Neuroinflammation in frontotemporal lobar degeneration revealed by 11 C-PBR28 PET.

This study used 11 C-PBR28 positron emission tomography (PET) imaging to determine whether levels of 18-kDa translocator protein (TSPO), an inflammation-specific biomarker, are increased in frontotemporal lobar degeneration (FTLD) patients. 11 C-PBR28, 18 F-FDG, and 11 C-PIB brain PET scans, as well as magnetic resonance imaging (MRI), were conducted in four FTLD patients and 22 healthy controls. 11 C-PBR28 scans revealed that all FTLD patients showed increased TSPO binding versus controls. Significantly greater increases in TSPO were observed in the frontal, lateral temporal, parietal, and occipital cortices, topographically consistent with individual clinical phenotypes and with brain MRI and 18 F-FDG PET. Amyloid burden was not increased.

2-(4-Methylsulfonylphenyl)pyrimidines as Prospective Radioligands for Imaging Cyclooxygenase-2 with PET-Synthesis, Triage, and Radiolabeling.

Cyclooxygenase 2 (COX-2) is an inducible enzyme responsible for the conversion of arachidonic acid into the prostaglandins, PGG2 and PGH2. Expression of this enzyme increases in inflammation. Therefore, the development of probes for imaging COX-2 with positron emission tomography (PET) has gained interest because they could be useful for the study of inflammation in vivo, and for aiding anti-inflammatory drug development targeting COX-2. Nonetheless, effective PET radioligands are still lacking. We synthesized eleven COX-2 inhibitors based on a 2(4-methylsulfonylphenyl)pyrimidine core from which we selected three as prospective PET radioligands based on desirable factors, such as high inhibitory potency for COX-2, very low inhibitory potency for COX-1, moderate lipophilicity, and amenability to labeling with a positronemitter. These inhibitors, namely 6-methoxy-2-(4-(methylsulfonyl)phenyl-N-(thiophen-2ylmethyl)pyrimidin-4-amine (17), the 6-fluoromethyl analogue (20), and the 6-(2-fluoroethoxy) analogue (27), were labeled in useful yields and with high molar activities by treating the 6-hydroxy analogue (26) with [11C]iodomethane, [18F]2-fluorobromoethane, and [d2-18F]fluorobromomethane, respectively. [11C]17, [18F]20, and [d2-18F]27 were readily purified with HPLC and formulated for intravenous injection. These methods allow these radioligands to be produced for comparative evaluation as PET radioligands for measuring COX-2 in healthy rhesus monkey and for assessing their abilities to detect inflammation.

3-Substituted 1,5-Diaryl-1 H-1,2,4-triazoles as Prospective PET Radioligands for Imaging Brain COX-1 in Monkey. Part 2: Selection and Evaluation of [11C]PS13 for Quantitative Imaging.

In our preceding paper (Part 1), we identified three 1,5-bis-diaryl-1,2,4-triazole-based compounds that merited evaluation as potential positron emission tomography (PET) radioligands for selectively imaging cyclooxygenase-1 (COX-1) in monkey and human brain, namely, 1,5-bis(4-methoxyphenyl)-3-(alkoxy)-1 H-1,2,4-triazoles bearing a 3-methoxy (PS1), a 3-(2,2,2-trifluoroethoxy) (PS13), or a 3-fluoromethoxy substituent (PS2). PS1 and PS13 were labeled from phenol precursors by O-11C-methylation with [11C]iodomethane and PS2 by O-18F-fluoroalkylation with [2H2,18F]fluorobromomethane. Here, we evaluated these PET radioligands in monkey. All three radioligands gave moderately high uptake in brain, although [2H2,18F]PS2 also showed undesirable radioactivity uptake in skull. [11C]PS13 was selected for further evaluation, mainly based on more favorable brain kinetics than [11C]PS1. Pharmacological preblock experiments showed that about 55% of the radioactivity uptake in brain was specifically bound to COX-1. An index of enzyme density, VT, was well identified from serial brain scans and from the concentrations of parent radioligand in arterial plasma. In addition, VT values were stable within 80 min, suggesting that brain uptake was not contaminated by radiometabolites. [11C]PS13 successfully images and quantifies COX-1 in monkey brain, and merits further investigation for imaging COX-1 in monkey models of neuroinflammation and in healthy human subjects.

