Kinetic Modelling of [68Ga]Ga-DOTA-Siglec-9 in Porcine Osteomyelitis and Soft Tissue Infections.

BACKGROUND: [68Ga]Ga-DOTA-Siglec-9 is a positron emission tomography (PET) radioligand for vascular adhesion protein 1 (VAP-1), a protein involved in leukocyte trafficking. The tracer facilitates the imaging of inflammation and infection. Here, we studied the pharmacokinetic modelling of [68Ga]Ga-DOTA-Siglec-9 in osteomyelitis and soft tissue infections in pigs. METHODS: Eight pigs with osteomyelitis and soft tissue infections in the right hind limb were dynamically PET scanned for 60 min along with arterial blood sampling. The fraction of radioactivity in the blood accounted for by the parent tracer was evaluated with radio-high-performance liquid chromatography. One- and two-tissue compartment models were used for pharmacokinetic evaluation. Post-mortem soft tissue samples from one pig were analysed with anti-VAP-1 immunofluorescence. In each analysis, the animal's non-infected left hind limb was used as a control. RESULTS: Tracer uptake was elevated in soft tissue infections but remained low in osteomyelitis. The kinetics of [68Ga]Ga-DOTA-Siglec-9 followed a reversible 2-tissue compartment model. The tracer metabolized quickly; however, taking this into account, produced more ambiguous results. Infected soft tissue samples showed endothelial cell surface expression of the Siglec-9 receptor VAP-1. CONCLUSION: The kinetics of [68Ga]Ga-DOTA-Siglec-9 uptake in porcine soft tissue infections are best described by the 2-tissue compartment model.

Preclinical evaluation of potential infection-imaging probe [68 Ga]Ga-DOTA-K-A9 in sterile and infectious inflammation.

The development of bacteria-specific infection radiotracers is of considerable interest to improve diagnostic accuracy and enabling therapy monitoring. The aim of this study was to determine if the previously reported radiolabelled 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (DOTA) conjugated peptide [68 Ga]Ga-DOTA-K-A9 could detect a staphylococcal infection in vivo and distinguish it from aseptic inflammation. An optimized [68 Ga]Ga-DOTA-K-A9 synthesis omitting the use of acetone was developed, yielding 93 ± 0.9% radiochemical purity. The in vivo infection binding specificity of [68 Ga]Ga-DOTA-K-A9 was evaluated by micro positron emission tomography/magnetic resonance imaging of 15 mice with either subcutaneous Staphylococcus aureus infection or turpentine-induced inflammation and compared with 2-deoxy-2-[18 F]fluoro-D-glucose ([18 F]FDG). The scans showed that [68 Ga]Ga-DOTA-K-A9 accumulated in all the infected mice at injected doses ≥3.6 MBq. However, the tracer was not found to be selective towards infection, since the [68 Ga]Ga-DOTA-K-A9 also accumulated in mice with inflammation. In a concurrent in vitro binding evaluation performed with a 5-carboxytetramethylrhodamine (TAMRA) fluorescence analogue of the peptide, TAMRA-K-A9, the microscopy results suggested that TAMRA-K-A9 bound to an intracellular epitope and therefore preferentially targeted dead bacteria. Thus, the [68 Ga]Ga-DOTA-K-A9 uptake observed in vivo is presumably a combination of local hyperemia, vascular leakiness and/or binding to an epitope present in dead bacteria.

Exploring the radiosynthesis and in vitro characteristics of [68 Ga]Ga-DOTA-Siglec-9.

