Amyloid-Targeting PET Tracer [18F]Flutemetamol Accumulates in Atherosclerotic Plaques.

Atherosclerosis is characterized by the accumulation of oxidized lipids in the artery wall, which triggers an inflammatory response. Oxidized low-density lipoprotein (ox-LDL) presents amyloid-like structural properties, and different amyloid species have recently been recognized in atherosclerotic plaques. Therefore, we studied the uptake of the amyloid imaging agent [18F]Flutemetamol in atherosclerotic plaques. The binding of [18F]Flutemetamol to human carotid artery plaque was studied in vitro. In vivo uptake of the tracer was studied in hypercholesterolemic IGF-II/LDLR-/-ApoB100/100 mice and C57BL/6N controls. Tracer biodistribution was studied in vivo with PET/CT, and ex vivo by gamma counter and digital ex vivo autoradiography. The presence of amyloid, ox-LDL, and macrophages in the plaques was examined by immunohistochemistry. [18F]Flutemetamol showed specific accumulation in human carotid plaque, especially in areas positive for amyloid beta. The aortas of IGF-II/LDLR-/-ApoB100/100 mice showed large thioflavin-S-positive atherosclerotic plaques containing ox-LDL and macrophages. Autoradiography revealed 1.7-fold higher uptake in the plaques than in a lesion-free vessel wall, but no difference in aortic tissue uptake between mouse strains were observed in the in vivo PET/CT. In conclusion, [18F]Flutemetamol binds to amyloid-positive areas in human atherosclerotic plaques. Further studies are warranted to clarify the uptake mechanisms, and the potential of the tracer for in vivo imaging of atherosclerosis in patients.

Evaluation of gadolinium-based contrast agents in juvenile CD-1 mice including behavioral evaluations.

INTRODUCTION: Seven gadolinium-based contrast agents (GBCAs), four linear and three macrocyclic, were evaluated for potential effects on development, including behavior of juvenile CD-1 mice. METHODS: The GBCAs were administered via intravenous injection once daily on postnatal day (PND) 9, 12, 15, 18, and 21 (PND 1 was the day of delivery) at doses up to twice the human equivalent clinical dose (i.e., 0.63 mmol Gd/kg for gadoxetate disodium and 2.5 mmol Gd/kg for the other GBCAs). Mice were bled for evaluation of exposure (plasma) to gadolinium (Gd) on PND 9, 12, and 70. At scheduled euthanasia, the liver, spleen, brain, skin (dorsal surface), bone (left femur), and kidneys were excised from up to six mice/sex/group on PND 10, 22, or 70 for the determination of Gd levels and histopathological analysis. All mice were monitored for toxicity, growth and survival, sexual maturation, and behavior. CONCLUSION: Gd was quantifiable in the brain tissues with levels declining over time. There was no long-term effect on the growth and development for mice exposed to any of the GBCAs. There was no impact on neurodevelopment as assessed by brain histology and validated neurobehavioral tests, including a functional observational battery, motor activity, and learning and memory as evaluated in the Morris water maze. For all GBCAs, the highest dose tested represented the no-observable-adverse-effect level in juvenile mice.

Evaluation of gadolinium-based contrast agents in pregnant CD-1 mice and subsequent in utero exposure of the developing offspring, including behavioral evaluations.

INTRODUCTION: The offspring of CD-1 mice exposed during pregnancy to one of seven gadolinium-based contrast agents (GBCAs) were evaluated for potential effects on postnatal development and behavior. The GBCAs, comprising four linear (gadopentetate dimeglumine, gadodiamide, gadobenate dimeglumine, and gadoxetate disodium) and three macrocyclic (gadoterate meglumine, gadoteridol, and gadobutrol), were administered via intravenous injection once daily from Gestation Day 6 through 17 following confirmed mating (Day 0) at doses of at least twice the human equivalent recommended clinical dose (i.e., 0.63 mmol Gd/kg for gadoxetate disodium and 2.5 mmol Gd/kg for the other GBCAs). All dams were allowed to deliver naturally. F0 generation females were monitored for maternal toxicity and gadolinium (Gd) levels in blood and brain. Offspring were evaluated for Gd levels in blood and brain at birth and on Day 70 postpartum. F1 generation mice were evaluated for survival and growth preweaning. Selected pups/litter were evaluated postweaning for sexual maturation, growth, and behavior. Gd was quantifiable in the brain of the F1 offspring on PND 1, with levels declining over time. There was no long-term effect of any GBCA on the growth and development of any offspring. There was no impact on neurodevelopment, as assessed by brain histology and validated neurobehavioral tests, including a battery of functional observational tests, motor activity, and learning and memory as evaluated in the Morris water maze. CONCLUSION: At the end of the postweaning period, the highest dose tested was considered the no-observable-adverse-effect level (NOAEL) in the F0 and F1 offspring for all tested GBCAs.