3-Substituted 1,5-Diaryl-1 H-1,2,4-triazoles as Prospective PET Radioligands for Imaging Brain COX-1 in Monkey. Part 1: Synthesis and Pharmacology.

Cyclooxygenase-1 (COX-1) is a key enzyme in the biosynthesis of proinflammatory thromboxanes and prostaglandins and is found in glial and neuronal cells within brain. COX-1 expression is implicated in numerous neuroinflammatory states. We aim to find a direct-acting positron emission tomography (PET) radioligand for imaging COX-1 in human brain as a potential biomarker of neuroinflammation and for serving as a tool in drug development. Seventeen 3-substituted 1,5-diaryl-1 H-1,2,4-triazoles were prepared as prospective COX-1 PET radioligands. From this set, three 1,5-(4-methoxyphenyl)-1 H-1,2,4-triazoles, carrying a 3-methoxy (5), 3-(1,1,1-trifluoroethoxy) (20), or 3-fluoromethoxy substituent (6), were selected for radioligand development, based mainly on their high affinities and selectivities for inhibiting human COX-1, absence of carboxyl group, moderate computed lipophilicities, and scope for radiolabeling with carbon-11 ( t1/2 = 20.4 min) or fluorine-18 ( t1/2 = 109.8 min). Methods were developed for producing [11C]5, [11C]20, and [ d2-18F]6 from hydroxy precursors in a form ready for intravenous injection for prospective evaluation in monkey with PET.

Distinct patterns of increased translocator protein in posterior cortical atrophy and amnestic Alzheimer's disease.

We sought to determine whether patients with posterior cortical atrophy (PCA) demonstrate a pattern of binding to translocator protein 18 kDa, a marker of microglial activation, that is distinct from that in patients with amnestic presentation of Alzheimer's disease (AD). Eleven PCA patients, 11 amnestic AD patients, and 15 age-matched controls underwent positron emission tomography with 11C-PBR28 to measure translocator protein 18 kDa. PCA patients showed greater 11C-PBR28 binding than controls in occipital, posterior parietal, and temporal regions. In contrast, amnestic AD patients showed greater 11C-PBR28 binding in inferior and medial temporal cortex. Increased 11C-PBR28 binding overlapped with reduced cortical volume for both PCA and amnestic AD patients, and with areas of reduced glucose metabolism in PCA patients. While both patient groups showed diffuse amyloid binding, PCA patients showed greater binding than amnestic AD patients in bilateral occipital cortex. These results suggest that microglial activation is closely associated with neurodegeneration across different subtypes of AD.

The 5-HT1A Receptor PET Radioligand 11C-CUMI-101 Has Significant Binding to α1-Adrenoceptors in Human Cerebellum, Limiting Its Use as a Reference Region.

Prazosin, a potent and selective α1-adrenoceptor antagonist, displaces 25% of 11C-CUMI-101 ([O-methyl-11C]2-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)dione) binding in monkey cerebellum. We sought to estimate the percentage contamination of 11C-CUMI-101 binding to α1-adrenoceptors in human cerebellum under in vivo conditions. In vitro receptor-binding techniques were used to measure α1-adrenoceptor density and the affinity of CUMI-101 for these receptors in human, monkey, and rat cerebellum. METHODS: Binding potential (maximum number of binding sites × affinity [(1/dissociation constant]) was determined using in vitro homogenate binding assays in human, monkey, and rat cerebellum. 3H-prazosin was used to determine the maximum number of binding sites, as well as the dissociation constant of 3H-prazosin and the inhibition constant of CUMI-101. RESULTS: α1-adrenoceptor density and the affinity of CUMI-101 for these receptors were similar across species. Cerebellar binding potentials were 3.7 for humans, 2.3 for monkeys, and 3.4 for rats. CONCLUSION: Reasoning by analogy, 25% of 11C-CUMI-101 uptake in human cerebellum reflects binding to α1-adrenoceptors, suggesting that the cerebellum is of limited usefulness as a reference tissue for quantification in human studies.

