Silvia Trespidi

Induction and suppression of cytochrome P450 isoenzymes and generation of oxygen radicals by procymidone in liver, kidney and lung of CD1 mice

Although chronic administration of procymidone (a widely used dicarboximide fungicide) leads to an increased incidence of liver tumors in mice, short-term genotoxicity studies proved negative. As cytochrome P450 (CYP) induction has been linked to non-genotoxic carcinogenesis, we investigated whether procymidone administration causes induction of CYP-dependent monooxygenases in liver, kidney and lung microsomes of male Swiss Albino CD1 mice after single or repeated (daily for three consecutive days) i.p. treatment with either 400 or 800 (1/10 or 1/20 of the DL(50)) mgkg(-1) b.w. procymidone. CYP content and CYP3A1/2, 1A1, 1A2, 2B1/2, 2E1, 2A, 2D9 and 2C11 supported oxidations were studied using either the regio- and stereo-selective hydroxylation of testosterone as multibiomarker or highly specific substrates as probes of various CYPs. While a single dose was uneffective, multiple procymidone administration lead to marked inductions of various monooxygenases: CYP3A1/2 in liver and lung (as measured by N-demethylation of aminopyrine and testosterone 6 beta-hydroxylase); CYP2E1 in liver (p-nitrophenol hydroxylation); CYP1A1 in liver and kidney (deethylation of ethoxyresorufin). Several hydroxylations were induced in the liver, including the CYP2A-linked 7 alpha (14-fold) as well as 6 alpha (22-fold), 6 beta, 16 beta and 2 beta hydroxylases. The pattern of inductions/suppressions recorded in the three different tissues suggests that procymidone exerts complex effects on the CYP profile. Tissue-specific trends included a large number of inductions in the liver and suppressions in the lung. The main inductions were corroborated by immunoblotting analyses and Northern blotting showed that inductions of CYP3A1/2, CYP2E1 and CYP1A1/2 were paralleled by increased mRNA levels. It was also found that CYP over-expression generates large amounts of reactive oxygen species (ROS), especially in liver. These data may explain why in vitro short-term genotoxicity studies on procymidone were negative, whereas in vivo long-term carcinogenesis studies turned out positive: long-term CYP induction (e.g. oxygen centered free radicals over-production) can have a co-carcinogenic and/or promoting potential.

Retro-orbital injection is an effective route for radiopharmaceutical administration in mice during small-animal PET studies.

Background and aimSmall-animal PET is acquiring importance for pre-clinical studies. In rodents, radiotracers are usually administrated via the tail vein. This procedure can be very difficult and time-consuming as soft tissue extravasations are very frequent and tail scars can prevent repeated injections after initial failure. The aim of our study was to compare the retro-orbital (RO) versus tail vein intravenous (i.v.) administration of 18F-FDG and 11C-choline in mice for small-animal PET studies. MethodsWe evaluated four healthy female ICR CD1 mice according to the following protocol. Day 1: each animal underwent an i.v. injection of 28 MBq of 11C-choline. PET scan was performed after 10 min and 40 min. Day 2: each animal received an RO injection of 28 MBq of 11C-choline. A PET scan was performed after 10 min and 40 min. Day 3: each animal received an i.v. injection of 28 MBq of 18F-FDG. A PET scan was performed after 60 min and 120 min. Day 4: each animal received an RO injection of 28 MBq of 18F-FDG. A PET scan was performed after 60 min and 120 min. Administration and image acquisition were performed under gas anaesthesia. For FDG studies the animals fasted for 2 h and were kept asleep for 20–30 min after injection, to avoid muscular uptake. Images were reconstructed with 2-D OSEM. For each scan ROIs were drawn on liver, kidneys, lung, brain, heart brown fat and muscles, and the SUV was calculated. We finally compared choline i.v. standard acquisition to choline RO standard acquisition; choline i.v. delayed acquisition to choline RO delayed acquisition; FDG i.v. standard acquisition to FDG RO standard acquisition; FDG i.v. delayed acquisition to FDG RO delayed acquisition. ResultsThe RO injections for both 18F-FDG and 11C-choline were comparable to the intravenous injection of 18F-FDG for the standard and delayed acquisitions. ConclusionThe RO administration in mice represents a technical advantage over intravenous administration in being an easier and faster procedure. However, its use requires high specific activity while its value in peptides and other receptor-specific radiopharmaceuticals needs further assessment.

