Dextromethorphan moderates reward deficiency associated with central serotonin transporter availability in 3,4-methylenedioxy-methamphetamine-treated animals.

BACKGROUND: The neurotoxicity of 3,4-methylenedioxy-methamphetamine (MDMA) to the serotonergic system is well-documented. Dextromethorphan (DM), an antitussive drug, decreased morphine- or methamphetamine (MA)-induced reward in rats and may prevent MDMA-induced serotonergic deficiency in primates, as indicated by increased serotonin transporter (SERT) availability. We aimed to investigate the effects of DM on reward, behavioral sensitization, and neurotoxicity associated with loss of SERT induced by chronic MDMA administration in rats. METHODS: Conditioned place preference (CPP) and locomotor activity tests were used to evaluate drug-induced reward and behavioral sensitization; 4-[ 18 F]-ADAM/animal-PET and immunohistochemistry were used to explore the effects of DM on MDMA-induced loss of SERT. RESULTS: MDMA significantly reduced SERT binding in the rat brain; however, co-administration of DM significantly restored SERT, enhancing the recovery rate at day 14 by an average of ~23% compared to the MDMA group. In confirmation of the PET findings, immunochemistry revealed MDMA reduced SERT immunoactivity in all brain regions, whereas DM markedly increased the serotonergic fiber density after MDMA induction. CONCLUSION: Behavioral tests and in vivo longitudinal PET imaging demonstrated the CPP indexes and locomotor activities of the reward system correlate negatively with PET 4-[ 18 F]ADAM SERT activity in the reward system. Our findings suggest MDMA induces functional abnormalities in a network of brain regions important to decision-making processes and the motivation circuit. DM may exert neuroprotective effects to reverse MDMA-induced neurotoxicity.

Amitriptyline Accelerates SERT Binding Recovery in a Rat 3,4-Methylenedioxymethamphetamine (MDMA) Model: In Vivo 4-[18F]-ADAM PET Imaging.

Numerous studies have confirmed that 3,4-Methylenedioxymethamphetamine (MDMA) produces long-lasting changes to the density of the serotonin reuptake transporter (SERT). Amitriptyline (AMI) has been shown to exert neuroprotective properties in neuropathologic injury. Here, we used a SERT-specific radionuclide, 4-[18F]-ADAM, to assess the longitudinal alterations in SERT binding and evaluate the synergistic neuroprotective effect of AMI in a rat MDMA model. In response to MDMA treatment regimens, SERT binding was significantly reduced in rat brains. Region-specific recovery rate (normalized to baseline) in the MDMA group at day 14 was 71.29% ± 3.21%, and progressively increased to 90.90% ± 7.63% at day 35. AMI dramatically increased SERT binding in all brain regions, enhancing average ~18% recovery rate at day 14 when compared with the MDMA group. The immunochemical staining revealed that AMI markedly increased the serotonergic fiber density in the cingulate and thalamus after MDMA-induction, and confirmed the PET findings. Using in vivo longitudinal PET imaging, we demonstrated that SERT recovery was positively correlated with the duration of MDMA abstinence, implying that lower SERT densities in MDMA-induced rats reflected neurotoxic effects and were (varied) region-specific and reversible. AMI globally accelerated the recovery rate of SERT binding and increased SERT fiber density with possible neuroprotective effects.

Preliminary Results on the Long-Term Effects of Dextromethorphan on MDMA-Mediated Serotonergic Deficiency and Volumetric Changes in Primates Based on 4-[18F]-ADAM PET/MRI.

Alterations to the serotonergic system due to 3,4-methylenedioxymethamphetamine (MDMA) (ecstasy) consumption have been extensively documented. However, knowledge of the reversibility of these neurotoxic effects based on in vivo evaluations of serotonin transport (SERT) availability remains limited. This study aimed to evaluate the long-term neurotoxicity of MDMA after 66 months abstinence and explored whether Dextromethorphan, a non-competitive N-methyl-D-aspartate (NMDA) receptor, could attenuate MDMA-induced neurotoxicity using 4-[18F]-ADAM, an imaging ligand that selectively targets SERT, with positron emission tomography technology (PET). Nine monkeys (Macaca cyclopis) were used in this study: control, MDMA, and DM + MDMA. Static 4-[18F]-ADAM PET was performed at 60 and 66 months after drug treatment. Serotonin transport (SERT) availability was presented as the specific uptake ratios (SURs) of 4-[18F]-ADAM in brain regions. Voxel-based region-specific SERT availability was calculated to generate 3D PET/MR images. Structural Magnetic Resonance Imaging (MRI) volumetric analysis was also conducted at 60 months. Significantly decreased 4-[18F]-ADAM SURs were observed in the striatum and thalamus of the MDMA group at 60 and 66 months compared to controls; the midbrain and frontal cortex SURs were similar at 60 and 66 months in the MDMA and control groups. All eleven brain regions showed significantly lower (∼13%) self-recovery rates over time; the occipital cortex and cingulate recovered to baseline by 66 months. DM attenuated MDMA-induced SERT deficiency on average, by ∼8 and ∼1% at 60 and 66 months, respectively; whereas significant differences were observed between the thalamus and amygdala of the MDMA and DM + MDMA groups at 66 months. Compared to controls, the MDMA group exhibited significantly increased (∼6.6%) gray matter volumes in the frontal cortex, occipital cortex, caudate nucleus, hippocampus, midbrain, and amygdala. Moreover, the gray matter volumes of the occipital cortex, hippocampus and amygdala correlated negatively with the 4-[18F]-ADAM SURs of the same regions. DM (n = 2) did not appear to affect MDMA-induced volumetric changes. The 4-[18F]-ADAM SURs, lower self-recovery rate and increased volumetric values indicate the occipital cortex, hippocampus and amygdala still exhibit MDMA-induced neurotoxicity after 66 months' abstinence. Moreover, DM may prevent MDMA-induced serotonergic deficiency, as indicated by increased 4-[18F]-ADAM SURs and SERT availability, but not volumetric changes.

