Masaki Tokunaga

Neuroimaging and Physiological Evidence for Involvement of Glutamatergic Transmission in Regulation of the Striatal Dopaminergic System

Aberrant neurotransmissions via glutamate and dopamine receptors have been the focus of biomedical research on the molecular basis of psychiatric disorders, but the mode of their interaction is yet to be uncovered. In this study, we demonstrated the pharmacological reversal of methamphetamine-stimulated dopaminergic overflow by suppression of group I metabotropic glutamate (mGlu) receptor in living primates and rodents. In vivo positron emission tomography (PET) was conducted on cynomolgus monkeys and rats using a full agonistic tracer for dopamine D2/3 receptor, [11C]MNPA [(R)-2-11CH3O-N-n-propylnorapomorphine], and fluctuation of kinetic data resulting from anesthesia was avoided by scanning awake subjects. Excessive release of dopamine induced by methamphetamine and abolishment of this alteration by treatment with an antagonist of group I mGlu receptors, 2-methyl-6-(phenylethynyl)pyridine (MPEP), were measured in both species as decreased binding potential because of increased dopamine and its recovery to baseline levels, respectively. Counteraction of MPEP to the methamphetamine-induced dopamine spillover was also supported neurochemically by microdialysis of unanesthetized rat striatum. Moreover, patch-clamp electrophysiological assays using acute brain slices prepared from rats indicated that direct targets of MPEP mechanistically involved in the effects of methamphetamine are present locally within the striatum. Because MPEP alone did not markedly alter the baseline dopaminergic neurotransmission according to our PET and electrophysiological data, the present findings collectively extend the insights on dopamine–glutamate cross talk from extrastriatal localization of responsible mGlu receptors to intrastriatal synergy and support therapeutic interventions in case of disordered striatal dopaminergic status using group I mGlu receptor antagonists assessable by in vivo imaging techniques.

Quantification of central substance P receptor occupancy by aprepitant using small animal positron emission tomography.

Background: Central substance P receptors, termed NK-1 receptors, have been considered as therapeutic targets in the development of drugs against diverse conditions, including emesis, overactive bladder, and depression. Methods: Here, we applied small animal positron emission tomography (PET) and a radioligand for NK-1 receptors ([18F]FE-SPA-RQ) for measuring occupancies of these receptors by a selective antagonist (aprepitant) in order to examine the validity of this in vivo imaging system for preclinical characterization of candidate agents acting on NK-1 receptors, and as a tool for predicting optimal doses in humans. Results: PET in gerbils depicted high uptake in the striatum and dose-dependent displacement with increasing doses of aprepitant. Occupancies increased as a function of aprepitant plasma concentrations according to a one-site competition model, which agrees with reported occupancy-concentration relationships in clinical studies after correction for species differences in plasma protein-unbound aprepitant fractions. These occupancy data were further supported by ex vivo autoradiography of brain samples from aprepitant-treated gerbils. In a pilot study of a marmoset, we obtained more accurate determinations of NK-1 receptor occupancy, less affected by spillover of signals from extracranial tissues than in gerbil experiments. Conclusions: These findings support the utility of small animals and quantitative PET in the development of drugs targeting NK-1 receptors.

Imaging for metabotropic glutamate receptor subtype 1 in rat and monkey brains using PET with [18F]FITM

PurposeIn this study, we evaluate the utility of 4-[18F]fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide ([18F]FITM) as a positron emission tomography (PET) ligand for imaging of the metabotropic glutamate receptor subtype 1 (mGluR1) in rat and monkey brains.MethodsIn vivo distribution of [18F]FITM in brains was evaluated by PET scans with or without the mGluR1-selective antagonist (JNJ16259685). Kinetic parameters of monkey PET data were obtained using the two-tissue compartment model with arterial blood sampling.ResultsIn PET studies in rat and monkey brains, the highest uptake of radioactivity was in the cerebellum, followed by moderate uptake in the thalamus, hippocampus and striatum. The lowest uptake of radioactivity was detected in the pons. These uptakes in all brain regions were dramatically decreased by pre-administration of JNJ16259685. In kinetic analysis of monkey PET, the highest volume of distribution (VT) was detected in the cerebellum (VT = 11.5).Conclusion[18F]FITM has an excellent profile as a PET ligand for mGluR1 imaging. PET with [18F]FITM may prove useful for determining the regional distribution and density of mGluR1 and the mGluR1 occupancy of drugs in human brains.

In-vivo visualization of key molecular processes involved in Alzheimer's disease pathogenesis: Insights from neuroimaging research in humans and rodent models.

