Synthesis and Preclinical Evaluation of 18F-Labeled Ketoprofen Methyl Esters for Cyclooxygenase-1 Imaging in Neuroinflammation.

Cyclooxygenase (COX) is a rate-limiting enzyme in the synthesis of proinflammatory prostanoids from arachidonic acid. In vivo imaging of COX by PET is a potentially powerful tool for assessing the inflammatory response to injury, infection, and disease. We previously reported on a promising PET probe for COX imaging, 11C-labeled ketoprofen methyl ester, which can detect COX-1 activation in models of neuroinflammation and neurodegenerative disorders. In the current study, we aimed to design a fluorine-substituted benzoyl group of ketoprofen (FKTP) and to evaluate its racemate and enantiomers (18F-labeled ketoprofen methyl ester, [18F]FKTP-Me) as PET proradiotracers, potential radiopharmaceuticals for in vivo PET study of COX-1. Methods: We performed nucleophilic aromatic 18F-fluorination to obtain the desired racemic radiolabeled probe, (RS)-[18F]FKTP-Me, at a radiochemical yield of 11%-13%. Subsequent high-performance liquid chromatography separation with a chiral column yielded the desired enantiomerically pure (R)- and (S)-[18F]FKTP-Me. We examined the in vivo properties of (RS)-, (R)-, and (S)-[18F]FKTP-Me in PET studies using rats in which hemispheric inflammation was induced by intrastriatally injecting a lipopolysaccharide. Results: Racemic (RS)-[18F]FKTP-Me and enantiomeric (R)- or (S)-[18F]FKTP-Me were synthesized with radiochemical and chemical purities of more than 99%. The metabolite analysis revealed that the racemic (RS)-[18F]FKTP-Me crossed the blood-brain barrier and entered the brain, where it was subsequently hydrolyzed to its pharmacologically active acid form. PET images revealed a high accumulation of (R)-, (S)-, and (RS)-[18F]FKTP in the inflamed regions in rat brain. Moreover, the accumulated radioactivity of (S)-[18F]FKTP-Me was higher than that of (RS)-[18F]FKTP-Me and (R)-[18F]FKTP-Me, which was correlated with the stereospecific inhibitory activity of FKTP against COX-1. Conclusion: From the results of this study, we conclude that racemic (RS)-[18F]FKTP-Me and its enantiomers could act as proradiotracers of neuroinflammation in rat brain by the association of their hydrolyzed acid forms with COX-1 in inflamed regions. In particular, (S)-[18F]FKTP-Me demonstrated suitable properties as a COX-1-specific probe in PET imaging of neuroinflammation.

Ex vivo imaging and analysis of ROS generation correlated with microglial activation in rat model with acute neuroinflammation induced by intrastriatal injection of LPS.

Neuroinflammation and oxidative stress are hallmarks of neurodegenerative diseases. Microglia, the major important regulators of neuroinflammation, are activated in response to excessive generation of reactive oxygen species (ROS) from damaged cells and resulting in elevated and sustained damages. However, the relationship between microglia and ROS-regulatory system in the early stages of neuroinflammation prior to the appearance of neuronal damages have not been elucidated in detail. In this study, we analyzed the time-dependent changes in ROS generation during acute neuroinflammation in rats that were given an intrastriatal injection of lipopolysaccharide (LPS). We evaluated the effects of minocycline, an anti-inflammatory antibiotic, and N,N'-dimethylthiourea (DMTU), a radical scavenger, to understand the correlation between activated microglia and ROS generation. Ex vivo fluorescence imaging using dihydroethidium (DHE) clearly demonstrated an increased ROS level in the infused side of striatum in the rats treated with LPS. The level of ROS was changed in time-dependent manner, and the highest level of ROS was observed on day 3 after the infusion of LPS. Immunohistochemical studies revealed that time-dependent changes in ROS generation were well correlated to the presence of activated microglia. The inhibition of microglial activation by minocycline remarkably reduced ROS levels in the LPS-injected striatum, which indicated that the increased ROS generation caused by LPS was induced by activated microglia. DMTU decreased ROS generation and resulted in remarkable inhibitory effect on microglial activation. This study demonstrated that ROS generation during acute neuroinflammation induced by LPS was considerably associated with microglial activation, in an intact rat brain. The results provides a basis for understanding the interaction of ROS-regulatory system and activated microglia during neuroinflammation underlying neurodegenerative diseases.