Synthesis and quality control of 68Ga citrate for routine clinical PET.

Introduction and aimScintigraphic imaging of infection and inflammation with 67Ga-citrate is an established and powerful diagnostic tool in the management of patients with infectious or inflammatory diseases. 68Ga is a short-lived positron-emitting radionuclide (half-life 67.6 min, positron energy 2.92 MeV), which allows better imaging qualities than 67Ga using the high spatial resolution and the quantitative features of PET. The aim of this study was to develop a method of synthesis for 68Ga citrate with high and reproducible radiochemical yield using a commercial 68Ga-labelling module. The resultant 68Ga citrate would be suitable for use in the detection of infectious or inflammatory diseases in routine clinical practice. MethodsA simplified method of producing 68Ga citrate is described. Radiochemical purity, pyrogen testing were performed as per the standard protocols. ResultsAfter performing 10 syntheses of 68Ga citrate, the radiochemical yield was 64.1±6.0% (mean±standard deviation) with an average activity of 971.2±103.4 MBq available for labelling. Radiochemical purity determined by instant thin-layer chromatography-silica gel was higher than 98%. All the synthesized products were found to be sterile and pyrogen-free. In this study, the quality control step provided good and reproducible results. This is worth noting, especially in view of the stringent new rules adopted in most European countries for the in-house good manufacturing practice (GMP) synthesis of radiopharmaceuticals. ConclusionThe high radiochemical yield and purity showed that this method is a reliable tool for the production of 68Ga citrate to be used in the detection of inflammatory and infectious diseases using high resolution and qualitative PET.

CAPTAN IMPAIRS CYP-CATALYZED DRUG METABOLISM IN THE MOUSE

To investigate whether the fungicide captan impairs CYP-catalyzed drug metabolism in murine liver, kidney and lung, the modulation of the regio- and stereo-selective hydroxylation of testosterone, including 6beta-(CYP3A), 6alpha-(CYP2A1 and CYP2B1) and 16alpha-(CYP2B9) oxidations was studied. Specific substrates as probes for different CYP isoforms such as p-nitrophenol (CYP2E1), pentoxyresorufin (CYP2B1), ethoxyresorufin (CYP1A1), aminopyrine (CYP3A), phenacetin and methoxyresorufin (CYP1A2), and ethoxycoumarin (mixed) were also considered. Daily doses of captan (7.5 or 15 mg/kg b.w., i.p.) were administered to different groups of Swiss Albino CD1 mice of both sexes for 1 or 3 consecutive days. While a single dose of this fungicide did not affect CYP-machinery, repeated treatment significantly impaired the microsomal metabolism; in the liver, for example, a general inactivating effect was observed, with the sole exception of testosterone 2alpha-hydroxylase activity which was induced up to 8.6-fold in males. In vitro studies showed that the mechanism-based inhibition was related to captan metabolites rather than the parental compound. In the kidney, both CYP3A- and CYP1A2-linked monooxygenases were significantly induced (2-fold) by this pesticide. Accelerated phenacetin and methoxyresorufin metabolism (CYP1A2) was also observed in the lung. Data on CYP3A (kidney) and CYP1A2 (kidney and lung) induction were corroborated by Western immunoblotting using rabbit polyclonal anti-CYP3A1/2 and CYP1A1/2 antibodies. By means of electron spin resonance (EPR) spectrometry coupled to a spin-trapping technique, it was found that the recorded induction generates a large amounts of the anion radical superoxide (O*2-) either in kidney or lung microsomes. These findings suggest that alterations in CYP-associated activities by captan exposure may result in impaired (endogenous) metabolism as well as of coadministered drugs with significant implications for their disposition. The adverse outcomes associated to CYP changes (e.g. cotoxicity, comutagenicity and promotion) may also have harmful consequences.

Taking EPR snapshots of the oxidative stress status in human blood

Assessment of oxidative stress status (OSS) in human tissues is still troublesome. Using an innovative EPR-radical-probe we successfully measured the instantaneous concentration of ROS directly in peripheral blood of athletes and normally active workers during 60 min controlled exercise. The probe employed was bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate, which quantitatively and instantaneously reacts with oxygen-centered radicals (including superoxide) to yield the parent nitroxide, which is sufficiently persistent to be measured by EPR. Our measurements suggest that while at rest normally active individuals may benefit more from antioxidant supplementation than athletes; conversely, during exercise athletes may benefit more from supplementation. Our method allows reliable, quick, and non-invasive quantitative determination of OSS in human peripheral blood.