Translocator protein ligands based on N-methyl-(quinolin-4-yl)oxypropanamides with properties suitable for PET radioligand development.

Modifications to an N-methyl-(quinolin-4-yl)oxypropanamide scaffold were explored to discover leads for developing new radioligands for PET imaging of brain TSPO (translocator protein), a biomarker of neuroinflammation. Whereas contraction of the quinolinyl portion of the scaffold or cyclization of the tertiary amido group abolished high TSPO affinity, insertion of an extra nitrogen atom into the 2-arylquinolinyl portion was effective in retaining sub-nanomolar affinity for rat TSPO, while also decreasing lipophilicity to within the moderate range deemed preferable for a PET radioligand. Replacement of a phenyl group on the amido nitrogen with an isopropyl group was similarly effective. Among others, compound 20 (N-methyl-N-phenyl-2-[2-(pyridin-2-yl)-1,8-naphthyridin-4-yloxy]propanamide) appears especially appealing for PET radioligand development, based on high selectivity and high affinity (Ki = 0.5 nM) for rat TSPO, moderate lipophilicity (logD = 2.48), and demonstrated amenability to labeling with carbon-11.

(11)C-PBR28 binding to translocator protein increases with progression of Alzheimer's disease.

This longitudinal study sought to determine whether the 18 kDa translocator protein (TSPO), a marker of neuroinflammation, increases over time in Alzheimer's disease. Positron emission tomography imaging with the TSPO radioligand (11)C-PBR28 was performed at baseline and after a median follow-up of 2.7 years in 14 amyloid-positive patients and 8 amyloid-negative controls. Patients had a greater increase in TSPO binding than controls in inferior parietal lobule, precuneus, occipital cortex, hippocampus, entorhinal cortex, and combined middle and inferior temporal cortex. TSPO binding in temporoparietal regions increased from 3.9% to 6.3% per annum in patients, but ranged from -0.5% to 1% per annum in controls. The change in TSPO binding correlated with cognitive worsening on clinical dementia rating scale-sum of boxes and reduced cortical volume. The annual rate of increased TSPO binding in temporoparietal regions was about 5-fold higher in patients with clinical progression (n = 9) compared with those who did not progress (n = 5). TSPO may serve as a biomarker of Alzheimer's progression and response to anti-inflammatory therapies.

[carbonyl-11C]4-Fluoro-N-methyl-N-(4-(6-(methylamino)pyrimidin-4-yl)thiazol-2-yl)benzamide ([11C]FIMX) is an effective radioligand for PET imaging of metabotropic glutamate receptor 1 (mGluR1) in monkey brain.

INTRODUCTION: Metabotropic glutamate subtype receptor 1 (mGluR1) is implicated in several neuropsychiatric disorders and is a target for drug development. [(18)F]FIMX ([(18)F]4-fluoro--N-methyl-N--(4-(6-(methylamino)pyrimidin-4-yl)thiazol-2-yl)benzamide) is an effective radioligand for imaging brain mGluR1 with PET. A similarly effective radioligand with a shorter half-life would usefully allow PET studies of mGluR1 at baseline and after pharmacological or other challenge on the same day. Here we describe the preparation of [(11)C]FIMX for evaluation in monkey with PET. METHODS: [(11)C]FIMX was prepared via Pd-promoted carbonylation of 1-fluoro-4-iodobenzene with [(11)C]carbon monoxide, aminolysis of the [(11)C]acyl-palladium complex with the requisite Boc-protected amine, and deprotection with HCl in THF. PET scans of [(11)C]FIMX injected into a monkey were performed at baseline and after preblock of mGluR1 with measurement of the arterial input function. RESULTS: The radiosynthesis required 42 min and gave [(11)C]FIMX in about 5% overall decay-corrected radiochemical yield and with a specific activity of about 100 GBq/µmol. PET in rhesus monkey at baseline showed that radioactivity peaked high in receptor-rich cerebellum and much lower in receptor-poor occipital cortex. Radioactivity in cerebellum declined to 32% of peak at 85 min. VT at baseline appeared stable in all brain regions after 60 min. Under mGluR1 pre-blocked condition, radioactivity uptake in all regions declined more rapidly to a low level. Receptor pre-block reduced VT from 13.0 to 1.5 in cerebellum and from 2.9 to 1.4 in occipital cortex. CONCLUSION: [(11)C]FIMX is an effective radioligand for imaging mGluR1 in monkey with PET.