Influence of Linker Molecules in Hexavalent RGD Peptides on Their Multivalent Interactions with Integrin αvß3.

Multivalent RGD peptides have been used as an excellent targeting vector to integrin αvß3-positive tumors. However, little attention has been paid to the influence of linker molecules in multivalent RGD peptides on their dissociation kinetics from tumor cells. In this study, we evaluated the dissociation kinetics of 99mTc-labeled hexavalent RGD peptides which have (CH2-CH2-O)n (n = 4, [99mTc][Tc(L1)6]+ and n = 12, [99mTc][Tc(L2)6]+) or (DPro-Gly)n (n = 1, [99mTc][Tc(L3)6]+; n = 6, [99mTc][Tc(L4)6]+; and n = 9, [99mTc][Tc(L5)6]+) as a linker molecule. The results showed that [99mTc][Tc(L4)6]+ and [99mTc][Tc(L5)6]+ displayed slower dissociation kinetics and [99mTc][Tc(L4)6]+ showed exceptionally high in vitro cellular uptake (203.1 ± 16.7% dose/mg protein) and the highest tumor to blood ratio (138.1 ± 26.3 at 4 h p.i.) in tumor bearing nude mice. These findings indicate that the use of appropriate length of (DPro-Gly)n would maximize the binding of multivalent RGD peptides to clustered integrin αvß3.

Evaluation of intracellular processes in quinolinic acid-induced brain damage by imaging reactive oxygen species generation and mitochondrial complex I activity.

PURPOSE: Our study aimed to elucidate the intracellular processes associated with quinolinic acid (QA)-induced brain injury by acquiring semiquantitative fluorescent images of reactive oxygen species (ROS) generation and positron emission tomography (PET) images of mitochondrial complex I (MC-I) activity. METHODS: Ex vivo fluorescent imaging with dihydroethidium (DHE) and PET scans with 18F-BCPP-EF were conducted at 3 h and 24 h after QA injection into the rat striatum. Immunohistochemical studies were performed 24 h after QA injection into the rat brain using monoclonal antibodies against neuronal nuclei (NeuN) and CD11b. RESULTS: A strong DHE-derived fluorescent signal was detected in a focal area within the QA-injected striatum 3 h after QA injection, and increased fluorescent signal spread throughout the striatum and parts of the cerebral cortex after 24 h. By contrast, 18F-BCPP-EF uptake in the QA-injected rat brain was unchanged after 3 h and markedly decreased after 24 h, not only in the striatum but also in the cerebral hemisphere. The fluorescent signal in the striatum 24 h after QA injection colocalised with microglial marker expression. CONCLUSIONS: We successfully obtained functional images of focal ROS generation during the early period of excitotoxic injury, and microglial ROS generation and mitochondrial dysfunction were observed during the progression of the inflammatory response. Both ex vivo DHE imaging and in vivo 18F-BCPP-EF-PET were sufficiently sensitive to detect the respective processes of QA-induced brain damage. Our study contributes to the functional imaging of multiple events during the pathological process.

A Simple Ex Vivo Semiquantitative Fluorescent Imaging Utilizing Planar Laser Scanner: Detection of Reactive Oxygen Species Generation in Mouse Brain and Kidney.

