitis was induced in Sprague-Dawley rats by intrarectal injection of TNBS (n = 10) while controls received vehicle (n = 10). SPECT-CT with intravenous injection of 10 MBq of 67Ga-Citrate was performed at day 2. SPECT-CT criteria were colon wall thickness and maximal wall signal intensity. Laboratory parameters were assessed: colon weight:length ratio, colon cyclooxygenase-2 expression by western blot and histological inflammatory score. RESULTS: Colon weight/length ratio, colon COX-2 expression and histological inflammatory score were significantly higher in the TNBS group than in the control group (P = 0.0296, P < 0.0001, P = 0.0007 respectively). Pixel max tend to be higher in the TNBS group than in the control group but did not reach statistical significance (P = 0.0662). Maximal thickness is significantly increased in the TNBS group compared to the control group (P = 0.0016) while colon diameter is not (P = 0.1904). Maximal thickness and colon diameter were correlated to colon COX-2 expression (P = 0.0093, P = 0.009 respectively) while pixel max was not (P = 0.22). Maximal thickness was significantly increased when inflammation was histologically observed (P = 0.0043) while pixel max and colon diameter did not (P = 0.2452, P = 0.3541, respectively). CONCLUSION: SPECT-CT is feasible and easily distinguished control from colitic rats.

Photodynamic therapy using methylene blue in lung adenocarcinoma xenograft and hamster cheek pouch induced squamous cell carcinoma.

BACKGROUND: Photodynamic therapy (PDT) is used to treat early proximal bronchial cancer during a flexible bronchoscopy. The technique relies on the excitation of a photosensitizer by an appropriate wavelength, which is delivered into the bronchus in close contact with the tumor. OBJECTIVE: To assess methylene blue (MB) as a PDT agent for the treatment of respiratory tract cancer in animal models. METHODS: MB-induced PDT was performed on 7 subcutaneous NCI-H460 lung adenocarcinoma xenografts in nude mice and 9 induced squamous cell cancer in the hamster cheek pouch model. In mice, PDT was carried out on right-sided tumors after intratumoral injection of methylene blue 1% (w/v) and illumination at 630nm at 200J/cm (Diomed PDT 630), with the left tumor used as control (illumination alone or MB alone). The tumoral volume was assessed before and 15 days after PDT. RESULTS: Fourteen xenografts were treated in mice, including seven treated with MB-PDT, producing a 52% mean tumor volume regression (1568mm(3)vs. 544mm(3)) compared to seven control cases in which tumor volume increased (p=0.007; Mann-Whitney test). Nine cheek pouch induced carcinomas were treated in the hamster group, with a mean volume decrease of 85.8% (from 44.8% to 100%) (initial mean volume=210mm(3)vs. post PDT mean volume=97mm(3)). Histology analysis showed 4/9 complete responses. CONCLUSION: Intratumoral MB appears efficient as PDT agent for cancer treatment in animal models. Further studies are needed to assess the safety and efficacy of MB-associated PDT for the treatment of lung cancer in humans.

Bevacizumab enhances efficiency of radiotherapy in a lung adenocarcinoma rodent model: Role of αvß3 imaging in determining optimal window.

INTRODUCTION: Earlier studies indicated that bevacizumab could favorably be combined with radiation. However excessive damage to tumor vasculature can result in radioresistance and clinical data suggest that treatment sequencing may be important when combining bevacizumab with radiation. The aim of this study was to evaluate whether αvß3 scintigraphic imaging could provide information to determine the optimal combination schedule of bevacizumab and radiotherapy on a lung adenocarcinoma model in mice. METHODS: The tumor volume and angiogenesis changes induced after bevacizumab and radiation treatment were evaluated using (99m)Tc-RGD on a microSPECT/CT. First, we determined the optimal dose regimen for bevacizumab and radiotherapy alone. Second, the combined effects of bevacizumab and radiation were evaluated according to the combination timing (radiation 2, 24, 48 hours after bevacizumab and 48 hours before bevacizumab). RESULTS: The optimal dose regimen is 20mg/kg for bevacizumab and 12.5 Gy for radiotherapy with a significant decrease of tumoral uptake and volume at day 9 compared to the controls (+8.8%, +7.7%, and +44% volume, respectively, and +9.8%, +3.8%, and +207% uptake, respectively). Scintigraphic imaging showed a significant increased RGD tumor uptake two hours after bevacizumab treatment compared to 24 hours and controls (p=0.02). When bevacizumab treatment was combined with radiation, the best combination appears to be the administration of bevacizumab two hours prior to radiation with better results than single treatments (p < 0.05). On the contrary, bevacizumab given 24 hours prior to radiation led to less tumor growth delay compared to a single agent, without significant difference compared to the controls. Histological results confirmed these data with an increased percentage of necrosis (p=0.04) and a decrease of angiogenesis (p=0.04) in the optimal combination group. CONCLUSIONS: The RGD tracer helps us identify the vascular normalization window and it shows a supra-additive effect of bevacizumab when administered two hours before radiotherapy.