Neuroinflammation in Temporal Lobe Epilepsy Measured Using Positron Emission Tomographic Imaging of Translocator Protein.

IMPORTANCE: Neuroinflammation may play a role in epilepsy. Translocator protein 18 kDa (TSPO), a biomarker of neuroinflammation, is overexpressed on activated microglia and reactive astrocytes. A preliminary positron emission tomographic (PET) imaging study using carbon 11 ([11C])-labeled PBR28 in patients with temporal lobe epilepsy (TLE) found increased TSPO ipsilateral to seizure foci. Full quantitation of TSPO in vivo is needed to detect widespread inflammation in the epileptic brain. OBJECTIVES: To determine whether patients with TLE have widespread TSPO overexpression using [11C]PBR28 PET imaging, and to replicate relative ipsilateral TSPO increases in patients with TLE using [11C]PBR28 and another TSPO radioligand, [11C]DPA-713. DESIGN, SETTING, AND PARTICIPANTS: In a cohort study from March 2009 through September 2013 at the Clinical Epilepsy Section of the National Institute of Neurological Disorders and Stroke, participants underwent brain PET and a subset had concurrent arterial sampling. Twenty-three patients with TLE and 11 age-matched controls were scanned with [11C]PBR28, and 8 patients and 7 controls were scanned with [11C]DPA-713. Patients with TLE had unilateral temporal seizure foci based on ictal electroencephalography and structural magnetic resonance imaging. Participants with homozygous low-affinity TSPO binding were excluded. MAIN OUTCOMES AND MEASURES: The [11C]PBR28 distribution volume (VT) corrected for free fraction (fP) was measured in patients with TLE and controls using FreeSurfer software and T1-weighted magnetic resonance imaging for anatomical localization of bilateral temporal and extratemporal regions. Side-to-side asymmetry in patients with TLE was calculated as the ratio of ipsilateral to contralateral [11C]PBR28 and [11C]DPA-713 standardized uptake values from temporal regions. RESULTS: The [11C]PBR28 VT to fp ratio was higher in patients with TLE than in controls for all ipsilateral temporal regions (27%-42%; P < .05) and in contralateral hippocampus, amygdala, and temporal pole (approximately 30%-32%; P < .05). Individually, 12 patients, 10 with mesial temporal sclerosis, had asymmetrically increased hippocampal [11C]PBR28 uptake exceeding the 95% confidence interval of the controls. Binding of [11C]PBR28 was increased significantly in thalamus. Relative [11C]PBR28 and [11C]DPA-713 uptakes were higher ipsilateral than contralateral to seizure foci in patients with TLE ([11C]PBR28: 2%-6%; [11C]DPA-713: 4%-9%). Asymmetry of [11C]DPA-713 was greater than that of [11C]PBR28 (F = 29.4; P = .001). CONCLUSIONS AND RELEVANCE: Binding of TSPO is increased both ipsilateral and contralateral to seizure foci in patients with TLE, suggesting ongoing inflammation. Anti-inflammatory therapy may play a role in treating drug-resistant epilepsy.

Synthesis and evaluation of translocator 18 kDa protein (TSPO) positron emission tomography (PET) radioligands with low binding sensitivity to human single nucleotide polymorphism rs6971.

