Pierre Bohn

Simultaneous positron emission tomography (PET) assessment of metabolism with 18F-fluoro-2-deoxy-d-glucose (FDG), proliferation with 18F-fluoro-thymidine (FLT), and hypoxia with 18fluoro-misonidazole (F-miso) before and during radiotherapy in patients with non-small-cell lung cancer (NSCLC): A pilot study

OBJECTIVES To investigate the changes in tumour proliferation (using FLT), metabolism (using FDG), and hypoxia (using F-miso) during curative (chemo-) radiotherapy (RT) in patients with non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS Thirty PET scans were performed in five patients (4 males, 1 female) that had histological proof of NSCLC and were candidates for curative-intent RT. Three PET-CT (Biograph S16, Siemens) scans were performed before (t(0)) and during (around dose 46 Gy, t(46)) RT with minimal intervals of 48 h between each PET-CT scan. The tracers used were (18)fluoro-2deoxyglucose (FDG) for metabolism, (18)fluorothymidine (FLT) for proliferation, and (18)F-misonidasole (F-miso) for hypoxia. The 3 image sets obtained at each time point were co-registered (rigid: n=9, elastic: n=1, Leonardo, TrueD, Siemens) using FDG PET-CT as reference. VOIs were delineated (40% SUV(max) values were used as a threshold) for tumours and lymph nodes on FDG PET-CT, and they were automatically pasted on FLT and F-miso PET-CT images. ANOVA and correlation analyses were used for comparison of SUV(max) values. RESULTS Four tumours and twelve nodes were identified on initial FDG PET-CT images. FLT SUV(max) values were significantly lower (p<0.0006) at t(46) in both tumours and nodes. The decrease in FDG SUV(max) values had a trend towards significance (p=0.048). F-Miso SUV(max) values were significantly higher in tumours than in nodes (p=0.02) and did not change during radiotherapy (p=0.39). A significant correlation was observed between FLT and FDG uptake (r=0.56, p<10(-4)) when all data were pooled together, and they remained similar when the before and during RT data were analysed separately. FDG and F-miso uptakes were significantly correlated (r=0.59, p=0.0004) when all data were analysed together. The best fit was obtained after adjusting for lesion type (tumour vs. node). This correlation was observed for the SUV(max) measured during RT (r=0.70, p=0.008) but not for the pre-RT data (r=0.19, p=0.35). The weak correlation between FLT and F-miso uptakes only became significant (r=0.66, p=0.002) when the analysis was restricted to the data acquired during RT. CONCLUSION Three different PET acquisitions can be performed quasi-simultaneously (4-7 days) before and during radiotherapy in patients with NSCLC. Our results at 46 Gy suggest that a fast decrease in the proliferation of both tumours and nodes exists during radiotherapy with differences in metabolism (borderline significant decrease) and hypoxia (stable).

Design of silicon-based misonidazole analogues and 18F-radiolabelling

INTRODUCTION Development of new (18)F-labeled tracers for positron emission tomography (PET) imaging is increasingly important. Herein, we described the synthesis of silicon analogues of [(18)F]fluoromisonidazole in order to develop new radiolabelled compounds for the detection of tumour hypoxic domain. Their stabilities and their in vivo biodistribution were evaluated. METHODS (18)F-labeled silicon-based misonidazole analogues were synthesized by alkylating 2-nitroimidazole with alkyloxy-(3-chloropropyl)dialkyl or diarylsilane. These intermediates were labeled with [(18)F]F(-) with a mixture of K(18)F and Kryptofix (K222) in acetonitrile as standard condition. PET imaging was performed using a dedicated small animal PET scanner. RESULTS (18)F-labeled silicon-based misonidazole analogues were easily synthesized in three steps. The hydrolytic and radiolytic stability of these new fluorosilanes depend on the steric hindrance at the silicon center. Indeed, partial uptake of dimethylfluorosilane [(18)F]2a(1-(3-(Fluorodimethylsilyl)propyl)-2-nitro-1H-imidazole) in tumor hypoxic area was observed but defluorination also appeared. Moreover, PET studies indicated that, owing to its high lipophilicity, the most stable dinaphtylfluorosilane [(18)F]2d is retained mainly by the lungs. CONCLUSION We have described an efficient and versatile approach for the synthesis of (18)F-labeled, silicon-based misonidazole analogues. PET imaging of one of these compounds revealed that hypoxia could be detected. Controlling the biodistribution of (18)F-labeled silicon-based misonidazole analogues will require additional studies.