Vascular adhesion protein 1 is a leukocyte homing-associated glycoprotein, which upon inflammation rapidly translocates from intracellular sources to the endothelial cell surface. It has been discovered that the cyclic peptide residues 283-297 of sialic acid-binding IgG-like lectin 9 (Siglec-9) "CARLSLSWRGLTLCPSK" bind to vascular adhesion protein 1 and hence makes the radioactive analogues of this compound ([68 Ga]Ga-DOTA-Siglec-9) interesting as a noninvasive visualizing marker of inflammation. Three different approaches to the radiosynthesis of [68 Ga]Ga-DOTA-Siglec-9 are presented and compared with previously published methods. A simple, robust radiosynthesis of [68 Ga]Ga-DOTA-Siglec-9 with a yield of 62% (non decay-corrected) was identified, and it had a radiochemical purity >98% and a specific radioactivity of 35 MBq/nmol. Furthermore, the protein binding and stability of [68 Ga]Ga-DOTA-Siglec-9 were analyzed in vitro in mouse, rat, rabbit, pig, and human plasma and compared with in vivo pig results. The plasma in vitro protein binding of [68 Ga]Ga-DOTA-Siglec-9 was the lowest in the pig followed by rabbit, human, rat, and mouse. It was considerably higher in the in vivo pig experiments. The in vivo stability in pigs was lower than the in vitro stability. Despite considerable species differences, the observed characteristics of [68 Ga]Ga-DOTA-Siglec-9 are suitable as a positron emission tomography tracer.

Effects of Long-term Anesthesia, Blood Sampling, Transportation, and Infection Status on Hearts and Brains in Pigs Inoculated with Staphylococcus aureus and Used for Imaging Studies.

Laboratory animals are widely used in imaging studies, including infection, heart, and brain research. Compared with rodents, pigs are especially useful because of their large organ sizes, ability to tolerate long-term anesthesia, and substantial blood volume, which allows repeated blood sampling. These factors are particularly important in positron emission tomography studies of potential new radioactive tracers, because the scans often are prolonged; in addition, kinetic studies involving repeated blood sampling may be performed to establish the optimal scan time. However, protracted studies may affect the cardiovascular system, brain, and other organs. This raises the question of how to monitor and counteract the effects of longterm anesthesia in pigs in a typical experimental setting yet prevent introducing bias into the experiment. To address this question, we investigated the effects of long-term anesthesia (maximum, 18 h), repeated blood sampling (maximum of 20 mL blood per kilogram body weight), and road transportation (as long as 1.5 h between 2 imaging centers) on key variables of lung, heart, and brain function in the context of a well-established pig model of Staphylococcus aureus infection. Pulse rate, oxygen saturation, body temperature, arterial pressure of CO2, and urine production were stable during anesthesia for at least 16 h, whereas blood glucose slowly decreased. Hct and leukocyte count decreased due to repeated blood sampling. During road transportation, blood lactate levels increased 5 fold and arterial pressure of O2 decreased by 50%. Repeated CT scans, necropsy results, and histopathology findings documented progressive lung changes and acute cardiac necrosis. No lesions indicative of hypoxia were found in brain. The study data show that the typical monitoring parameters do not fully depict the cardiovascular state of pigs during prolonged anesthesia. We recommend streamlining experimental protocols for imaging studies in pigs to avoid organ pathology.

Receptor occupancy of mirtazapine determined by PET in healthy volunteers.

RATIONALE: Molecular tools are needed for assessing anti-depressant actions by positron emission tomography (PET) in the living human brain. OBJECTIVES: This study determined whether [(11)C]mirtazapine is an appropriate molecular tool for use with PET to estimate the magnitude of neuroreceptor occupancy produced by daily intake of mirtazapine. METHODS: This study used a randomised, double-blind, placebo-controlled, parallel-group, within-subject design. Eighteen healthy volunteers were PET-scanned twice with [(11)C]mirtazapine; once under baseline condition and again after receiving either placebo or mirtazapine (7.5 or 15 mg) for 5 days. We determined kinetic parameters of [(11)C]mirtazapine in brain regions by the simplified reference region method and used binding potential values to calculate receptor occupancy produced by mirtazapine. RESULTS: Serum concentrations of mirtazapine ranged from 33 to 56 nmol/l after five daily doses of 7.5 mg mirtazapine and were between 41 and 74 nmol/l after 15 mg mirtazapine. Placebo treatment failed to alter the binding potential of [(11)C]mirtazapine from baseline values, whereas daily intake of mirtazapine markedly decreased the binding potential in cortex, amygdala and hippocampus. Receptor occupancy ranged from 74 to 96% in high-binding regions of the brain after five daily doses of 7.5 mg or 15 mg mirtazapine, whereas 17-48% occupancy occurred in low-binding regions. CONCLUSIONS: [(11)C]Mirtazapine together with PET can determine the degree of receptor occupancy produced by daily doses of mirtazapine in regions of the living human brain.