Diverse age-associated neurodegenerative disorders are featured at a molecular level by depositions of self-aggregating molecules, as represented by amyloid beta peptides (Abeta) and tau proteins in Alzheimers disease, and cascade-type chain reactions are supposedly commenced with biochemical aberrancies of these amyloidogenic components. Mutagenesis and multiplication of the genes encoding Abeta, tau and other pathogenic initiators may accelerate the incipient process at the cascade top, rationalizing generations of transgenic and knock-in animal models of these illnesses. Meanwhile, these genetic manipulations do not necessarily compress the timelines of crucial intermediate events linking amyloidogenesis and neuronal lethality, resulting in an incomplete recapitulation of the diseases. Requirements for modeling the entire cascade can be illustrated by a side-by-side comparison of humans and animal models with the aid of imaging-based biomarkers commonly applicable to different species. Notably, key components in a highly reactive state are assayable by probe-assisted neuroimaging techniques exemplified by positron emission tomography (PET), providing critical information on the in-vivo accessibility of these target molecules. In fact, multispecies PET studies in conjunction with biochemical, electrophysiological and neuropathological tests have revealed putative neurotoxic subspecies of Abeta assemblies, translocator proteins accumulating in aggressive but not neuroprotective microglia, and functionally active neuroreceptors available to endogenous neurotransmitters and exogenous agonistic ligands. Bidirectional translational studies between human cases and model strains based on this experimental paradigm are presently aimed at clarifying the tau pathogenesis, and would be expanded to analyses of disrupted calcium homeostasis and mitochondrial impairments. Since reciprocal causalities among the key processes have indicated an architectural interchangeability between cascade and network connections as an etiological representation, longitudinal imaging assays with manifold probes covering the cascade from top to bottom virtually delineate the network dynamics continuously altering in the course of the disease and its treatment, and therefore expedite the evaluation and optimization of therapeutic strategies intended for suppressing the neurodegenerative pathway over its full length.

In Vivo SPECT Imaging of Amyloid-β Deposition with Radioiodinated Imidazo[1,2-a]Pyridine Derivative DRM106 in a Mouse Model of Alzheimer's Disease

Noninvasive determination of amyloid-β peptide (Aβ) deposition has important significance for early diagnosis and medical intervention for Alzheimers disease (AD). In the present study, we investigated the availability of radiolabeled DRM106 (123/125I-DRM106 [6-iodo-2-[4-(1H-3-pyrazolyl)phenyl]imidazo[1,2-a]pyridine]), a compound with sufficient affinity for the synthesis of human Aβ fibrils and satisfactory metabolic stability, as a SPECT ligand in living brains. Method: The sensitivity of 125I-DRM106 for detecting Aβ deposition was compared with that of 125I-IMPY (2-(4′-dimethylaminophenyl)-6-iodo-imidazo[1,2-a]pyridine), a well-known amyloid SPECT ligand, by ex vivo autoradiographic analyses in 18-mo-old amyloid precursor protein transgenic mice. To verify the sensitivity and quantitation of radiolabeled DRM106 for in vivo imaging, we compared the detectability of Aβ plaques with 123I-DRM106 and a well-known amyloid PET agent, 11C-labeled Pittsburgh compound B (11C-PiB), in 29-mo-old transgenic mice and age-matched nontransgenic littermates. Additionally, we compared the binding characteristics of 125I-DRM106 with those of 11C-PiB and 11C-PBB3, which selectively bind to Aβ plaques and preferentially to tau aggregates, respectively, in postmortem AD brain sections. Results: Ex vivo autoradiographic analysis showed that measurement with 125I-DRM106 has a higher sensitivity for detecting Aβ accumulation than with 125I-IMPY in transgenic mice. SPECT imaging with 123I-DRM106 also successfully detected Aβ deposition in living aged transgenic mice and showed strong correlation (R = 0.95, P < 0.01) in quantitative analysis for Aβ plaque detection by PET imaging with 11C-PiB, implying that sensitivity and quantitation of SPECT imaging with 123I-DRM106 are almost as good as 11C-PiB PET for the detectability of Aβ deposition. Further, the addition of nonradiolabeled DRM106 fully blocked the binding of 125I-DRM106 and 11C-PiB, but not 11C-PBB3, to AD brain sections, and 125I-DRM106 showed a lower binding ratio of the diffuse plaque–rich lateral temporal cortex to the dense-cored/neuritic plaque–rich hippocampal CA1 area, compared with 11C-PiB. Conclusion: All of these data demonstrated the high potential of 123I-DRM106 for amyloid imaging in preclinical and clinical application, and it might more preferentially detect dense-cored/neuritic amyloid deposition, which is expected to be closely associated with neuropathologic changes of AD.

Synthesis and evaluation of novel radioligands for positron emission tomography imaging of the orexin-2 receptor.

Orexin receptors (OXRs) in the brain have been implicated in diverse physiological and neuropsychiatric conditions. Here we describe the design, synthesis, and evaluation of OXR ligands related to (1R,2S)-2-(((2-methyl-4-methoxymethylpyrimidin-5-yl)oxy)methyl)-N-(5-fluoropyridin-2-yl)-2-(3-fluorophenyl)cyclopropanecarboxamide (9a) applicable to positron emission tomography (PET) imaging. Structural features were incorporated to increase binding affinity for OXRs, to enable carbon-11 radiolabeling, and to adjust lipophilicity considered optimal for brain penetration and low nonspecific binding. 9a displayed nanomolar affinity for OXRs, and autoradiography using rat brain sections showed that specific binding of [(11)C]9a was distributed primarily to neocortical layer VI and hypothalamus, consistent with reported localizations of orexin-2 receptors (OX2Rs). In vivo PET study of [(11)C]9a demonstrated moderate uptake of radioactivity into rat and monkey brains under deficiency or blockade of P-glycoprotein, and distribution of PET signals in the brain was in agreement with autoradiographic data. Our approach and findings have provided significant information for development of OX2R PET tracers.