New huprine derivatives functionalized at position 9 as highly potent acetylcholinesterase inhibitors.

A series of 24 huprine derivatives diversely functionalized at position 9 have been synthesized and evaluated for their inhibitory activity against human recombinant acetylcholinesterase (AChE). These derivatives were prepared in one to five steps from huprine 1 bearing an ester function at position 9. Ten analogues (1, 2, 6–9, 13–15, and 23) are active in the low nanomolar range (IC50 <5 nM), very close to the parent compound huprine X. Compounds 2, 6, and 7 show a very good selectivity for AChE, with AChE inhibitory activities 700–1160‐fold higher than those for butyrylcholinesterase (BChE). The inhibitory potency of these compounds decreases with the steric bulk of the substituents at position 9. According to docking simulations, small substituents fit into the acyl‐binding pocket, whereas the larger ones stick out of the active site gorge of AChE. Determination of the kinetic parameters of three of the most potent huprines (2, 6, and 7) showed that most of the difference in KD is accounted by a decrease in kon, which is correlated to the increase of the substituent size. A first in vivo evaluation has been performed in mice for the most active compound 2 (IC50=1.1 nM) and showed a rather weak toxicity (LD50=40 mg kg−1) and an ability to cross the blood–brain barrier with doses above 15 mg kg−1.

In vivo molecular microimaging of pulmonary aspergillosis

The early diagnosis of invasive pulmonary aspergillosis (IPA) is challenging. Fibered confocal fluorescence microscopy (FCFM) is a new technique that allows in vivo imaging of the lung microstructure during bronchoscopy. In this study, we investigated the ability of FCFM to detect a fluorescent peptide-tracer bound to Aspergillus fumigatus in experimental IPA in 13 immunosuppressed, non-neutropenic rats. Subpleural IPA microabscesses were imaged through a transthoracic window using FCFM in vivo after i.v. injection of the c(CGGRLGPFC)-NH2([FITC]) peptide (n = 7) or saline. Results were compared to 10 immunosuppressed, non-infected rats and to six immunosuppressed Geosmithia argillacea-infected rats with and without i.v. injection of the peptide. The peptide in vitro specifically labeled A. fumigatus grown under biofilm growth conditions but not G. argillacea. In vivo, FCFM showed a local infiltration of fluorescent host cells in both Aspergillus and Geosmithia infections. Lung/inner thoracic wall fluorescence intensity ratio (FI) did not differ before and after peptide administration on healthy lung areas, on non-specific inflammatory areas, or on Geosmithia micro-abscesses. In contrast, FI increased from 1.05 without peptide to 1.83 after peptide injection on Aspergillus micro-abscesses (p < 0.0001). In peptide-injected rats, FI from IPA foci was higher than from non-specific inflammation or from Geosmithia abscesses (p ≤ 0.002). Using c(CGGRLFPC)-NH2([FITC]) peptide, FCFM allows the in vivo specific imaging of pulmonary aspergillosis. These data provide the basis for the in vivo diagnosis of human pulmonary aspergillosis using alveolar confocal endomicroscopy.

In vivo and in situ imaging of experimental invasive pulmonary aspergillosis using fibered confocal fluorescence microscopy

Invasive pulmonary aspergillosis (IPA) is a highly fatal disease in immunosuppressed patients. In this study, we assessed fibered confocal fluorescence microscopy (FCFM), a new endoscopic technique that enables in vivo microscopic imaging of the distal lung, as a tool for in vivo imaging of IPA. IPA was induced in immunosuppressed rats using a wild strain of Aspergillus fumigatus (n = 6) or a fluorescent transformed TAG-RFP A. fumigatus strain (n = 10). Subpleural areas of pulmonary infection were imaged in vivo using FCFM employing a transthoracic approach. Results were compared to three immunosuppressed control groups, i.e., non-inoculated rats (n = 4), rats inoculated with sterile Phospate-buffer saline (PBS; n = 5), and rats inoculated with Geosmithia argillacea (n = 6). Only hyphae of TAG-RFP A. fumigatus were detectable both in vitro and in vivo by FCFM. In vivo, a local infiltration of fluorescent alveolar macrophages was observed with FCFM in IPA areas in all fungal infections groups, but also in focal inflammatory areas in the immunosuppressed PBS group. A specific fibrillar fluorescence was observed in IPA areas with the TAG-RFP A. fumigatus group, with a 83% sensitivity, a 100% specificity, a 100% positive predictive value and 94% negative predictive value. FCFM provides a new tool to study host-aspergillus interactions in vivo.