The imaging of translocator 18 kDa protein (TSPO) in living human brain with radioligands by positron emission tomography (PET) has become an important means for the study of neuroinflammatory conditions occurring in several neuropsychiatric disorders. The widely used prototypical PET radioligand [(11)C](R)-PK 11195 ([(11)C](R)-1; [N-methyl-(11)C](R)-N-sec-butyl-1-(2-chlorophenyl)-N-methylisoquinoline-3-carboxamide) gives a low PET signal and is difficult to quantify, whereas later generation radioligands have binding sensitivity to a human single nucleotide polymorphism (SNP) rs6971, which imposes limitations on their utility for comparative quantitative PET studies of normal and diseased subjects. Recently, azaisosteres of 1 have been developed with improved drug-like properties, including enhanced TSPO affinity accompanied by moderated lipophilicity. Here we selected three of these new ligands (7-9) for labeling with carbon-11 and for evaluation in monkey as candidate PET radioligands for imaging brain TSPO. Each radioligand was readily prepared by (11)C-methylation of an N-desmethyl precursor and was found to give a high proportion of TSPO-specific binding in monkey brain. One of these radioligands, [(11)C]7, the direct 4-azaisostere of 1, presents many radioligand properties that are superior to those reported for [(11)C]1, including higher affinity, lower lipophilicity, and stable quantifiable PET signal. Importantly, 7 was also found to show very low sensitivity to the human SNP rs6971 in vitro. Therefore, [(11)C]7 now warrants evaluation in human subjects with PET to assess its utility for imaging TSPO in human brain, irrespective of subject genotype.

Development of N-methyl-(2-arylquinolin-4-yl)oxypropanamides as leads to PET radioligands for translocator protein (18 kDa).

Translocator protein (18 kDa), known as TSPO, is a recognized biomarker of neuroinflammation. Radioligands with PET accurately quantify TSPO in neuroinflammatory conditions. However, the existence of three human TSPO genotypes that show differential affinity to almost all useful TSPO PET radioligands hampers such studies. There is an unmet need for genotype-insensitive, high-affinity, and moderately lipophilic TSPO ligands that may serve as leads for PET radioligand development. To address this need, we varied the known high-affinity TSPO ligand (l)-N,N-diethyl-2-methyl-3-(2-phenylquinolin-4-yl)propanamide in its aryl scaffold, side chain tether, and pendant substituted amido group while retaining an N-methyl group as a site for labeling with carbon-11. From this effort, oxygen-tethered N-methyl-aryloxypropanamides emerged as new high-affinity TSPO ligands with attenuated lipophilicity, including one example with attractive properties for PET radioligand development, namely N-methyl-N-phenyl-2-{[2-(pyridin-2-yl)quinolin-4-yl]oxy}propanamide (22a; rat Ki=0.10 nM; human TSPO genotypes Ki=1.4 nM; clogD=4.18).

(11)C-CUMI-101, a PET radioligand, behaves as a serotonin 1A receptor antagonist and also binds to α(1) adrenoceptors in brain.

UNLABELLED: The PET radioligand (11)C-CUMI-101 was previously suggested as a putative agonist radioligand for the serotonin 1A (5-hydroxytryptamine 1A [5-HT1A]) receptor in recombinant cells expressing human 5-HT1A receptor. However, a recent study showed that CUMI-101 behaved as a potent 5-HT1A receptor antagonist in rat brain. CUMI-101 also has moderate affinity (Ki = 6.75 nM) for α1 adrenoceptors measured in vitro. The current study examined the functional properties and selectivity of CUMI-101, both in vitro and in vivo. METHODS: The functional assay was performed using (35)S-GTPγS (GTP is guanosine triphosphate) in primate brains. The cross-reactivity of CUMI-101 with α1 adrenoceptors was performed using in vitro radioligand binding studies in rat, monkey, and human brains as well as in vivo PET imaging in mouse, rat, and monkey brains. RESULTS: CUMI-101 did not stimulate (35)S-GTPγS binding in primate brain, in contrast to 8-OH-DPAT, a potent 5-HT1A receptor agonist. Instead, CUMI-101 behaved as a potent 5-HT1A receptor antagonist by dose-dependently inhibiting 8-OH-DPAT-stimulated (35)S-GTPγS binding. Both in vitro and in vivo studies showed that CUMI-101 had significant α1 adrenoceptor cross-reactivity. On average, across all 3 species examined, cross-reactivity was highest in the thalamus (>45%) and lowest in the neocortex and cerebellum (<10%). PET imaging further confirmed that only preblocking with WAY-100635 plus prazosin decreased (11)C-CUMI-101 brain uptake to that of self-block. CONCLUSION: CUMI-101 behaves as a 5-HT1A receptor antagonist in primate brain, with significant, regional-dependent α1 adrenoceptor cross-reactivity, limiting its potential use as a PET radioligand in humans.