Kinetic Modelling of Infection Tracers [18F]FDG, [68Ga]Ga-Citrate, [11C]Methionine, and [11C]Donepezil in a Porcine Osteomyelitis Model.

Introduction: Positron emission tomography (PET) is increasingly applied for infection imaging using [18F]FDG as tracer, but uptake is unspecific. The present study compares the kinetics of [18F]FDG and three other PET tracers with relevance for infection imaging. Methods: A juvenile porcine osteomyelitis model was used. Eleven pigs underwent PET/CT with 60-minute dynamic PET imaging of [18F]FDG, [68Ga]Ga-citrate, [11C]methionine, and/or [11C]donepezil, along with blood sampling. For infectious lesions, kinetic modelling with one- and two-tissue-compartment models was conducted for each tracer. Results: Irreversible uptake was found for [18F]FDG and [68Ga]Ga-citrate; reversible uptake was found for [11C]methionine (two-tissue model) and [11C]donepezil (one-tissue model). The uptake rate for [68Ga]Ga-citrate was slow and diffusion-limited. For the other tracers, the uptake rate was primarily determined by perfusion (flow-limited uptake). Net uptake rate for [18F]FDG and distribution volume for [11C]methionine were significantly higher for infectious lesions than for correspondingly noninfected tissue. For [11C]donepezil in pigs, labelled metabolite products appeared to be important for the analysis. Conclusions: The kinetics of the four studied tracers in infection was characterized. For clinical applications, [18F]FDG remains the first-choice PET tracer. [11C]methionine may have a potential for detecting soft tissue infections. [68Ga]Ga-citrate and [11C]donepezil were not found useful for imaging of osteomyelitis.

Detection of alpha2-adrenergic receptors in brain of living pig with 11C-yohimbine.

UNLABELLED: There have been few radiotracers for imaging adrenergic receptors in brain by PET, but none has advanced for use in human studies. We developed a radiosynthesis for the alpha(2)-adrenergic antagonist (11)C-yohimbine and characterized its binding in living pigs. As a prelude to human studies with (11)C-yohimbine, we determined the whole-body distribution of (11)C-yohimbine and calculated its dosimetry. METHODS: Yorkshire x Landrace pigs weighing 35-40 kg were used in the study. Baseline and postchallenge PET recordings of (11)C-yohimbine in pig brain were conducted for 90 min, concurrent with arterial blood sampling, and with yohimbine and RX821002 as pharmacologic interventions. (15)O-Water scans were performed to detect changes in cerebral perfusion. The PET images were manually coregistered to an MR atlas of the pig brain. Maps of the (11)C-yohimbine distribution volume ([V(d)] mL g(-1)) in brain were calculated relative to the arterial input function. RESULTS: Whole-body scans with (11)C-yohimbine revealed high accumulation of radioactivity in kidney, intestine, liver, and bone. The estimated human dose was 5.6 mSv/GBq, a level commonly accepted in human PET studies. Brain imaging showed baseline values of V(d) ranging from 1.9 in medulla, 3.0 in cerebellum, and to 4.0 in frontal cortex. Coinjection with nonradioactive yohimbine (0.07 mg/kg) reduced V(d) globally to approximately 1.5-2 mL g(-1). A higher yohimbine dose (1.6 mg/kg) was without further effect on self-displacement. Very similar results were obtained by displacement with the more selective alpha(2)-adrenergic antagonist RX821002 at doses of 0.15 and 0.7 mg/kg. Cerebral blood flow was globally increased 43% after administration of RX821002. Notable features of (11)C-yohimbine are a lack of plasma metabolism over 90 min and a rapid approach to equilibrium binding in brain. CONCLUSION: The new radiotracer (11)C-yohimbine seems well suited for PET investigations of alpha(2)-adrenergic receptors in brain and peripheral structures, with the caveat that displaceable binding was present in cerebellum and throughout the brain.