Exploration of the structure-activity relationship of a novel tetracyclic class of TSPO ligands-potential novel positron emitting tomography imaging agents.

A series of novel TSPO ligands based on the tetracyclic class of translocator protein (TSPO) ligands first described by Okubo et al. was synthesised and evaluated as potential positron emitting tomography (PET) ligands for imaging TPSO in vivo. Fluorine-18 labelling of the molecules was achieved using direct radiolabelling or synthon based labelling approaches. Several of the ligands prepared have promising profiles as potential TSPO PET imaging ligands.

Acute effect of the anti-addiction drug bupropion on extracellular dopamine concentrations in the human striatum: an [11C]raclopride PET study.

Bupropion is an effective medication in treating addiction and is widely used as an aid to smoking cessation. Bupropion inhibits striatal dopamine reuptake via dopamine transporter blockade, but it is unknown whether this leads to increased extracellular dopamine levels at clinical doses in man. The effects of bupropion on extracellular dopamine levels in the striatum were investigated using [(11)C]raclopride positron emission tomography (PET) imaging in rats administered saline, 11 or 25 mg/kg bupropion i.p. and in healthy human volunteers administered either placebo or 150 mg bupropion (Zyban Sustained-Release). A cognitive task was used to stimulate dopamine release in the human study. In rats, bupropion significantly decreased [(11)C]raclopride specific binding in the striatum, consistent with increases in extracellular dopamine concentrations. In man, no significant decreases in striatal [(11)C]raclopride specific binding were observed. Levels of dopamine transporter occupancy in the rat at 11 and 25 mg/kg bupropion i.p. were higher than predicted to occur in man at the dose used. Thus, these data indicate that, at the low levels of dopamine transporter occupancy achieved in man at clinical doses, bupropion does not increase extracellular dopamine levels. These findings have important implications for understanding the mechanism of action underlying bupropions' therapeutic efficacy and for the development of novel treatments for addiction and depression.

Further evaluation of the carbon11-labeled D(2/3) agonist PET radiotracer PHNO: reproducibility in tracer characteristics and characterization of extrastriatal binding.

[(11)C]-(+)-PHNO is a new dopamine D(2/3) receptor agonist radiotracer which has been successfully used to measure D(2/3) receptor availability in experimental animals and man. Here we report in vivo evaluation in the rat of the biodistribution, metabolism, specificity, selectivity, and dopamine sensitivity of carbon11-labeled PHNO ([(11)C]-3-PHNO) produced by an alternative radiochemical synthesis method. [(11)C]-3-PHNO showed rapid metabolism and clearance from most peripheral organs and tissues. [(11)C]-3-PHNO, but not its polar metabolite, readily crossed the blood-brain barrier and showed high levels of uptake in the D(2/3)-rich striatum. Pretreatment with unlabeled PHNO and the D(2/3) receptor antagonist raclopride indicated that binding in the striatum was specific and selective to D(2/3) receptors. PET studies in anesthetized rats revealed significant reductions in [(11)C]-3-PHNO binding in the striatum following amphetamine administration, indicating sensitivity to increases in endogenous dopamine concentrations. D(2/3) antagonist pretreatment additionally indicated moderate levels of [(11)C]-3-PHNO specific binding in several extrastriatal brain areas-most notably the olfactory bulbs and tubercles, thalamus, and hypothalamus. Of particular interest, approximately 30% of [(11)C]-3-PHNO signal in the cerebellum-a region often used as a "low-binding" reference region for PET quantification-was attributable to specific signal. These data demonstrate that [(11)C]-3-PHNO shows similar tracer characteristics to [(11)C]-(+)-PHNO, but additionally indicate that radiolabeled PHNO may be used to estimate D(2/3) receptor availability in select extrastriatal brain regions with PET.