Automated Synthesis and Initial Evaluation of (4'-Amino-5',8'-difluoro-1'H-spiro[piperidine-4,2'-quinazolin]-1-yl)(4-[18F]fluorophenyl)methanone for PET/MR Imaging of Inducible Nitric Oxide Synthase.

Background: Inducible nitric oxide synthase (iNOS) plays a crucial role in neuroinflammation, especially microglial activity, and may potentially represent a useful biomarker of neuroinflammation. In this study, we carefully defined a strategic plan to develop iNOS-targeted molecular PET imaging using (4'-amino-5',8'-difluoro-1'H-spiro[piperidine-4,2'-quinazolin]-1-yl)(4-fluorophenyl)methanone ([18F]FBAT) as a tracer in a mouse model of lipopolysaccharide- (LPS-) induced brain inflammation. Methods: An in vitro model, murine microglial BV2 cell line, was used to assess the uptake of [18F]FBAT in response to iNOS induction at the cellular level. In vivo whole-body dynamic PET/MR imaging was acquired in LPS-treated (5 mg/kg) and control mice. Standard uptake value (SUV), total volume of distribution (V t), and area under the curve (AUC) based on the [18F]FBAT PET signals were determined. The expression of iNOS was confirmed by immunohistochemistry (IHC) of brain tissues. Results: At the end of synthesis, the yield of [18F]FBAT was 2.2-3.1% (EOS), radiochemical purity was >99%, and molar radioactivity was 125-137 GBq/µmol. In vitro, [18F]FBAT rapidly and progressively accumulated in murine microglial BV2 cells exposed to LPS; however, [18F]FBAT accumulation was inhibited by aminoguanidine, a selective iNOS inhibitor. In vivo biodistribution studies of [18F]FBAT showed a significant increase in the liver and kidney on LPS-treated mice. At 3 h postinjection of LPS, in vivo, the [18F]FBAT accumulation ratios at 30 min post intravenous (i.v.) radiotracer injection for the whole brain, cortex, cerebellum, and brainstem were 2.16 ± 0.18, 1.53 ± 0.25, 1.41 ± 0.21, and 1.90 ± 0.12, respectively, compared to those of mice not injected with LPS. The mean area under the curve (AUC0-30min), total volume of distribution (V t, mL/cm3), and K i (influx rate) of [18F]FBAT were 1.9 ± 0.21- and 1.4 ± 0.22-fold higher in the 3 h LPS group, respectively, than in the control group. In the pharmacokinetic two-compartment model, the whole brain K i of [18F]FBAT was significantly higher in mice injected with LPS compared to the control group. Aminoguanidine, selective iNOS inhibitor, pretreatment significantly reduced the AUC0-30min and V t values in LPS-induced mice. Quantitative analysis of immunohistochemically stained brain sections confirmed iNOS was preferentially upregulated in the cerebellum and cortex of mice injected with LPS. Conclusion: An automated robotic method was established for radiosynthesis of [18F]FBAT, and the preliminary in vitro and in vivo results demonstrated the feasibility of detecting iNOS activity/expression in LPS-treated neuroinflammation by noninvasive imaging with [18F]FBAT PET/MRI.

GMP-compliant fully automated radiosynthesis of [18F]FEPPA for PET/MRI imaging of regional brain TSPO expression.