OBJECTIVE: Oxidative stress plays an important role in the onset of many neuronal and peripheral disorders. We examined the feasibility of obtaining semiquantitative fluorescent images of reactive oxygen species (ROS) generation in mouse brain and kidney utilizing a planar laser scanner and dihydroethidium (DHE). METHODS: To investigate ROS generation in brain, sodium nitroprusside was injected into the striatum. Dihydroethidium was injected into the tail vein. After DHE injection, tissue slices were analyzed utilizing a planar laser scanner. For kidney study, cis-diamminedichloroplatinum [II] (cisplatin) was intraperitoneally administrated into mice. RESULTS: Clear and semiquantitative fluorescent images of ROS generation in the mouse brain and kidney were obtained. Furthermore, the fluorescence intensity was stable and not affected by fading. Sodium nitroprusside induced approximately 6 times the fluorescence accumulation in the brain. Cisplatin caused renal injury in all mice, and in comparison with control mice, more than 10 times fluorescence accumulation was observed in the renal medulla with tubular necrosis and vacuolization. CONCLUSIONS: We successfully obtained ex vivo semiquantitative fluorescent images of ROS generation utilizing a planar laser scanner and DHE. This simple method is useful for ROS detection in several ROS-related animal models and would be applicable to a variety of biochemical processes.

Unexpected decrease in in vivo binding of [3H]QNB in the mouse cerebral cortex in the developing brain - A comparison with [11C]NMPB.

INTRODUCTION: Significant discrepancies between in vitro and in vivo binding of the muscarinic receptor ligand - 3H-labeled Quinuclidinyl Benzilate (QNB) - have been well documented. Discernable in vivo cerebellar [3H]QNB binding has been observed in mouse brain, despite the maximum number of binding sites (Bmax) being low. In order to understand this unique in vivo binding phenomenon, the binding of two muscarinic receptor ligands - [3H]QNB and N-[11C]methylpiperidyl Benzilate ([11C]NMPB) - were compared in vivo and in vitro in 3- and 8-week-old mice. METHOD: In vitro binding parameters of [3H]QNB were determined using brain homogenates. The time course of radioactivity concentration (TACs) in the cerebral cortex and cerebellum was measured following injection of [3H]QNB and [11C]NMPB with or without 3 mg/kg of carrier QNB in 3- and 8 week old mice using a dual tracer administration technique. A graphical method was employed for the quantitative analysis of in vivo binding of these radioligands. RESULTS: In vitro, the available number of binding sites for cerebral cortical muscarinic receptors increased by 17% during the developmental period studied. Paradoxically, in vivo, we observed a decrease of [3H]QNB binding in the cerebral cortex, while [11C]NMPB binding was markedly increased. In vivo saturation analysis of [3H]QNB in 3-week-old mice revealed an apparent positive cooperativity of binding in the cerebral cortex. CONCLUSIONS: Our results support the hypothesis that microenvironmental factors proximal to muscarinic receptors cause a local decrease in the cortical free-ligand concentration of [3H]QNB and that this 'ligand barrier' is modulated during brain development. ADVANCES IN KNOWLEDGE: The present study demonstrates that the combined use of radiolabeled QNB and NMPB has the potential to reveal the important effects of receptor microenvironmental factors on receptor function in the living brain.

[Positron Emission Tomography Imaging of Microglia in Diagnosis of Dementia].

Neuroinflammation which involves microglial activation is believed to be closely associated with the progression of neurodegeneration in Alzheimer's disease. The activation of microglia is receiving rising attention as a diagnostic and therapeutic target; however, it is important to distinguish its state and phenotype of the microglia. Positron emission tomography (PET) is a non-invasive method that can help visualize the changes of molecules associated with microglial activation in neurological disease under in vivo conditions. In this review, recent PET preclinical and clinical studies, and new molecular targets for the imaging of microglia are described.

Exploratory human PET study of the effectiveness of (11)C-ketoprofen methyl ester, a potential biomarker of neuroinflammatory processes in Alzheimer's disease.