Comment on " In Vivo [18F]GE-179 Brain Signal Does Not Show NMDA-Specific Modulation with Drug Challenges in Rodents and Nonhuman Primates".

Schoenberger and colleagues ( Schoenberger et al. ( 2018 ) ACS Chem. Neurosci. 9 , 298 - 305 ) recently reported attempts to demonstrate specific binding of the positron emission tomography (PET) radiotracer, [18F]GE-179, to NMDA receptors in both rats and Rhesus macaques. GE-179 did not work as expected in animal models; however, we disagree with the authors' conclusion that "the [18F]GE-179 signal seems to be largely nonspecific". It is extremely challenging to demonstrate specific binding for the use-dependent NMDA receptor intrachannel ligands such as [18F]GE-179 in animals via traditional blocking, due to its low availability of target sites ( Bmax'). Schoenberger and colleagues anesthetized rats and Rhesus monkeys using isoflurane, which has an inhibitory effect on NMDA receptor function and thus would be expected to further reduce the Bmax'. The extent of glutamate release achieved in the provocation experiments is uncertain, as is whether a significant increase in NMDA receptor channel opening can be expected under anesthesia. Prior data suggest that the uptake of disubstituted arylguanidine-based ligands such as GE-179 can be reduced by phencyclidine binding site antagonists, if injection is performed in the absence of ketamine and isoflurane anesthesia, e.g., with GE-179's antecedent, CNS 5161 ( Biegon et al. ( 2007 ) Synapse 61 , 577 - 586 ), and with GMOM ( van der Doef et al. ( 2016 ) J. Cereb. Blood Flow Metab. 36 , 1111 - 1121 ). However, the extent of nonspecific uptake remains uncertain.

Modulation of striatal dopamine release by 5-HT2A and 5-HT2C receptor antagonists: [11C]raclopride PET studies in the rat.

RATIONALE: Antagonism at serotonin 5-HT2A and 5-HT2C receptors modulates cortical and striatal dopamine (DA) release and may underlie some aspects of the clinical efficacy of 'atypical' antipsychotic compounds. However, it is not known whether 5-HT2A/2C receptor-mediated modulation of DA release can be quantified with non-invasive neurochemical imaging, as would be required for investigation of these processes in man. OBJECTIVE: The objective of the study was to perform a feasibility study in the rat in order to determine whether 5-HT2A/2C modulation of DA release can be observed using positron emission tomography (PET) imaging. MATERIALS AND METHODS: Rats were administered with either vehicle, a combined 5-HT2A/2C antagonist (ketanserin, 3 mg/kg i.p.), or the more selective 5-HT2C antagonist SB 206,553 (10 mg/kg i.p.) 30 min before administration of the PET DA D2 receptor radiotracer [11C]raclopride ( approximately 11 MBq) and were then scanned for 60 min using a quad-high-density avalanche chamber small animal tomograph. Using the same technique, modulation of amphetamine (4 mg/kg)-induced decreases in [11C]raclopride binding by 5-HT2A antagonism (SR 46349B, 0.2 mg/kg i.v.) was also determined. RESULTS: Consistent with the increase in DA release measured by others using microdialysis, 5-HT2C antagonism markedly reduced striatal [11C]raclopride binding (p < 0.003), while amphetamine-induced reductions in striatal [11C]raclopride binding (p < 0.001) were attenuated by 5-HT2A antagonist administration (p = 0.04). CONCLUSIONS: These results inform the feasibility of monitoring 5-HT2A/2C receptor-mediated modulation of DA systems in man using PET and, more generally, demonstrate that D2 radiotracer PET imaging may be used to monitor the efficacy of new DA modulators in attenuating stimulated DA release.