BACKGROUND: Expression of translocator protein (TSPO) on the outer mitochondrial membrane of activated microglia is strongly associated with neuroinflammation. The second-generation PET ligand [18F]FEPPA specifically binds TSPO to enable in vivo visualization and quantification of neuroinflammation. We optimized a fully automated radiosynthesis method and evaluated the utility of [18F]FEPPA, the second-generation PET ligand specifically binds TSPO, in a mouse model of systemic LPS challenge to detect TSPO-associated signals of central and peripheral inflammation. In vivo dynamic PET/MR imaging was performed in LPS-induced and control mice after [18F]FEPPA administration. The relationship between the [18F]FEPPA signal and the dose of LPS was assessed. The cytokine levels (i.e., TNF-α, Il-1ß, Il-6) in LPS-induced mice were measured by RT-PCR. Standard uptake value (SUV), total volume of distribution (VT) and area under the curve (AUC) were determined based on the metabolite-uncorrected plasma input function. Western blotting and immunostaining were used to measure TSPO expression in the brain. RESULTS: The fully automated [18F]FEPPA radiosynthesis produced an uncorrected radiochemical yield of 30 ± 2% within 80 min, with a radiochemical purity greater than 99% and specific activity of 148.9‒216.8 GBq/µmol. Significant differences were observed in the brain after [18F]FEPPA administration: SUV, VT and AUC were 1.61 ± 0.1, 1.25 ± 0.12 and 1.58 ± 0.09-fold higher in LPS-injected mice than controls. TNF-α, Il-1ß and Il-6 mRNA levels were also elevated in the brains of LPS-injected mice. Western blotting revealed TSPO (p < 0.05) and Iba-1 (p < 0.01) were upregulated in the brain after LPS administration. In LPS-injected mice, TSPO immunoactivity colocalized with Iba-1 in the cerebrum and TSPO was significantly overexpressed in the hippocampus and cerebellum. The peripheral organs (heart, lung) of LPS-injected mice had higher [18F]FEPPA signal-to-noise ratios than control mice. CONCLUSIONS: Based on the current data on ligand specificity and selectivity in central tissues using 7 T PET/MR imaging, we demonstrate that [18F]FEPPA accumulations significant increased in the specific brain regions of systemic LPS-induced neuroinflammation (5 mg/kg). Future investigations are needed to determine the sensitivity of [18F]FEPPA as a biomarker of neuroinflammation as well as the correlation between the PET signal intensity and the expression levels of TSPO.

Synthesis and comparison of 4-[18F]F-ADAM, 2-[18F]F-ADAM, N-Desmethyl-4-[18F]F-ADAM and [18F]F-AFM as serotonin transporter imaging agents.

4-[(18)F]F-ADAM (1a), 2-[(18)F]F-ADAM (2a), N-Desmethyl-4-[(18)F]F-ADAM (3a) and [(18)F]F-AFM (4a ) were synthesized in 1.7, 3.9, 2.9 and 0.6% yield (EOS), respectively, in a synthesis time of ~120 min from EOB. PET studies in rats showed that the maximum specific uptake ratios of 1a, 2a, 3a and 4a in midbrain were 3.86, 0.73, 0.35 and 2.23, respectively. Thus, in terms of radiochemical yield, specific binding and in vivo stability, 4-[(18)F]F-ADAM may be the most appropriate SERT imaging agent for human studies.

Validation of 4-[18F]-ADAM as a SERT imaging agent using micro-PET and autoradiography.

Serotonin transporters (SERTs) have been implicated in various neuropsychiatric disorders. We aim to validate 4-[(18)F]-ADAM (N,N-dimethyl-2-(2-amino-4-[(18)F]fluorophenylthio)benzylamine) as a SERT imaging agent in rats using micro-positron emission tomography (micro-PET) and autoradiography. Sixty to ninety min after injecting 4-[(18)F]-ADAM, specific uptake ratios (SURs) were determined by micro-PET measurements in various brain regions of normal control rats. For n=3, the SUR in the midbrain was 4.94+/-0.16, for the hypothalamus it was 4.39+/-0.031 and for the caudate it was 4.18+/-0.53. The retention of 4-[(18)F]-ADAM in the hypothalamus and midbrain regions increased rapidly between 5 to 10 min after injection and declined thereafter. The SURs determined by autoradiography were: 9.31+/-1.41 for the midbrain, 7.15+/-1.45 for the hypothalamus and 5.22+/-1.14 for the caudate putamen. Both micro-PET and autoradiography studies revealed a dose-dependent progressive inhibition of radioligand uptake in the frontal cortex, caudate putamen and hypothalamus in rats treated with 0.01 to 0.25 mg/kg paroxetine. A decrease in 4-[(18)F]-ADAM uptake of approximately 84% was observed in the midbrain of rats pretreated with 0.25 mg/kg paroxetine as compared to controls (4.94+/-0.16 versus 0.80+/-0.17, n=3). Both 5,7-dihydroxytryptamine and p-chloroamphetamine-treated rats showed pronounced reduction in 4-[(18)F]-ADAM binding when compared to normal controls. Rats pretreated with p-chloroamphetamine exhibited significant inhibition of 4-[(18)F]-ADAM uptake in brain regions rich in SERT over a period of four weeks. Thus, 4-[(18)F]-ADAM is a SERT-specific radioligand that may be useful for evaluating neuropsychiatric conditions involving serotonergic dysfunction.