Impact of contamination with long-lived radionuclides on PET kinetics modelling in multitracer studies.

INTRODUCTION: An important issue in multitracer studies is the separation of signals from the different radiotracers. This is especially the case when an early tracer has a long physical half-life and kinetic modelling has to be performed, because the early tracer can confer a long-lived contaminating background not only to images but also to a measured input function derived from blood samples. In this study, we examined data from a sequential multitracer infection study involving In (t1/2=2.8 days), investigating the influence on gamma counting of blood samples and on the kinetic modelling of subsequent PET tracers. Blood sample counts were corrected by recounting the samples a few days later. A more optimal choice of energy window was also explored. The effect of correction versus noncorrection was investigated using a two-tissue kinetic model with irreversible uptake (K1, k2, k3). RESULTS: K1 was least affected and k3 was most affected by the contamination, corresponding to the effect being relatively larger on the late part of the blood input function. A narrower energy window reduced the problem, but this will not be possible for all types of contaminating background. CONCLUSION: Gamma counting of blood samples can lead to a contaminating background not observed in PET imaging and this background can affect kinetic modelling. If the contaminating tracer has a much longer half-life than the foreground tracer, then the problem can be solved by late recounting of the samples.

Biodistribution of the radionuclides (18)F-FDG, (11)C-methionine, (11)C-PK11195, and (68)Ga-citrate in domestic juvenile female pigs and morphological and molecular imaging of the tracers in hematogenously disseminated Staphylococcus aureus lesions.

Approximately 5-7% of acute-care patients suffer from bacteremia. Bacteremia may give rise to bacterial spread to different tissues. Conventional imaging procedures as X-ray, Computed Tomography (CT), Magnetic Resonance Imaging (MRI), and ultrasound are often first-line imaging methods for identification and localization of infection. These methods are, however, not always successful. Early identification and localization of infection is critical for the appropriate and timely selection of therapy. The aim of this study was thus; a head to head comparison of (18)F-fluorodeoxyglucose ((18)F-FDG) positron emission tomography (PET) to PET with tracers that potentially could improve uncovering of infectious lesions in soft tissues. We chose (11)C-methionine, (11)C-PK11195, and (68)Ga-citrate as tracers and besides presenting their bio-distribution we validated their diagnostic utility in pigs with experimental bacterial infection. Four juvenile 14-15 weeks old female domestic pigs were scanned seven days after intra-arterial inoculation in the right femoral artery with a porcine strain of S. aureus using a sequential scanning protocol with (18)F-FDG, (11)C-methionine, (11)C-PK11195 and (68)Ga-citrate. This was followed by necropsy of the pigs consisting of gross pathology, histopathology and microbial examination. The pigs primarily developed lesions in lungs and neck muscles. (18)F-FDG had higher infection to background ratios and accumulated in most infectious foci caused by S. aureus, while (11)C-methionine and particularly (11)C-PK11195 and (68)Ga-citrate accumulated to a lesser extent in infectious foci. (18)F-FDG-uptake was seen in the areas of inflammatory cells and to a much lesser extent in reparative infiltration surrounding necrotic regions.

Utility of 11C-methionine and 11C-donepezil for imaging of Staphylococcus aureus induced osteomyelitis in a juvenile porcine model: comparison to autologous 111In-labelled leukocytes, 99m Tc-DPD, and 18F-FDG.