Imaging the GABA-benzodiazepine receptor subtype containing the alpha5-subunit in vivo with [11C]Ro15 4513 positron emission tomography.

There is evidence of marked variation in the brain distribution of specific subtypes of the GABA-benzodiazepine receptor and that particular subtypes mediate different functions. The alpha5-containing subtype is highly expressed in the hippocampus, and selective alpha5 inverse agonists (which decrease tonic GABA inhibition) are being developed as potential memory-enhancing agents. Evidence for such receptor localization and specialization in humans in vivo is lacking because the widely used probes for imaging the GABA-benzodiazepine receptors, [11C]flumazenil and [123I]iomazenil, appear to reflect binding to the alpha1 subtype, based on its distribution and affinity of flumazenil for this subtype. The authors characterized for positron emission tomography (PET) a radioligand from Ro15 4513, the binding of which has a marked limbic distribution in the rat and human brain in vivo. Competition studies in vivo in the rat revealed that radiolabeled Ro15 4513 uptake was reduced to nonspecific levels only by drugs that have affinity for the alpha5 subtype (flunitrazepam, RY80, Ro15 4513, L655,708), but not by the alpha1 selective agonist, zolpidem. Quantification of [11C]Ro15 4513 PET was performed in humans using a metabolite-corrected plasma input function. [11C]Ro15 4513 uptake was relatively greater in limbic areas compared with [11C]flumazenil, but lower in the occipital cortex and cerebellum. The authors conclude that [11C]Ro15 4513 PET labels in vivo the GABA-benzodiazepine receptor containing the alpha5 subtype in limbic structures and can be used to further explore the functional role of this subtype in humans.

Occupancy of agonist drugs at the 5-HT1A receptor.

Drugs acting on the 5-HT1A receptor are used in the treatment of depression, generalized anxiety disorder, and schizophrenia. This study investigated 5-HT1A receptor occupancy by the 5-HT1A agonist drugs flesinoxan (a highly selective probe for the 5-HT1A receptor) and ziprasidone (a novel atypical antipsychotic drug). Using a within-subject design, 14 healthy volunteers each received two positron emission tomography scans using the selective 5-HT1A antagonist radiotracer [11C]WAY-100635. One scan constituted a baseline, while the other followed either 1 mg flesinoxan or 40 mg ziprasidone orally. In addition, rats were pretreated with intravenous flesinoxan at doses ranging from 0.001 to 5 mg/kg then [11C]WAY-100635 binding measured ex vivo. Cerebral cortical and hippocampal regions of interest, and cerebellar reference regions were sampled to estimate 5-HT1A receptor occupancy (inferred from reductions in specific radioligand binding). In man, occupancy was not significant despite volunteers experiencing side effects consistent with central serotonergic activity. The mean cerebral cortex occupancy (+/- 1 SD) for flesinoxan was 8.7% (+/- 13%), and for ziprasidone 4.6% (+/- 17%). However, in rats, flesinoxan achieved significant and dose-related occupancy (17-57%) at 0.25 mg/kg and above. We conclude that 5-HT1A receptor agonists produce detectable occupancy only at higher doses that would produce unacceptable levels of side effects in man, although lower doses are sufficient to produce pharmacological effects. The development of agonist radiotracers may increase the sensitivity of detecting agonist binding, as 5-HT1A antagonists bind equally to low- and high-affinity receptor states, while agonists bind preferentially to the high-affinity state.

In vivo multimodal imaging of stem cell transplantation in a rodent model of Parkinson's disease.

Stem cell therapy in the nervous system aims to replace the lost neurons and provide functional recovery. However, it is imperative that we understand the in vivo behaviour of these cells post-implantation. We report visualisation of iron oxide labelled bone marrow-derived stem cells (BMSCs) implanted into the striatum of hemi-parkinsonian rats by magnetic resonance imaging (MRI). Functional efficacy of the donor cells was monitored in vivo using the positron emission tomography (PET) radioligand [11C]raclopride. The cells were visible for 28 days by in vivo MRI. BMSCs provided functional recovery demonstrated by a decreased binding of [11C]raclopride. Although, histology confirmed the persistence of donor cells, no tyrosine hydroxylase positive cells were present. This suggests that BMSCs may have a limited paracrine effect and influence functional recovery. We demonstrate, using multimodal imaging, that we can not only track BMSCs but also establish their effects in a pre-clinical model of Parkinson's disease.