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.

Development of TASP0410457 (TASP457), a novel dihydroquinolinone derivative as a PET radioligand for central histamine H3 receptors

BackgroundHistamine H3 receptor (H3R) is a potential therapeutic target of sleep- and cognition-related disorders. The purpose of the present study is to develop a novel positron emission tomography (PET) ligand for H3Rs from dihydroquinolinone derivatives, which we previously found to have high affinity with these receptors.MethodsSix compounds were selected from a dihydroquinolinone compound library based on structural capability for 11C labeling and binding affinity for H3Rs. Their in vivo kinetics in the rat brain were examined in a comparative manner by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Chemicals with appropriate kinetic properties were then labeled with 11C and evaluated in rats and monkeys using PET.ResultsOf the six compounds, TASP0410457 (also diminutively called TASP457) and TASP0434988 exhibited fast kinetics and relatively high brain uptakes in ex vivo LC-MS/MS and were selected as candidate PET imaging agents. PET data in rat brains were mostly consistent with LC-MS/MS findings, and rat and monkey PET scans demonstrated that [11C]TASP0410457 was superior to [11C]TASP0434988 for high-contrast H3R PET imaging. In the monkey brain PET, distribution volume for [11C]TASP0410457 could be quantified, and receptor occupancy by a nonradioactive compound was measurable using this radioligand. The specific binding of [11C]TASP0410457 to H3Rs was confirmed by autoradiography using rat and monkey brain sections.ConclusionsWe developed [11C]TASP0410457 as a radioligand enabling a robust quantification of H3Rs in all brain regions and demonstrated the utility of ex vivo LC-MS/MS and in vivo PET assays for selecting appropriate imaging tracers. [11C]TASP0410457 will help to examine the implication of H3Rs in neuropsychiatric disorders and to characterize emerging therapeutic agents targeting H3Rs.

Development of Novel PET Probes for Central 2-Amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic Acid Receptors

We document the development of PET probes for central AMPA receptors and their application to in vivo animal imaging. An initial screening of perampanel derivatives was performed to identify probe candidates. Despite the high autoradiographic contrast yielded by several radioligands, rat PET scans did not support their in vivo suitability. Further focused derivatization and a second screening by ex vivo LC-MS measurements led to the selection of 2-[1-(3-methylaminophenyl)-2-oxo-5-(pyrimidin-2-yl)-1,2-dihydropyridin-3-yl]benzonitrile, 21a, and its analogues as candidates. [(11)C]21a was shown by autoradiography to specifically bind to the neocortex and hippocampus, consistent with AMPA receptor localization. PET imaging with [(11)C]21a demonstrated moderate uptake of radioactivity in rat and monkey brains, with the retention of radiosignals being consistent with that from the autoradiogram data, and the uptake was blocked by pretreatment with unlabeled 21a in a dose-dependent manner. The current approach has facilitated the discovery of a PET probe potentially suitable for translational research and development focused on AMPA receptors.

A Case Report of Steroid‐resistant Antineutrophil Cytoplasmic Antibody‐related Vasculitis Successfully Treated by Mizoribine in a Hemodialysis Patient

Abstract:  Mizoribine (MZR) has shown to be effective against antineutrophil cytoplasmic antibody (ANCA)‐related vasculitis; however, no reports have described the successful treatment of steroid‐resistant ANCA‐related vasculitis with MZR in patients with renal insufficiency requiring hemodialysis. We herein report the case of a 39‐year‐old man undergoing hemodialysis in whom MZR successfully lowered the myeloperoxidase (MPO)–ANCA titer accompanied by remission of interstitial pneumonia, together with the pharmacokinetics of MZR. The patient developed severe renal insufficiency and interstitial pneumonia, and was started on hemodialysis. Although prednisolone was administered followed by azathioprine, the MPO–ANCA level and interstitial pneumonia showed insufficient improvement. Azathioprine was replaced by MZR and the administered dose of MZR was determined by measuring serum concentrations of MZR at the start of the dialysis session; this was because we confirmed that MZR could only be removed via dialysis, and that the serum concentration of MZR was maintained until the next dialysis session. The maintenance dose was finally set at MZR 75 mg after each dialysis. Subsequently, the ANCA titer decreased and interstitial pneumonia resolved without any MZR‐related side effects. This case demonstrates that MZR is safe and effective, even in patients with steroid‐resistant ANCA‐related vasculitis undergoing hemodialysis, and can be monitored by measuring serum concentrations of MZR.