INTRODUCTION: Neuroinflammatory processes play an important role in the pathogenesis of Alzheimer's disease (AD). As a biomarker of neuroinflammatory processes, we designed (11)C-labeled ketoprofen methyl ester ([(11)C]KTP-Me) to increase the blood-brain barrier permeability of ketoprofen (KTP), a selective cyclooxygenase-1 (COX-1) inhibitor. Animal studies indicated that [(11)C]KTP-Me enters the brain and accumulates in activated microglia of inflammatory lesions. In a first-in-human study, we reported that [(11)C]KTP-Me is a safe positron emission tomography (PET) tracer and enters the brain; the radioactivity is washed out from normal cerebral tissue. Here we explored the efficacy of [(11)C]KTP-Me as a diagnostic biomarker of neuroinflammatory processes in AD. METHODS: [(11)C]KTP-Me was synthesized by rapid C-[(11)C]methylation of [(11)C]CH3I and the corresponding arylacetate precursor. Nine subjects (four healthy subjects, two Pittsburgh compound-B (PiB)-positive patients with mild cognitive impairment (MCI), and three PiB-positive AD patients) underwent a dynamic brain PET scan for 70min after injection. We evaluated differences in cortical retention and washout rate in the brain between healthy subjects and MCI/AD patients. RESULTS: A brain distribution pattern reflecting blood flow in the early-phase image was seen in both healthy subjects and MCI/AD patients. Cortical activity gradually cleared in all groups. However, we observed no obvious difference in the washout rate between healthy subjects and MCI/AD patients or between MCI and AD patients. CONCLUSIONS: [(11)C]KTP-Me cannot be useful as a potential diagnostic biomarker for MCI/AD. Further improvements in binding affinity and specificity, etc., are needed to be a diagnostic biomarker of neuroinflammation in AD. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: [(11)C]KTP-Me is a new tracer that targets COX-1. [(11)C]KTP-Me is expected to be a diagnostic biomarker of neuroinflammation in AD in the future. The effectiveness was limited in a small number of AD patients. Therefore, further studies are needed to clarify the usefulness of [(11)C]KTP-Me.

Detection of Cyclooxygenase-1 in Activated Microglia During Amyloid Plaque Progression: PET Studies in Alzheimer's Disease Model Mice.

UNLABELLED: Cyclooxygenase (COX), a prostanoid-synthesizing enzyme, is considered to be involved in the neuroinflammatory process of neurodegenerative diseases. However, the role of COX in the progression of neurodegeneration is not well understood. We hypothesized that in vivo imaging of COX by PET will contribute to elucidation of the function of COX during the neurodegenerative process in Alzheimer's disease (AD). (11)C-labeled ketoprofen methyl ester (racemic (RS)-(11)C-KTP-Me) developed recently by our group is a useful PET probe for in vivo imaging of COX-1 during neuroinflammation. The (S)-enantiomer of ketoprofen is known to be pharmacologically more active than the (R)-enantiomer. We thus synthesized (11)C-labeled (S)-ketoprofen methyl ester ((S)-(11)C-KTP-Me) as an improved PET probe specific for COX-1 and applied it for investigation of the changes in COX-1 during the progression of AD in a mouse model. METHODS: The specificity of (S)-(11)C-KTP-Me for COXs was examined in PET studies with rats that had intrastriatal injection of lipopolysaccharide. To determine the details of changes in COX-1 during progression of amyloid-ß (Aß) plaque formation in amyloid precursor protein transgenic (APP-Tg) mice, we performed immunohistochemical studies and ex vivo autoradiography with (S)-(11)C-KTP-Me. RESULTS: PET studies using hemispheric lipopolysaccharide injection into rats revealed that the sensitivity of (S)-(11)C-KTP-Me in neuroinflammation was much higher than that of (RS)-(11)C-KTP-Me and (R)-(11)C-KTP-Me; these results closely corresponded to the inhibitory activities of each enantiomer against COX-1 estimated by an in vitro assay. In APP-Tg mice, (S)-(11)C-KTP-Me administration resulted in progressive and significant increases in accumulation of radioactivity in the brain from 16 to 24 mo old in accordance with the histopathologic appearance of abundant Aß plaques and activated microglia, whereas few changes in radioactivity accumulation and few Aß plaques were seen in age-matched wild-type control mice. High-radioactivity accumulation by (S)-(11)C-KTP-Me was markedly observed in the frontal cortex and hippocampus in which COX-1-expressing activated microglia tightly surrounded and enclosed large and more intensely stained Aß plaques, indicating neuroinflammation that originated with Aß. CONCLUSION: (S)-(11)C-KTP-Me is a potent PET probe that is highly selective for COX-1. Studies using APP-Tg mice demonstrated that (S)-(11)C-KTP-Me could detect activated microglia that are associated with amyloid plaque progression, suggesting the involvement of COX-1 in the neuroinflammatory process in AD.