The aim of this study was to compare 11C-methionine and 11C-donepezil positron emission tomography (PET) with 111In-labeled leukocyte and 99m Tc-DPD (Tc-99m 3,3-diphosphono-1,2-propanedicarboxylic acid) single-photon emission computed tomography (SPECT), and 18F-fluorodeoxyglucose (18F-FDG) PET to improve detection of osteomyelitis. The tracers' diagnostic utility where tested in a juvenile porcine hematogenously induced osteomyelitis model comparable to osteomyelitis in children. Five 8-9 weeks old female domestic pigs were scanned seven days after intra-arterial inoculation in the right femoral artery with a porcine strain of Staphylococcus aureus. The sequential scan protocol included Computed Tomography, 11C-methionine and 11C-donepezil PET, 99m Tc-DPD and 111In-labelled leukocytes scintigraphy, and 18F-FDG PET. This was followed by necropsy of the pigs and gross pathology, histopathology, and microbial examination. The pigs developed a total of 24 osteomyelitic lesions, 4 lesions characterized as contiguous abscesses and pulmonary abscesses (in two pigs). By comparing the 24 osteomyelitic lesions, 18F-FDG accumulated in 100%, 111In-leukocytes in 79%, 11C-methionine in 79%, 11C-donepezil in 58%, and 99m Tc-DPD in none. Overall, 18F-FDG PET was superior to 111In-leukocyte SPECT and 11C-methionine in marking infectious lesions.

(68)Ga-labeled phage-display selected peptides as tracers for positron emission tomography imaging of Staphylococcus aureus biofilm-associated infections: Selection, radiolabelling and preliminary biological evaluation.

INTRODUCTION: Staphylococcus aureus is a major cause of skin and deep-sited infections, often associated with the formation of biofilms. Early diagnosis and initiated therapy is essential to prevent disease progression and to reduce complications that can be serious. Imaging techniques are helpful combining anatomical with functional data in order to describe and characterize site, extent and activity of the disease. The purpose of the study was to identify and (68)Ga-label peptides with affinity for S. aureus biofilm and evaluate their potential as bacteria-specific positron emission tomography (PET) imaging agents. METHODS: Phage-displayed dodecapeptides were selected using an in vitro grown S. aureus biofilm as target. One cyclic (A8) and two linear (A9, A11) dodecapeptides were custom synthesized with 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (DOTA) conjugated via a lysine linker (K), and for A11 also a glycine-serine-glycine spacer (GSG). The (68)Ga-labeling of A8-K-DOTA, A9-K-DOTA, and A11-GSGK-DOTA were optimized and in vitro bacterial binding was evaluated for (68)Ga-A9-K-DOTA and (68)Ga-A11-GSGK-DOTA. Stability of (68)Ga-A9-K-DOTA was studied in vitro in human serum, while the in vivo plasma stability was analyzed in mice and pigs. Additionally, the whole-body distribution kinetics of (68)Ga-A9-K-DOTA was measured in vivo by PET imaging of pigs and ex vivo in excised mice tissues. RESULTS: The (68)Ga-A9-K-DOTA and (68)Ga-A11-GSGK-DOTA remained stable in product formulation, whereas (68)Ga-A8-K-DOTA was unstable. The S. aureus binding of (68)Ga-A11-GSGK-DOTA and (68)Ga-A9-K-DOTA was observed in vitro, though blocking of the binding was not possible by excess of cold peptide. The (68)Ga-A9-K-DOTA was degraded slowly in vitro, while the combined in vivo evaluation in pigs and mice showed a rapid blood clearance and renal excretion of the (68)Ga-A9-K-DOTA. CONCLUSION: The preliminary in vitro and in vivo studies of the phage-display S. aureus biofilm-selected (68)Ga-A9-K-DOTA showed desirable features for a novel bacteria-specific imaging agent, despite of relative fast blood degradation in vivo.

Comparison of autologous (111)In-leukocytes, (18)F-FDG, (11)C-methionine, (11)C-PK11195 and (68)Ga-citrate for diagnostic nuclear imaging in a juvenile porcine haematogenous staphylococcus aureus osteomyelitis model.