Radiolabelling, quality control and radiochemical purity assessment of the Octreotide analogue 68Ga DOTA NOC

Somatostatin receptors 1-5 are over expressed in neuroendocrine tumours (NETs). 68Ga-labelled [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-1-Nal3-Octreotide (DOTA NOC), a recent synthesized somatostatin analogue, shows high affinity for those receptors. Herein, modifications of a commercial module for the labelling of DOTA NOC with 68Ga, as well as the assessment of time course of the radiochemical purity variation are described. The evaluation of radiochemical stability was done by two different chromatographic methods: reversed-phase radio HPLC and fast TLC analysis. Labelled compound has been found radiochemically stable within 3h from the end of labelling (EOL) and radiochemical purity was always higher than 99%. After 73 labelling sessions the system showed great reproducibility and high radiochemical yield.

Small animal PET for the evaluation of an animal model of genital infection

Background:  [18F]‐FDG is a widely used tracer for the non‐invasive evaluation of hypermetabolic processes like cancer and inflammation. However, [18F]‐FDG is considered inaccurate for the diagnosis of urinary tract and genital infections because of its urinary excretion. Since the 1970s, Gallium scintigraphy is a well established test that has been used for the evaluation of inflammation and infection in human patients.

Development of a modular system for the synthesis of PET [11C]labelled radiopharmaceuticals

[((11))C]labelled radiopharmaceuticals as N-[(11)C]methyl-choline ([(11)C]choline), l-(S-methyl-[(11)C])methionine ([(11)C]methionine) and [(11)C]acetate have gained increasing importance in clinical PET and for the routine production of these radiopharmaceuticals, simple and reliable modules are needed to produce clinically relevant radioactivity. On the other hand, flexible devices are needed not only for the routine synthesis but also for more complex applications as the development of new tracers. The aim of this work was the adaptation of an Eckert Ziegler modular system for easy routine synthesis of [(11)C]choline, [(11)C]methionine and [(11)C]acetate using components that account for straightforward scaling up and upgrades.

Reliability and reproducibility of N-[11C]methyl-choline and L-(S-methyl-[11C])methionine solid-phase synthesis: a useful and suitable method in clinical practice.

Background and objectiveN-[11C]methyl-choline ([11C]choline) and L-(S-methyl-[11C])methionine ([11C]methionine) are PET radiopharmaceuticals which have gained interest as oncological tracers. The increasing demand of these radiopharmaceuticals needs robust methods of synthesis with high and reproducible yield which provide enough activity for multiple patient administration in a short synthesis time. MethodsDifferent synthetic approaches have been described in the literature but exhaustive reports on performance and reliability of different methods have not been described yet. Results and conclusionIn the present study, we demonstrated the reliability and reproducibility of the solid-phase [11C]methylation method for the synthesis of [11C]choline and [11C]methionine as a suitable tool for the routine clinical use.

C-Methionine PET/CT in Central Nervous System Tumours: A Review

Central nervous system (CNS) malignant tumors are usually treated with different combinations of surgery, radiotherapy and chemotherapy to achieve a radical treatment. After therapy, conventional imaging procedures (CT and MR) are useful to detect disease relapse but a correct differential diagnosis in presence post-surgical fibrosis, radionecrosis or oedema (benign conditions) may sometime be difficult. CNS shows physiologically an intense uptake of FDG and consequently, small and/or hypo/isometabolic malignant lesions are very difficult to detect, as they may be masked by the hypermetabolic surrounding tissue. 11C-Methionine is a radiolabeled aminoacid that physiologically do not accumulate into the brain, nor in case of benign conditions such as fibrosis, necrosis or oedema. On the other side, malignant lesions (although low grade) present an increased 11C-Methionine uptake due to an increased proteic metabolism and vascular permeability. Therefore 11CMethionine has optimal tumor-to-background ratio, making this radiotracer very useful to study CNS malignant tumors. The most appropriate indication for 11C-Methionine PET is the presence of non conclusive conventional imaging procedures, not allowing to discriminate between benign conditions and disease relapse. Other applications of 11C-Methionine PET are the definition of the radiotherapy field, the localization of the most metabolic area inside a mass to guide the biopsy and the early evaluation of radiotherapy effect, but all these applications are marginal and not yet validated.