In vitro and in vivo evaluation of (11)C-SD5024, a novel PET radioligand for human brain imaging of cannabinoid CB1 receptors.

We recently developed a novel cannabinoid subtype-1 (CB1) receptor radioligand (11)C-SD5024 for brain imaging. This study aimed to evaluate (11)C-SD5024 both in vitro and in vivo and compare it with the other CB1 receptor ligands previously used in humans, i.e., (11)C-MePPEP, (11)C-OMAR, (18)F-MK-9470, and (18)F-FMPEP-d2. In vitro experiments were performed to measure dissociation constant (Ki) in the human brain and to measure the lipophilicity of the five CB1 receptor ligands listed above. In vivo specific binding in monkeys was measured by comparing total distribution volume (VT) at baseline and after full receptor blockade. The kinetics of (11)C-SD5024 in humans were evaluated in seven healthy subjects with compartmental modeling. SD5024 showed Ki=0.47nM, which was at an intermediate level among the five CB1 receptor ligands. Lipophilicity (LogD7.4) was 3.79, which is appropriate for brain imaging. Monkey scans showed high proportion of specific binding: ~80% of VT. In humans, (11)C-SD5024 showed peak brain uptake of 1.5-3 standardized uptake value, which was slightly higher than that of (11)C-OMAR and (18)F-MK-9470. One-compartment model showed good fitting, consistent with the vast majority of brain uptake being specific binding found in the monkey. Regional VT values were consistent with known distribution of CB1 receptors. VT calculated from 80 and 120min of scan data was strongly correlated (R(2)=0.97), indicating that 80min provided adequate information for quantitation and that the influence of radiometabolites was low. Intersubject variability for VT of (11)C-SD5024 was 22%, which was low among the five radioligands and indicated precise measurement. In conclusion, (11)C-SD5024 has appropriate affinity and lipophilicity, high specific binding, moderate brain uptake, and provides good precision to measure the binding. The results suggest that (11)C-SD5024 is slightly better than or equivalent to (11)C-OMAR and that both are suitable for clinical studies, especially those that involve two scans in one day.

In vivo radioligand binding to translocator protein correlates with severity of Alzheimer's disease.