Human whole-body biodistribution and dosimetry of a new PET tracer, [(11)C]ketoprofen methyl ester, for imagings of neuroinflammation.

INTRODUCTION: Neuroinflammatory processes play an important role in the pathogenesis of Alzheimer's disease and other brain disorders, and nonsteroidal anti-inflammatory drugs (NSAIDs) are considered therapeutic candidates. As a biomarker of neuroinflammatory processes, (11)C-labeled ketoprofen methyl ester ([(11)C]KTP-Me) was designed to allow cerebral penetration of ketoprofen (KTP), an active form of a selective cyclooxygenase-1 inhibitor that acts as an NSAID. Rat neuroinflammation models indicate that [(11)C]KTP-Me enters the brain and is retained in inflammatory lesions, accumulating in activated microglia. [(11)C]KTP-Me is washed out from normal tissues, leading to the present first-in-human exploratory study. METHODS: [(11)C]KTP-Me was synthesized by rapid C-[(11)C]methylation of [(11)C]CH3I and the corresponding arylacetate precursor, purified with high-performance liquid chromatography, and prepared as an injectable solution including PEG400, providing radiochemical purity of >99% and specific activity of >25GBq/µmol at injection. Six young healthy male humans were injected with [(11)C]KTP-Me and scanned with PET camera to determine the early-phase brain time course followed by three whole-body scans starting 8, 20, and 40 min post-injection, together with sequential blood sampling and labeled metabolite analysis. RESULTS: No adverse effects were observed during PET scanning after [(11)C]KTP-Me injection. [(11)C]KTP-Me was rapidly metabolized to (11)C-labeled ketoprofen ([(11)C]KTP) within 2-3 min and was gradually cleared from blood. The radioactivity entered the brain with an average peak cortical SUV of 1.5 at 2 min. The cortical activity was gradually washed out. Whole-body images indicated that the urinary bladder was the major excretory pathway. The organ with the highest radiation dose was the urinary bladder (average dose of 41µGy/MBq, respectively). The mean effective dose was 4.7µSv/MBq, which was comparable to other (11)C-labeled radiopharmaceuticals. CONCLUSION: [(11)C]KTP-Me demonstrated a favorable dosimetry, biodistribution, and safety profile. [(11)C]KTP-Me entered the human brain, and the radioactivity was washed out from cerebral tissue. These data warrant further exploratory studies on patients with neuroinflammation.

Assessment of radioligands for PET imaging of cyclooxygenase-2 in an ischemic neuronal injury model.

Cyclooxygenase-2 (COX-2) plays crucial roles in progressive neuronal death in ischemic brain injury. In the present study, we evaluated two radiolabeled COX-2 selective inhibitors, [11C]celecoxib and [11C]rofecoxib, as positron emission tomography (PET) tracers for COX-2 imaging in normal and ischemic mouse brains. We also took advantage of our newly-generated antibody highly selective for mouse COX-2 to prove accumulation of the radioligands in regions enriched with COX-2. In vitro autoradiography demonstrated specific binding of high-concentration [11C]rofecoxib but not [11C]celecoxib to the cerebellum and brain stem of normal brains wherein COX-2 immunoreactivity in neurons was most abundantly observed. Meanwhile, both of these radioligands failed to detect COX-2 expression in PET assays despite their excellent brain permeability. Hypoperfusion-induced ischemia caused marked necrotic neuron death accompanied by gliosis and enhancement of neuronal COX-2 immunoreactivity in the hippocampus. Correspondingly, in vitro autoradiographic binding of [11C]rofecoxib was increased in the injured hippocampus compared to the uninjured contralateral region, but failed in living brains of ischemia model likewise. Our work provides the rationale for monitoring COX-2 as a biomarker reflecting ischemic brain injuries and demonstrates that [11C]rofecoxib, not [11C]celecoxib, is useful for in vitro assays of COX-2, but its affinity would be insufficient for in vivo PET visualization.