Low sensitivity of the positron emission tomography ligand [11C]diprenorphine to agonist opiates.

Previously, we reported minimal opioid receptor occupancy following a clinical dose of the micro-opioid agonist, methadone, measured in vivo using positron emission tomography (PET) with [(11)C]diprenorphine and subsequently used rats to obtain experimental data in support of a high receptor reserve hypothesis (Melichar et al., 2005). Here, we report on further preclinical studies investigating opioid receptor occupancy with oxycodone (micro- and kappa-receptor agonist), morphine (micro-receptor agonist), and buprenorphine (partial agonist at the micro-receptor and antagonist at the delta- and kappa-receptors), each given at antinociceptive doses. In vivo binding of [(11)C]diprenorphine was not significantly reduced after treatment with the full agonists but was reduced by approximately 90% by buprenorphine. In addition, given that [(11)C]diprenorphine is a non-subtype-specific PET tracer, there was no regional variation that might feasibly be interpreted as due to differences in opioid subtype distribution. The data support minimal competition between the high-efficacy agonists and the non-subtype-selective antagonist radioligand and highlight the limitations of [(11)C]diprenorphine PET to monitor in vivo occupancy. Alternative means may be needed to address clinical issues regarding opioid receptor occupancy that are required to optimize treatment strategies.

Effect of reduction in endogenous dopamine on extrastriatal binding of [11C]FLB 457 in rat brain--an ex vivo study.

Carbon-11 labeled FLB 457 has been used successfully as a selective, high affinity PET ligand for the quantification of extrastriatal D2-like receptors in man. This study was carried out in rats to investigate regional values for maximal binding and ED50 (a measure of apparent K(d)) for the radioligand in vivo in control animals and in a group pretreated with the neuronal impulse flow inhibitor, gamma-butyrolactone. The aims were to obtain further information regarding the specific activity needed to ensure tracer kinetics and to investigate baseline occupancy by dopamine (DA), each relevant to optimal clinical use of the radioligand. Regional B(max) values were consistent with the distribution of D2-like receptors in rat brain. Of interest, 60% of the binding in cerebellum, often used as a low-binding "reference region" for PET quantification, was saturable, with B(max) only 2- to 3-fold less than that in neocortex, hippocampus, and thalamus. ED50 values were in the range 2-3 nmol/kg, confirming minimal receptor occupancy by the tracer in human PET, using high but achievable specific activities. In the majority of extrastriatal tissues, reduction in synaptic DA did not significantly decrease the apparent K(d), except in cortical regions, where the extent of the effect suggested a low ( approximately 10%), but measurable baseline receptor occupancy by DA.

Temporal characterisation of amphetamine-induced dopamine release assessed with [11C]raclopride in anaesthetised rodents.

Competition between endogenous neurotransmitters and radiolabelled tracers, as measured by positron emission tomography (PET), may provide a measure of endogenous neurotransmitter flux in vivo. For example, carbon-11 labelled raclopride has been effectively used to monitor dopamine release following pharmacological and behavioural manipulations. The current study describes a rodent model of amphetamine-induced [11C]raclopride reduction, which allowed the characterisation of the dose-response and temporal dynamics of this reduction over a 24-h time course. Over the range studied, a monotonic dose-response relationship between amphetamine dose and [11C]raclopride reduction was observed. When compared with previously published microdialysis data, an approximate 16% reduction in [11C]raclopride binding potential was associated with a approximately 25-fold increase in extracellular dopamine. A reduction of 20-30% in raclopride binding was observed 30 min after amphetamine injection (4 mg/kg i.p.). This reduction in [11C]raclopride binding persisted for 4 h but returned to baseline by 8 h. The data suggest a persistent amphetamine-induced raclopride displacement in rodents and reinforce findings from nonhuman primates that a simple competitive occupancy model may not adequately explain the temporal characteristics of the amphetamine-induced decrease in radiotracer binding.