Neuroinflammation is a pathological hallmark of Alzheimer's disease, but its role in cognitive impairment and its course of development during the disease are largely unknown. To address these unknowns, we used positron emission tomography with (11)C-PBR28 to measure translocator protein 18 kDa (TSPO), a putative biomarker for inflammation. Patients with Alzheimer's disease, patients with mild cognitive impairment and older control subjects were also scanned with (11)C-Pittsburgh Compound B to measure amyloid burden. Twenty-nine amyloid-positive patients (19 Alzheimer's, 10 mild cognitive impairment) and 13 amyloid-negative control subjects were studied. The primary goal of this study was to determine whether TSPO binding is elevated in patients with Alzheimer's disease, and the secondary goal was to determine whether TSPO binding correlates with neuropsychological measures, grey matter volume, (11)C-Pittsburgh Compound B binding, or age of onset. Patients with Alzheimer's disease, but not those with mild cognitive impairment, had greater (11)C-PBR28 binding in cortical brain regions than controls. The largest differences were seen in the parietal and temporal cortices, with no difference in subcortical regions or cerebellum. (11)C-PBR28 binding inversely correlated with performance on Folstein Mini-Mental State Examination, Clinical Dementia Rating Scale Sum of Boxes, Logical Memory Immediate (Wechsler Memory Scale Third Edition), Trail Making part B and Block Design (Wechsler Adult Intelligence Scale Third Edition) tasks, with the largest correlations observed in the inferior parietal lobule. (11)C-PBR28 binding also inversely correlated with grey matter volume. Early-onset (<65 years) patients had greater (11)C-PBR28 binding than late-onset patients, and in parietal cortex and striatum (11)C-PBR28 binding correlated with lower age of onset. Partial volume corrected and uncorrected results were generally in agreement; however, the correlation between (11)C-PBR28 and (11)C-Pittsburgh Compound B binding was seen only after partial volume correction. The results suggest that neuroinflammation, indicated by increased (11)C-PBR28 binding to TSPO, occurs after conversion of mild cognitive impairment to Alzheimer's disease and worsens with disease progression. Greater inflammation may contribute to the precipitous disease course typically seen in early-onset patients. (11)C-PBR28 may be useful in longitudinal studies to mark the conversion from mild cognitive impairment or to assess response to experimental treatments of Alzheimer's disease.

Is metabotropic glutamate receptor 5 upregulated in prefrontal cortex in fragile X syndrome?

BACKGROUND: Fragile X syndrome (FXS) is a common inherited form of intellectual disability caused by loss of function of the fragile X mental retardation protein. Recent animal studies suggest that upregulated downstream signaling by metabotropic glutamate receptor 5 (mGluR5) might be an important mechanism for cognitive and behavioral abnormalities associated with FXS. However, mGluR5 density in human FXS remains unknown. METHODS: Receptor binding and protein expression were measured in the postmortem prefrontal cortex of 14 FXS patients or carriers and 17 age- and sex-matched control subjects without neurological disorders. In-vitro binding assays were performed using [3H]-labeled 3-methoxy-5-pyridin-2-ylethynylpyridine (MPEPy), a selective and high-affinity negative allosteric modulator of mGluR5, to measure receptor density and the radioligand's dissociation constant, which is inversely proportional to affinity. Immunoblotting was also performed, to measure mGluR5 protein expression. RESULTS: The mGluR5 density increased with marginal significance (+16%; P = 0.058) in the prefrontal cortex of FXS patients or carriers compared with matched healthy controls. No significant change in dissociation constant (-4%; P = 0.293) was observed. Immunoblotting found a significant elevation (+32%; P = 0.048) in mGluR5 protein expression. CONCLUSIONS: Both mGluR5 binding density and protein expression were increased in the brains of FXS patients or carriers, but only expression was significantly different, which could be because of the small sample size and moderate variability. Another important caveat is that the effects of psychotropic medications on mGluR5 expression are largely unknown. Future in-vivo measurement of mGluR5 with positron emission tomography might characterize the role of this receptor in the pathophysiology of FXS and facilitate trials of mGluR5-oriented treatments for this disorder.

A genetic polymorphism for translocator protein 18 kDa affects both in vitro and in vivo radioligand binding in human brain to this putative biomarker of neuroinflammation.

Second-generation radioligands for translocator protein (TSPO), an inflammation marker, are confounded by the codominant rs6971 polymorphism that affects binding affinity. The resulting three groups are homozygous for high-affinity state (HH), homozygous for low-affinity state (LL), or heterozygous (HL). We tested if in vitro binding to leukocytes distinguished TSPO genotypes and if genotype could affect clinical studies using the TSPO radioligand [(11)C]PBR28. In vitro binding to leukocytes and [(11)C]PBR28 brain imaging were performed in 27 human subjects with known TSPO genotype. Specific [(3)H]PBR28 binding was measured in prefrontal cortex of 45 schizophrenia patients and 47 controls. Leukocyte binding to PBR28 predicted genotype in all subjects. Brain uptake was ∼40% higher in HH than HL subjects. Specific [(3)H]PBR28 binding in LL controls was negligible, while HH controls had ∼80% higher binding than HL controls. After excluding LL subjects, specific binding was 16% greater in schizophrenia patients than controls. This difference was insignificant by itself (P=0.085), but was significant after correcting for TSPO genotype (P=0.011). Our results show that TSPO genotype influences PBR28 binding in vitro and in vivo. Correcting for this genotype increased statistical power in our postmortem study and is recommended for in vivo positron emission tomography studies.