The food reaching test: a sensitive test of behavioral improvements by deep brain stimulation in MPTP-treated monkey.

We modified an objective behavioral test, namely the food reaching test (FRT), for quantitative assessment of motor performance improved by deep brain stimulation (DBS) of the subthalamic nucleus (STN) in the Parkinsonian monkeys. The symptomatic features and their severity in 3 monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were evaluated with a subjective monkey Parkinson's disease rating scale (PDRS). We then performed STN-DBS with the minimum current intensity that stopped the tremor. The time required for the monkeys to pick up all 5 pieces of potato (FRT time) was measured as a major index to evaluate bradykinesia. The success rate was adopted as another index for assessing overall motor impairments. Although both FRT time and PDRS score were similarly improved by STN-DBS, change of FRT time appeared more sensitive than that of PDRS scores. FRT is an easily trained behavioral test with high objectivity and sensitivity that can be applied for assessing motor performance in MPTP-treated monkeys during experiments in a restrained condition such as functional imaging of the brain.

In vivo expression of cyclooxygenase-1 in activated microglia and macrophages during neuroinflammation visualized by PET with 11C-ketoprofen methyl ester.

UNLABELLED: Cyclooxygenase (COX)-1 and -2 are prostanoid-synthesizing enzymes that play important roles in the regulation of neuroinflammation and in the development of neurodegenerative disorders. However, the specific functions of these isoforms are still unclear. We recently developed (11)C-labeled ketoprofen methyl ester as a PET probe that targets the COXs for imaging neuroinflammation, though its responsible isoform is yet to be determined. In the present study, we performed ex vivo and in vivo imaging studies with (11)C-ketoprofen methyl ester and determined the contributions of the COX isoforms during the neuroinflammatory process. METHODS: To identify the COX isoform responsible for (11)C-ketoprofen methyl ester in the brain, we examined the ex vivo autoradiography of (11)C-ketoprofen methyl ester using COX-deficient mice. Time-dependent changes in accumulation of (11)C-ketoprofen methyl ester during the neuroinflammatory process were evaluated by PET in rats with hemispheric neuroinflammation induced by intrastriatal injection of lipopolysaccharide or quinolinic acid. In both rat models, cell-type specificity of COX isoform expression during neuroinflammation was identified immunohistochemically. RESULTS: Ex vivo autoradiographic analysis of COX-deficient mice revealed a significant reduction of (11)C-ketoprofen methyl ester accumulation only in COX-1-deficient mice, not COX-2-deficient mice. PET of rats after intrastriatal injection of lipopolysaccharide showed a significant increase in accumulation of (11)C-ketoprofen methyl ester in the inflamed area. This increase was evident at the early phase of 6 h, peaked at day 1, and then returned to basal levels by day 7. In addition, immunohistochemical analysis revealed that the population of activated microglia and macrophages was elevated at the early phase with COX-1 expression but not COX-2. A significant increase in (11)C-ketoprofen methyl ester accumulation was also observed at day 1 after intrastriatal injection of quinolinic acid, with increased COX-1-expressing activated microglia and macrophages. CONCLUSION: We have identified (11)C-ketoprofen methyl ester as a COX-1-selective PET probe, and using this, we have also demonstrated a time-dependent expression of COX-1 in activated microglia and macrophages during the neuroinflammatory process in the living brain. Thus, COX-1 may play a crucial role in the pathology of neuroinflammation and might be a critical target for the diagnosis and therapy of neurodegenerative disorders.

Establishment of in vivo brain imaging method in conscious mice.