The aim of this study was to compare (111)In-labeled leukocyte single-photon emission computed tomography (SPECT) and (18)F-fluorodeoxyglucose ((18)F-FDG) positron emission tomography (PET) to PET with tracers that potentially could improve detection of osteomyelitis. We chose (11)C-methionine, (11)C-PK11195 and (68)Ga-citrate and validated their diagnostic utility in a porcine haematogenous osteomyelitis model. Four juvenile 14-15 weeks old female pigs were scanned seven days after intra-arterial inoculation in the right femoral artery with a porcine strain of Staphylococcus aureus using a sequential scan protocol with (18)F-FDG, (68)Ga-citrate, (11)C-methionine, (11)C-PK11195, (99m)Tc-Nanocoll and (111)In-labelled autologous leukocytes. This was followed by necropsy of the pigs and gross pathology, histopathology and microbial examination. The pigs developed a total of five osteomyelitis lesions, five lesions characterized as abscesses/cellulitis, arthritis in three joints and five enlarged lymph nodes. None of the tracers accumulated in joints with arthritis. By comparing the 10 infectious lesions, (18)F-FDG accumulated in nine, (111)In-leukocytes in eight, (11)C-methionine in six, (68)Ga-citrate in four and (11)C-PK11195 accumulated in only one lesion. Overall, (18)F-FDG PET was superior to (111)In-leukocyte SPECT in marking infectious and proliferative, i.e. hyperplastic, lesions. However, leukocyte SPECT was performed as early scans, approximately 6 h after injection of the leukocytes, to match the requirements of the 18 h long scan protocol. (11)C-methionine and possibly (68)Ga-citrate may be useful for diagnosis of soft issue lesions.

Kinetics of the uptake and distribution of the dopamine D(2,3) agonist (R)-N-[1-(11)C]n-propylnorapomorphine in brain of healthy and MPTP-treated Göttingen miniature pigs.

The binding of radioligand agonists to dopamine receptors in living brain can be informative about the abundance of receptors which are coupled to intracellular second messenger systems. Therefore, we developed a radiosynthesis for the dopamine D(2,3) partial agonist (R)-N- [1-(11)C]n-propylnorapomorphine ([(11)C]NPA). The uptake of this tracer in brain of anesthetized Göttingen miniature pigs was recorded by positron emission tomography (PET) and analyzed by compartmental analysis using the metabolite-corrected arterial input, and using reference tissue methods. [(11)C]NPA had a blood-brain unidirectional clearance of approximately 0.35 ml g(-1) min(-1) and an apparent distribution volume of 6 ml g(-1) in cerebellum. The ligand had a binding potential of 1.5 in striatum, comparable to that reported previously for the receptor antagonist [(11)C]raclopride in the same strain of animals. Significant binding was detected in the hypophysis, thalamus, and medial forebrain bundle. The binding in striatum was of comparable magnitude in normal pigs and in pigs with a documented 50% dopamine depletion produced by MPTP-intoxication. Deep brain stimulation of the subthalamus was without conspicuous effect on the binding of [(11)C]NPA in vivo. Results of this preliminary study indicate that this tracer meets many requirements for assaying dopamine agonist binding sites by PET.

Oxidative metabolism of astrocytes is not reduced in hepatic encephalopathy: a PET study with [(11)C]acetate in humans.

In patients with impaired liver function and hepatic encephalopathy (HE), consistent elevations of blood ammonia concentration suggest a crucial role in the pathogenesis of HE. Ammonia and acetate are metabolized in brain both primarily in astrocytes. Here, we used dynamic [(11)C]acetate PET of the brain to measure the contribution of astrocytes to the previously observed reduction of brain oxidative metabolism in patients with liver cirrhosis and HE, compared to patients with cirrhosis without HE, and to healthy subjects. We used a new kinetic model to estimate uptake from blood to astrocytes and astrocyte metabolism of [(11)C]acetate. No significant differences of the rate constant of oxidation of [(11)C]acetate (k 3) were found among the three groups of subjects. The net metabolic clearance of [(11)C]acetate from blood was lower in the group of patients with cirrhosis and HE than in the group of healthy subjects (P < 0.05), which we interpret to be an effect of reduced cerebral blood flow rather than a reflection of low [(11)C]acetate metabolism. We conclude that the characteristic decline of whole-brain oxidative metabolism in patients with cirrhosis with HE is not due to malfunction of oxidative metabolism in astrocytes. Thus, the observed decline of brain oxidative metabolism implicates changes of neurons and their energy turnover in patients with HE.