Binding of a tritiated inverse agonist to cannabinoid CB1 receptors is increased in patients with schizophrenia.

This study sought to determine whether cannabinoid-1 (CB(1)) receptor binding was altered in the postmortem dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia (schizophrenia; n=47) compared to controls (n=43). The CB(1) receptor inverse agonist radioligand [(3)H]MePPEP was used to measure specific binding to CB(1) receptors. The specific binding of [(3)H]MePPEP to CB(1) receptors was 20% higher in patients with schizophrenia than in controls. Power analyses suggested that 53 subjects per group would be needed to detect a similar difference in vivo with positron emission tomography (PET) and the structurally related inverse agonist radioligand [(18)F]FMPEP-d(2) (80% statistical power, p<0.05).

Radiosynthesis and evaluation of an (18)F-labeled positron emission tomography (PET) radioligand for brain histamine subtype-3 receptors based on a nonimidazole 2-aminoethylbenzofuran chemotype.

A known chemotype of H(3) receptor ligand was explored for development of a radioligand for imaging brain histamine subtype 3 (H(3)) receptors in vivo with positron emission tomography (PET), namely nonimidazole 2-aminoethylbenzofurans, represented by the compound (R)-(2-(2-(2-methylpyrrolidin-1-yl)ethyl)benzofuran-5-yl)(4-fluorophenyl)methanone (9). Compound 9 was labeled with fluorine-18 (t(1/2) = 109.7 min) in high specific activity by treating the prepared nitro analogue (12) with cyclotron-produced [(18)F]fluoride ion. [(18)F]9 was studied with PET in mouse and in monkey after intravenous injection. [(18)F]9 showed favorable properties as a candidate PET radioligand, including moderately high brain uptake with a high proportion of H(3) receptor-specific signal in the absence of radiodefluorination. The nitro compound 12 was found to have even higher H(3) receptor affinity, indicating the potential of this chemotype for the development of further promising PET radioligands.

On quantitative relationships between drug-like compound lipophilicity and plasma free fraction in monkey and human.

Drug interactions with plasma proteins influence their pharmacokinetics and pharmacodynamics. We aimed to test whether a strong quantitative relationship exists between plasma free fraction (f(P) ) and lipophilicity for low molecular weight nonacidic drug-like compounds. We measured the n-octanol-buffer distribution coefficients at pH 7.4 ((m) logD) of 18 diverse radiotracers (<470 Da) used for brain imaging with positron emission tomography in vivo. Lipophilicities were also computed as (c) logD with two software packages. The f(P) values for monkeys and humans were determined by ultrafiltration and transformed into m logD(pr/pl) values representing the log(10) of the within phase partition of the radiotracers between plasma proteins and remaining plasma. (m) logD(pr/pl) correlated strongly with (m) logD for human ((m) logD(pr/pl) = 0.733(m) logD-0.780, r(2) = 0.74) and monkey ((m) logD(pr/pl) = 0.780(m) logD-1.15, r(2) = 0.83), but less strongly with (c) logD. These relationships were significantly different between species (P = 0.006). Removal of eight fluorinated compounds from the datasets raised r(2) to 0.81 and 0.91 for humans and monkeys, respectively. For the tested compounds, we conclude that n-octanol-buffer (pH 7.4) distribution strongly models that between plasma proteins and remaining plasma and moreover that (m) logD accounts for over 74% of compound (m) logD(pr/pl) and is a strong determinant of f(P).