UNLABELLED: In vivo imaging, such as PET, requires restriction of body movements and is generally conducted under sedation by anesthetic agents in studies using laboratory animals. Because anesthetics reduce neural activity and metabolism, physiologic neural functions are difficult to assess in animal PET studies. Therefore, use of an appropriate method in conscious animals is important and is a practical requirement for physiologic in vivo brain imaging studies. Here, we established an in vivo imaging system for conscious mice to reveal the physiologic regional cerebral glucose metabolic rate (rCMRglu) with (18)F-FDG PET. METHODS: We first developed a head holder to enable brain PET of a conscious mouse. To obtain optimal rCMRglu, we examined the effects of physical and psychologic stresses caused by ambient temperature, intravenous injection, and acclimation to the apparatus and immobile state. Finally, quantitative kinetic analysis was performed for rCMRglu based on a 2-tissue-compartment model with an input function of arterial blood sampling under both conscious and anesthetized (1.5% isoflurane) conditions. RESULTS: Increasing the ambient temperature increased uptake of (18)F-FDG in the brain significantly while reducing the uptake in skeletal muscle and brown adipose tissue that was caused by shivering. The reduction of brain (18)F-FDG uptake caused by tail holding and manual injection was significantly ameliorated by the use of an automated slow injection. Although brain uptake of (18)F-FDG varied at the first session of PET, uptake at the second and subsequent sessions was stable, even after long-term acclimation. After these beneficial changes, brain uptake of (18)F-FDG improved significantly, to approximately 260% above the preconditioned state, which is comparable with that obtained in mice that have been allowed to move freely about their home cages. Quantitative kinetic analyses revealed that isoflurane anesthesia lowered rCMRglu in the cerebral cortex, striatum, thalamus, and cerebellum by 66%, 59%, 62%, and 22%, respectively, mainly by reducing the k(3) value, a rate constant for phosphorylation by hexokinase. CONCLUSION: To our knowledge, this is the first study to report quantitative kinetic analysis of rCMRglu in mice that have been conscious throughout PET. This investigation will open avenues for research into in vivo functional brain molecular imaging in both normal and genetically manipulated mice.

General method for the (11)C-labeling of 2-arylpropionic acids and their esters: construction of a PET tracer library for a study of biological events involved in COXs expression.

Cyclooxygenase (COX) is a critical enzyme in prostaglandin biosynthesis that modulates a wide range of biological functions, such as pain, fever, and so on. To perform in vivo COX imaging by positron emission tomography (PET), we developed a method to incorporate (11)C radionuclide into various 2-arylpropionic acids that have a common methylated structure, particularly among nonsteroidal anti-inflammatory drugs (NSAIDs). Thus, we developed a novel (11)C-radiolabeling methodology based on rapid C-[(11)C]methylation by the reaction of [(11)C]CH(3)I with enolate intermediates generated from the corresponding esters under basic conditions. One-pot hydrolysis of the above [(11)C]methylation products also allows the synthesis of desired (11)C-incorporated acids. We demonstrated the utility of this method in the syntheses of six PET tracers, [(11)C]Ibuprofen, [(11)C]Naproxen, [(11)C]Flurbiprofen, [(11)C]Fenoprofen, [(11)C]Ketoprofen, and [(11)C]Loxoprofen. Notably, we found that their methyl esters were particularly useful as proradiotracers for a study of neuroinflammation. The microPET studies of rats with lipopolysaccharide (LPS)-induced brain inflammation clearly showed that the radioactivity of PET tracers accumulated in the inflamed region. Among these PET tracers, the specificity of [(11)C]Ketoprofen methyl ester was demonstrated by a blocking study. Metabolite analysis in the rat brain revealed that the methyl esters were initially taken up in the brain and then underwent hydrolysis to form pharmacologically active forms of the corresponding acids. Thus, we succeeded in general (11)C-labeling of 2-arylpropionic acids and their methyl esters as PET tracers of NSAIDs to construct a potentially useful PET tracer library for in vivo imaging of inflammation involved in COXs expression.

11C-PK11195 PET for the in vivo evaluation of neuroinflammation in the rat brain after cortical spreading depression.