Fast and simple one-step preparation of 68Ga citrate for routine clinical PET.

The imaging of infectious and inflammatory diseases using gallium-67 (67Ga) citrate scintigraphy has been a well-established diagnostic tool for decades. In recent times, interest has focused on PET using the short-lived positron emitting radioisotope 68Ga. 68Ga is not only more readily available, it also provides better quality images whose high resolution permits quantitative analyses, thus improving the management of patients suffering from infections or inflammation. The purpose of our study was to develop a fast and reliable synthesis protocol for the preparation of 68Ga citrate under good manufacturing practice aspects without the use of organic solvents. A commercially available synthesis module was used to perform 10 syntheses with an average yield of 768 ± 31 MBq (mean ± SD) within 10 min; 92.04 ± 1.23% of the radioactivity was located in the product vial, and the rest on the cation exchange cartridge (7.48 ± 1.23%) and in the waste vial (0.47 ± 0.28%). The radiochemical purity of the product determined by instant thin-layer chromatography was greater than 99%. The products have been proven to be sterile and pyrogen-free. Variations were made in several critical synthesis parameters, and the results are presented herein. By eliminating the use of organic solvents, the previously required quality control testing of the final product by gas chromatography can be abandoned. This novel, high-yielding method allows for a more efficient synthesis of 68Ga citrate with both shorter production time and high radiochemical purity.

A PET study of effects of chronic 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") on serotonin markers in Göttingen minipig brain.

The psychostimulant 3,4-methylendioxymethamphetamine (MDMA, "ecstasy") evokes degeneration of telencephalic serotonin innervations in rodents, nonhuman primates, and human recreational drug users. However, there has been no alternative to nonhuman primates for studies of the cognitive and neurochemical consequences of serotonin depletion in a large-bodied animal. Therefore, we used positron emission tomography (PET) with [(11)C]DASB to map the distribution of plasma membrane serotonin transporters in brain of Göttingen minipigs, first in a baseline condition, and again at 2 weeks after treatment with MDMA (i.m.), administered at a range of doses. In parallel PET studies, [(11)C]WAY-100635 was used to map the distribution of serotonin 5HT(1A) receptors. The acute MDMA treatment in awake pigs evoked 1 degrees C of hyperthermia. MDMA at total doses greater than 20 mg/kg administered over 2-4 days reduced the binding potential (pB) of [(11)C]DASB for serotonin transporters in porcine brain. A mean total dose of 42 mg/kg MDMA in four animals evoked a mean 32% decrease in [(11)C]DASB pB in mesencephalon and diencephalon, and a mean 53% decrease in telencephalic structures. However, this depletion of serotonin innervations was not associated with consistent alterations in the binding of [(11)C]WAY-100635 to serotonin 5HT(1A) receptors. Stereological cell counting of serotonin-positive neurons, which numbered 95,000 in the dorsal raphé nucleus of normal animals, was unaffected in MDMA-treated group. group.

Interaction between LSD and dopamine D2/3 binding sites in pig brain.

The psychoactive properties of the hallucinogen LSD have frequently been attributed to high affinity interactions with serotonin 5HT2 receptors in brain. Possible effects of LSD on dopamine D2/3 receptor availability have not previously been investigated in living brain. Therefore, we used PET to map the binding potential (pB) of [11C]raclopride in brain of three pigs, first in a baseline condition, and again at 1 and 4 h after administration of LSD (2.5 microg/kg, i.v.). There was a progressive treatment effect in striatum, where the pB was significantly reduced by 19% at 4 h after LSD administration. Concomitant maps of cerebral blood flow did not reveal significant changes in perfusion during this interval. Subsequent in vitro studies showed that LSD displaced [3H]raclopride (2 nM) from pig brain cryostat sections with an IC50 of 275 nM according to a one-site model. Fitting of a two-site model to the data suggested the presence of a component of the displacement curves with a subnanomolar IC50, comprising 20% of the total [3H]raclopride binding. In microdialysis experiments, LSD at similar and higher doses did not evoke changes in the interstitial concentration of dopamine or its acidic metabolites in rat striatum. Together, these results are consistent with a direct interaction between LSD and a portion of dopamine D2/3 receptors in pig brain, possibly contributing to the psychopharmacology of LSD.