Rational design of central selective acetylcholinesterase inhibitors by means of a “bio-oxidisable prodrug” strategy

This work deals with the design of a bio-oxidisable prodrug strategy for the development of new central selective acetylcholinesterase inhibitors. This prodrug approach is expected to reduce peripheral anticholinesterase activity responsible for various side effects observed with presently marketed AChE inhibitors. The design of these new AChE inhibitors in quinoline series is roughly based on cyclic analogues of rivastigmine. The key activation step of the prodrug involves an oxidation of an N-alkyl-1,4-dihydroquinoline 1 to the corresponding quinolinium salt 2 unmasking the positive charge required for binding to the catalytic anionic site of the enzyme. The synthesis of a set of 1,4-dihydroquinolines 1 and their corresponding quinolinium salts 2 is presented. An in vitro biological evaluation revealed that while all reduced forms 1 were unable to exhibit any anticholinesterase activity (IC50 > 10(6) nM), most of the quinolinium salts 2 displayed high AChE inhibitory activity (IC50 ranging from 6 microM to 7 nM). These preliminary in vitro assays validate the use of these cyclic analogues of rivastigmine in quinoline series as appealing chemical tools for further in vivo development of this bio-oxidisable prodrug approach.

Synthesis of new 18F-radiolabeled silicon-based nitroimidazole compounds

The syntheses of new nitroimidazole compounds using silicon-[(18)F]fluorine chemistry for the potential detection of tumor hypoxia are described. [(18)F]silicon-based compounds were synthesized by coupling 2-nitroimidazole with silyldinaphtyl or silylphenyldi-tert-butyl groups and labeled by fluorolysis or isotopic exchange. Dinaphtyl compounds (6, 10) were labeled in 56-71% yield with a specific activity of 45 GBq/μmol, however these compounds ([(18)F]7 and [(18)F]11) were not stable in plasma. Phenyldi-tert-butyl compounds were labeled in 70% yield with a specific activity of 3 GBq/μmol by isotopic exchange, or in 81% yield by fluorolysis of siloxanes with a specific activity of 45 GBq/μmol. The labeled compound [(18)F]18 was stable in plasma and excreted by the liver and kidneys in vivo. In conclusion, the fluorosilylphenyldi-tert-butyl (SiFA) group is more stable in plasma than fluorosilyldiphenyl moiety. Thus, compound [(18)F]18 is suitable for further in vivo assessments.

Synthesis of sulfonic acid derivatives by oxidative deprotection of thiols using tert-butyl hypochlorite.

Starting from alkyl halides or Michael acceptors, thioacetates were prepared in situ and further treated with t-BuOCl, affording the corresponding sulfonyl chlorides which were trapped with nucleophiles such as water, alcohol, or amines. The three steps can be achieved in a one-pot procedure. Oxidative deprotection also proved to be efficient with S-trityl and S-tert-butyl groups, making it a convenient route toward cysteic acid derivatives.

Synthesis and biological evaluation of a novel 99mTc labeled 2-nitroimidazole derivative as a potential agent for imaging tumor hypoxia

Tumor hypoxia plays a major role in reducing the efficacy of therapeutic modalities like chemotherapy and radiation therapy in combating cancer. In order to target hypoxic tissues, a tripeptide ligand having a 2-nitroimidazole moiety, as a bioreductive species, was synthesized. The latter was radiolabeled with (99m)Tc for imaging hypoxic regions of tumors and was characterized by means of its rhenium analogue. The biodistribution and scintigraphic image of the corresponding (99m)Tc-complex showed accumulation in tumor and these results suggest that it could be a marker for imaging tumor hypoxia.

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.