Safety and performance analysis of acriflavine and methylene blue for in vivo imaging of precancerous lesions using fibered confocal fluorescence microscopy (FCFM): an experimental study.

BACKGROUND: Fibered confocal fluorescence microscopy (FCFM) allows in vivo investigation of pulmonary microstructures. However, the bronchial epithelium can only be imaged using exogenous fluorophores. The objective of this study is to compare methylene blue (MB) and acriflavine genotoxicity and to assess FCFM performance for in vivo imaging of precancerous lesions. METHODS: Genotoxicity was assessed using the comet assay on both cultured human lymphocytes and NCI-H460 cells, which had been exposed to MB or acriflavine before being illuminated at 660 or 488 nm, respectively. FCFM was performed on precancerous lesions in the hamster cheek pouch model, following topical application of the fluorophores. FCFM data were analyzed according to histology. RESULTS: No genotoxicity was found using 0.01% (w/v) MB after illumination at 660 nm for 2 and 15 min (5 mW). Acriflavine exposure (0.025%) led to DNA damages, increasing from 2 to 15 min of light exposure at 448 nm in both lymphocytes (83.4 to 88%, p = 0.021) and NCI H460 cell populations (79.9 to 84.6%, p = 0.045). In total, 11 invasive carcinoma, 24 reserve cell hyperplasia, and 17 dysplasia lesions were imaged using FCFM in vivo. With both fluorophores, the cellular density increased from hyperplasia to high-grade dysplasia (p < 0.05). With MB, the cellular diameter significantly decreased (48.9 to 13.9 µm) from hyperplasia to carcinoma (p < 0.05). In this model, a cut-off diameter of 30 µm enabled the diagnosis of high-grade lesions with a sensitivity of 94.7% and a specificity of 97%. CONCLUSION: Methylene blue can be used safely to image precancerous lesions in vivo. This study does not support the use of acriflavine in humans.

Dihydroquinoline Carbamate Derivatives as "Bio-oxidizable" Prodrugs for Brain Delivery of Acetylcholinesterase Inhibitors: [¹¹C] Radiosynthesis and Biological Evaluation.

With the aim of improving the efficiency of marketed acetylcholinesterase (AChE) inhibitors in the symptomatic treatment of Alzheimer's disease, plagued by adverse effects arising from peripheral cholinergic activation, this work reports a biological evaluation of new central AChE inhibitors based on an original "bio-oxidizable" prodrug strategy. After peripheral injection of the prodrug 1a [IC50 > 1 mM (hAChE)] in mice, monitoring markers of central and peripheral cholinergic activation provided in vivo proof-of-concept for brain delivery of the drug 2a [IC50 = 20 nM (hAChE)] through central redox activation of 1a. Interestingly, peripheral cholinergic activation has been shown to be limited in time, likely due to the presence of a permanent positive charge in 2a promoting rapid elimination of the AChE inhibitor from the circulation of mice. To support these assumptions, the radiosynthesis with carbon-11 of prodrug 1a was developed for additional ex vivo studies in rats. Whole-body biodistribution of radioactivity revealed high accumulation in excretory organs along with moderate but rapid brain uptake. Radio-HPLC analyses of brain samples confirm rapid CNS penetration of [(11)C]1a, while identification of [(11)C]2a and [(11)C]3a both accounts for central redox activation of 1a and pseudoirreversible inhibition of AChE, respectively. Finally, Caco-2 permeability assays predicted metabolite 3a as a substrate for efflux transporters (P-gp inter alia), suggesting that metabolite 3a might possibly be actively transported out of the brain. Overall, a large body of evidence from in vivo and ex vivo studies on small animals has been collected to validate this "bio-oxidizable" prodrug approach, emerging as a very promising strategy in the rational design of selective central AChE inhibitors.