MDMA-evoked changes in [11C]raclopride and [11C]NMSP binding in living pig brain.

Positron emission tomography (PET) studies with radiolabeled dopamine D2-like receptor ligands reveal d-amphetamine-evoked increases in the competition from endogenous dopamine. However, the corresponding effects of methylenedioxymethamphetamine (MDMA, "Ecstasy"), which releases catecholamines and also serotonin, are unknown. Using PET, we measured the binding potentials (pBs) of the benzamide [11C]raclopride and the butyrophenone N-[11C]methylspiperone ([11C]NMSP) in brain of living pigs first in a baseline condition and at 45 and 165 min after infusion of (+/-)-MDMA-HCl (1 mg/kg, i.v.). Concomitant studies of cerebral blood flow did not reveal significant perfusion changes in the cerebellum reference region or in striatum, supporting the present use of reference tissue methods for the mapping of MDMA-evoked pB changes. Relative to the baseline pB of [11C]raclopride for dopamine D(2/3) receptors in striatum (pB = 1.5-2.2), MDMA-treatment reduced pB by 35% in the first posttreatment scan and by 22% in the second posttreatment scan, comparable to changes typically evoked by d-amphetamine at a similar dose. In most previous studies, the in vivo binding of butyrophenones has been nearly insensitive to d-amphetamine-evoked dopamine release. However, we found the baseline pB of [11C]NMSP for dopamine D2-like receptors in striatum (pB = 4-5) was decreased by 30% in the first post-MDMA scan and by 50% in the second post-MDMA scan, irrespective of assumptions about the extent of equilibrium binding attained during the 90-min-long PET recordings. Distinct properties of MDMA such as simultaneous release of dopamine and serotonin in brain may account for the present finding of progressive decline in the availability of [11C]NMSP binding sites in striatum.

[11C]-NS 4194 versus [11C]-DASB for PET imaging of serotonin transporters in living porcine brain.

In vitro, the novel diazabicyclononane NS 4194 has several thousand-fold selectivity for blocking the transport into rat brain synaptosomes of [(3)H]-serotonin in comparison to [(3)H]-dopamine or [(3)H]-noradrenaline. We have prepared [(11)C]-NS 4194 in order to test its properties for PET imaging of brain serotonin transporters in comparison with the well-documented tracer [(11)C]-DASB. Both compounds had rapid clearance from blood to brain of living pigs. The apparent equilibrium distribution volumes in cerebellum were 35 ml g(-1) for [(11)C]-NS 4194 and 11 ml g(-1) for [(11)C]-DASB. Pretreatment of pigs with citalopram did not reduce the uptake of either tracer in cerebellum, validating the use of that tissue as a nonbinding reference tissue for kinetic analysis of specific binding. The binding potential (pB) calculated for [(11)C]-NS 4194 using arterial input models was close to 0.5 in the telencephalon, and was 60% displaced by citalopram. However, the reference tissue method of Lammertsma was unsuited to calculate pB for this tracer, apparently due to its excessive nonspecific binding. In contrast to the relatively homogeneous binding of [(11)C]-NS 4194, the pB of [(11)C]-DASB ranged from 0.6 in frontal cortex to 2 in the mesencephalon when calculated by the method of Lammertsma. Parametric maps of the pB of [(11)C]-DASB showed a pattern consistent with the known distribution of serotonin transporters in pig brain in vitro, and there was a uniform displacement of 80% of the specific binding after citalopram treatment in vivo. In conclusion, [(11)C]-DASB is in several respects superior to [(11)C]-NS 4194 for the detection of serotonin uptake sites by PET.