UNLABELLED: Neurogenic inflammation triggered by extravasation of plasma protein has been hypothesized as a key factor in the generation of the pain sensation associated with migraine. The principal immune cell that responds to this inflammation is the parenchymal microglia of the central nervous system. METHODS: Using a PET technique with (11)C-(R)-[1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide] ((11)C-PK11195), a PET ligand for peripheral type-benzodiazepine receptor, we evaluated the microglial activation in the rat brain after generation of unilateral cortical spreading depression, a stimulation used to bring up an experimental animal model of migraine. RESULTS: We found a significant increase in the brain uptake of (11)C-PK11195, which was completely displaceable by the excess amounts of unlabeled ligands, in the ipsilateral hemisphere of the spreading depression-generated rats. Moreover, the binding potential of (11)C-PK11195 in the spreading depression-generated rats was significantly higher than that in the sham-operated control rats. CONCLUSION: These results suggest that as an inflammatory reaction, microglial cells are activated in response to the nociceptive stimuli induced by cortical spreading depression in the rat brain. Therefore, the (11)C-PK11195 PET technique could have a potential for diagnostic and therapeutic monitoring of neurologic disorders related to neuroinflammation such as migraine.

Distinct different intra-tumor distribution of FDG between early phase and late phase in mouse fibrosarcoma.

An early image of intra-tumor distribution of 14C-labeled fluorodeoxy glucose (14C-FDG) was compared with a late image of 18F-labeled FDG (18F-FDG) using mouse fibrosarcoma. Heterogeneous intra-tumor distribution of 14C-FDG was observed 1 minute post injection of the tracer, whereas relatively homogeneous distribution of 18F-FDG was seen 30 minutes later. 14C-FDG was particularly taken up in the peripheral part of the tumor immediately after the tracer injection. A gradual and significant increase in 18F-FDG accumulation with time was seen in the central part of tumor, which indicated an enhancement of anaerobic glycolysis. An initial uptake of 18F-FDG was also compared with distribution of 14C-iodoantipyrine and 14C-thymidine uptake. Intratumoral distribution of initial uptake of 18F-FDG showed almost the same regional distribution of 14C-iodoantipyrine. A similar distribution of 14C-thymidine as the initial uptake of 18F-FDG was also observed. These results indicated that a high initial FDG uptake area seemed to be highly proliferative. A significant difference in the intratumoral distribution of FDG between early phase and late phase seemed to be related to heterogeneous biological characteristics of tumor cells.

Effect of rolipram on relative 14C-deoxyglucose uptake in inflammatory lesions and skeletal muscle.

PURPOSE: 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) has become a useful imaging tool for inflammatory diseases. In this study we investigated the effects of rolipram, a selective phosphodiesterase type 4 inhibitor, on 14C-deoxyglucose (DG) uptake in inflammatory lesions and other normal tissues, and attempted to improve the inflammation/muscle ratio. METHODS: To induce inflammation, mice were inoculated with turpentine oil. Inflammation-bearing mice were pretreated with rolipram (3 mg/kg i.p. or i.v.), and the effect on 14C-DG uptake was measured using a tissue dissection method and autoradiography. The inflammatory tissue samples were stained with haematoxylin and eosin. RESULTS: Rolipram (3 mg/kg i.p.) significantly decreased 14C-DG uptake in normal tissues like brain, heart and skeletal muscle (brain 31%, heart 60%, skeletal muscle 61%). On the other hand, 14C-DG uptake in inflammatory lesions was not significantly altered by pretreatment with rolipram. The inflammation/muscle ratio of 14C-DG uptake (30 min after tracer injection) was enhanced from 1.1 to 2.8 by rolipram. An autoradiographic study revealed heterogeneous distributions of 14C-DG in the inflammatory lesions and skeletal muscle of animals that were not treated with rolipram. Pretreatment with rolipram significantly attenuated the intramuscular distribution of 14C-DG, producing a relatively homogeneous distribution of radioactivity. CONCLUSION: These results indicate that rolipram decreased 14C-DG uptake in skeletal muscle by activation of the adenosine 3',5'-cyclic monophosphate system, whereas 14C-DG uptake in inflammatory lesions was not significantly altered. Therefore, rolipram may be a valuable tool for improving the visualisation of inflammatory lesions in clinical PET studies employing FDG.