Development of a modular system for the synthesis of PET [(11)C]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.

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

INTRODUCTION AND AIM: Scintigraphic 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. METHODS: A simplified method of producing 68Ga citrate is described. Radiochemical purity, pyrogen testing were performed as per the standard protocols. RESULTS: After 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. CONCLUSION: The 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.

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

BACKGROUND: [(18)F]-FDG is a widely used tracer for the non-invasive evaluation of hypermetabolic processes like cancer and inflammation. However, [(18)F]-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. AIM: The aim of this study was to assess the feasibility of (68)Ga-Chloride small animal PET for the analysis of an animal model of genital infection, induced after the vaginal inoculum of Chlamydia muridarum. Material and Thirty mice were infected by placing 15 microl sucrose phosphate glutamic acid (SPG) 10(7) inclusion forming units of C. muridarum into the vaginal vault. As controls of inflammation, three animals were challenged with 15 microl of SPG and one healthy animal was used to assess the tracer biodistribution. Four animals died during the experiment. Eleven animals were evaluated with (68)Ga-Chloride small animal PET (GE, eXplore Vista) 3-5, 10-12, 17-19 days after infection, as well as three controls of inflammation and one healthy animal. Infection was monitored by obtaining cervical-vaginal swabs from all the animals on the day of each PET procedure. Moreover, five groups of three animals each were killed at 6, 13, 20, 27 and 34 days after infection were studied. RESULTS: (68)Ga-PET turned out positive in all the infected animals, concordantly to data obtained by the cervical swabs and by the ex vivo analysis. The tumour-to-background ratio (TBR) decreased over time as the inflammation tended to naturally extinguish. The controls showed a slightly increased uptake of tracer due to the aseptic inflammation caused by SPG and frequent cervical swabs. The healthy control did not show any pelvic uptake. CONCLUSION: (68)Ga-Chloride is a promising tracer for the assessment of genital infection in a mouse animal model.

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 OBJECTIVE: N-[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. METHODS: Different 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 CONCLUSION: In 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.

Epithelial and mesenchymal tumor compartments exhibit in vivo complementary patterns of vascular perfusion and glucose metabolism.

Glucose transport and consumption are increased in tumors, and this is considered a diagnostic index of malignancy. However, there is recent evidence that carcinoma-associated stromal cells are capable of aerobic metabolism with low glucose consumption, at least partly because of their efficient vascular supply. In the present study, using dynamic contrast-enhanced magnetic resonance imaging and [F-18]fluorodeoxyglucose (FDG) positron emission tomography (PET), we mapped in vivo the vascular supply and glucose metabolism in syngeneic experimental models of carcinoma and mesenchymal tumor. We found that in both tumor histotypes, regions with high vascular perfusion exhibited a significantly lower FDG uptake. This reciprocity was more conspicuous in carcinomas than in mesenchymal tumors, and regions with a high-vascular/low-FDG uptake pattern roughly overlapped with a stromal capsule and intratumoral large connectival septa. Accordingly, mesenchymal tumors exhibited a higher vascular perfusion and a lower FDG uptake than carcinomas. Thus, we provide in vivo evidence of vascular/metabolic reciprocity between epithelial and mesenchymal histotypes in tumors, suggesting a new intriguing aspect of epithelial-stromal interaction. Our results suggests that FDG-PET-based clinical analysis can underestimate the malignity or tumor extension of carcinomas exhibiting any trait of "mesenchymalization" such as desmoplasia or epithelial-mesenchymal transition.

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

BACKGROUND AND AIM: Small-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 (18)F-FDG and (11)C-choline in mice for small-animal PET studies. METHODS: We 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 (11)C-choline. PET scan was performed after 10 min and 40 min. Day 2: each animal received an RO injection of 28 MBq of (11)C-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 (18)F-FDG. A PET scan was performed after 60 min and 120 min. Day 4: each animal received an RO injection of 28 MBq of (18)F-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. RESULTS: The RO injections for both (18)F-FDG and (11)C-choline were comparable to the intravenous injection of F-FDG for the standard and delayed acquisitions. CONCLUSION: The 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.

FDG small animal PET permits early detection of malignant cells in a xenograft murine model.

PURPOSE: The administration of new anticancer drugs in animal models is the first step from in vitro to in vivo pre-clinical protocols. At this stage it is crucial to ensure that cells are in the logarithmic phase of growth and to avoid vascular impairment, which can cause inhomogeneous distribution of the drug within the tumour and thus lead to bias in the final analysis of efficacy. In subcutaneous xenograft murine models, positivity for cancer is visually recognisable 2-3 weeks after inoculation, when a certain amount of necrosis is usually already present. The aim of this study was to evaluate the accuracy of FDG small animal PET for the early detection of malignant masses in a xenograft murine model of human rhabdomyosarcoma. A second goal was to analyse the metabolic behaviour of this xenograft tumour over time. METHODS: We studied 23 nude mice, in which 7 x 10(6) rhabdomyosarcoma cells (RH-30 cell line) were injected in the dorsal subcutaneous tissues. Each animal underwent four FDG PET scans (GE, eXplore Vista DR) under gas anaesthesia. The animals were studied 2, 5, 14 and 20 days after inoculation. We administered 20 MBq of FDG via the tail vein. Uptake time was 60 min, and acquisition time, 20 min. Images were reconstructed with OSEM 2D iterative reconstruction and the target to background ratio (TBR) was calculated for each tumour. Normal subcutaneous tissue had a TBR of 0.3. Necrosis was diagnosed when one or more cold areas were present within the mass. All the animals were sacrificed and histology was available to verify PET results. PET results were concordant with the findings of necropsy and histology in all cases. RESULTS: The incidence of the tumour was 69.6% (16/23 animals); seven animals did not develop a malignant mass. Ten of the 23 animals had a positive PET scan 2 days after inoculation. Nine of these ten animals developed a tumour; the remaining animal became negative, at the third scan. The positive predictive value of the early PET scan was 90% (9/10 animals) while the negative predictive value was 46% (6/13 animals). In the whole group of animals, mean TBR increased scan by scan. There was a statistically significant difference in TBR between 2 and 20 days after inoculation. Necrosis was present at the second scan in two animals, at the third scan in six animals and at the fourth scan in 11 animals. CONCLUSION: The high positive predictive value of FDG PET 2 days after inoculation means that an animal with a first positive scan has a very high likelihood of developing a mass and can be treated at an early stage with an experimental drug. Animals negative at this point in time will never develop a mass or will eventually do so at a late phase. As 2 of the 16 (12.5%) positive animals had necrosis at the second scan, indicating a vascular mismatch, it may be argued that animals should be treated 2 days after inoculation to guarantee homogeneous vascularisation (thereby ensuring a good drug supply within the tumour) in all subjects.

A simple Tracerlab module modification for automated on-column [11C]methylation and [11C]carboxylation.

A modification of commercial [11C]methylation module which can be implemented for both on-column [11C]methylation and [11C]carboxylation in the same automated system is described. This module configuration was applied to the solid-phase synthesis of N-[11C]methyl-choline ([11C]choline) and L-(S-methyl-[11C])methionine ([11C]methionine), using [11C]CH(3)I as methylating agent, as well as to the synthesis of [11C]acetate by [11C]carboxylation with [11C]CO2 of methylmagnesium chloride with high and reproducible radiochemical yields in short reaction time, demonstrating to be a fast and reliable tool for the production of these [11C]radiopharmaceuticals for clinical use.

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.

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.