Fenfluramine evokes 5-HT2A receptor-mediated responses but does not displace [11C]MDL 100907: small animal PET and gene expression studies.

The in vivo binding of the 5-HT(2A) receptor-selective positron emission tomography (PET) ligand [(11)C]MDL 100907 and its sensitivity to endogenous 5-HT were quantified in rat brain using quad-HIDAC, a novel high-resolution PET camera for small animals. Specific binding of [(11)C]MDL 100907, estimated using volume of interest (VOI) to cerebellum ratios, corresponded well with both the known distribution of 5-HT(2A) receptors and tissue:cerebellum ratios obtained using ex vivo dissection. Specific binding was blocked by predosing with either nonradioactive MDL 100907 (0.2 or 0.4 mg/kg i.v.) or the 5-HT(2A/2C) receptor antagonist ketanserin (2 mg/kg i.v.), but was unaffected in rats pretreated with the 5-HT releasing agent, fenfluramine (10 mg/kg i.p.). In parallel studies, the same dose of fenfluramine was shown to be sufficient to cause an increase in the expression of the immediate early genes (IEG) c-fos and Arc mRNA in cortical regions with high 5-HT(2A) receptor density. This increase was blocked by MDL 100907 (0.2 mg/kg i.v.), confirming a 5-HT(2A) receptor-mediated effect. The results demonstrate that PET with [(11)C]MDL 100907 is insensitive to an increased concentration of synaptic 5-HT, implying that the ligand can be used clinically to monitor 5-HT(2A) receptor function or dysfunction in disease or during therapy, without the need to consider concomitant changes in neurotransmitter concentration.

Radioiodinated SB 207710 as a radioligand in vivo: imaging of brain 5-HT4 receptors with SPET.

Single-photon emission tomography (SPET) and positron emission tomography (PET), when coupled to suitable radioligands, are uniquely powerful for investigating the status of neurotransmitter receptors in vivo. The serotonin subtype-4 (5-HT(4)) receptor has discrete and very similar distributions in rodent and primate brain. This receptor population may play a role in normal cognition and memory and is perhaps perturbed in some neuropsychiatric disorders. SB 207710 [(1-butyl-4-piperidinylmethyl)-8-amino-7-iodo-1,4-benzodioxan-5-carboxylate] is a selective high-affinity antagonist at 5-HT(4) receptors. We explored radioiodinated SB 207710 as a possible radioligand for imaging 5-HT(4) receptors in vivo. Rats were injected intravenously with iodine-125 labelled SB 207710, euthanised at known times and dissected to establish radioactivity content in brain tissues. Radioactivity entered brain but cleared rapidly and to a high extent from blood and plasma. Between 45 and 75 min after injection, the ratios of radioactivity concentration in each of 12 selected brain tissues to that in receptor-poor cerebellum correlated with previous measures of 5-HT(4) receptor density distribution in vitro. The highest ratio was about 3.4 in striatum. SB 207710 was labelled with iodine-123 by an iododestannylation procedure. A cynomolgus monkey was injected intravenously with [(123)I]SB 207710 and examined by SPET. Maximal whole brain uptake of radioactivity was 2.3% of the injected dose at 18 min after radioligand injection. Brain images acquired between 9 and 90 min showed high radioactivity uptake in 5-HT(4) receptor-rich regions, such as striatum, and low uptake in receptor-poor cerebellum. At 169 min the ratio of radioactivity concentration in striatum to that in cerebellum was 4.0. In a second SPET experiment, the cynomolgus monkey was pretreated with a selective 5-HT(4) receptor antagonist, SB 204070, at 20 min before [(123)I]SB 207710 injection. Radioactivity in all brain regions was reduced almost to the level in cerebellum by 176 min after radioligand injection. These findings show that [(123)I]SB 207710 is an effective radioligand for imaging brain 5-HT(4) receptors in vivo.