MRI Signal Intensity and Parkinsonism in Manganese-Exposed Workers.

OBJECTIVE: T1-weighted brain magnetic resonance imaging (MRI) of the basal ganglia provides a noninvasive measure of manganese (Mn) exposure, and may also represent a biomarker for clinical neurotoxicity. METHODS: We acquired T1-weighted MRI scans in 27 Mn-exposed welders, 12 other Mn-exposed workers, and 29 nonexposed participants. T1-weighted intensity indices were calculated for four basal ganglia regions. Cumulative Mn exposure was estimated from work history data. Participants were examined using the Unified Parkinson's Disease Rating Scale motor subsection 3 (UPDRS3). RESULTS: We observed a positive dose-response association between cumulative Mn exposure and the pallidal index (PI) (ß = 2.33; 95% confidence interval [CI], 0.93 to 3.74). There was a positive relationship between the PI and UPDRS3 (ß = 0.15; 95% CI, 0.03 to 0.27). CONCLUSION: The T1-weighted pallidal signal is associated with occupational Mn exposure and severity of parkinsonism.

[18F]FDOPA positron emission tomography in manganese-exposed workers.

Occupational manganese (Mn) exposure is associated with the development of parkinsonism; however, the mechanism of neurotoxicity is unknown. Brain positron emission tomography (PET) imaging provides a non-invasive method of assessing dopamineric neuronal function. 6-[18F]fluoro-L-DOPA (FDOPA) PET reflects in-vivo nigrostriatal function, but results in Mn exposure are conflicting. The objective of this study was to investigate the association between Mn exposure secondary to occupational welding, FDOPA striatal uptake, and clinical parkinsonism as measured by Unified Parkinson Disease Rating Scale motor subscore 3 (UPDRS3) scores. FDOPA PET scans were acquired on 72 subjects (27 Mn-exposed welders, 14 other Mn-exposed workers, and 31 non-exposed subjects). We estimated cumulative welding exposure from detailed work histories, and a movement disorders specialist examined all subjects. Striatal volumes of interest were identified on aligned magnetic resonance imaging (MRI) for each subject. Specific striatal FDOPA uptake was calculated with a graphical analysis method. We used linear regression while adjusting for age to assess the association between welding exposure and FDOPA uptake in the caudate, anterior putamen, and posterior putamen. Compared to the non-exposed subjects, mean caudate FDOPA uptake was 0.0014min-1 (95% confidence interval [CI] 0.0008, 0.0020) lower in Mn-exposed welders and 0.0012min-1 (95% CI 0.0005, 0.0019) lower in other Mn-exposed workers (both p≤0.001). There was no clear dose-response association between caudate FDOPA uptake and Mn exposure or UPDRS3 scores. Mn-exposed welders and workers demonstrated lower caudate FDOPA uptake, indicating pre-synaptic dopaminergic dysfunction in Mn-exposed subjects that was not associated with clinical parkinsonism.

Synthesis, resolution, and in vitro evaluation of three vesicular acetylcholine transporter ligands and evaluation of the lead fluorine-18 radioligand in a nonhuman primate.

The vesicular acetylcholine transporter (VAChT) is a reliable biomarker for assessing cholinergic dysfunction associated with dementia. We recently reported three new potent and selective carbon-11 labeled VAChT radiotracers. Herein, we report the resolution with a Chiralcel OD column of three additional fluorine containing VAChT ligands in which a fluoroethoxy or fluoroethylamino moiety was substituted for the methoxy group. An in vitro competitive binding assay showed that (-)-7 had high potency for VAChT (Ki-VAChT = 0.31 ± 0.03 nM) and excellent selectivity for VAChT versus σ receptors (Ki-σ1 = 1870 ± 250 nM, Ki-σ2 = 5480 ± 140 nM). Three different radiolabeling approaches were explored; the radiosynthesis of (-)-[18F]7 was successfully accomplished via a stepwise two-pot, three-step method with moderate yield (11 ± 2%) and high radiochemical purity (>98%). PET imaging studies in a nonhuman primate indicated that (-)-[18F]7 rapidly entered the brain and accumulated in the VAChT-enriched striatum. The uptake of (-)-[18F]7 in the target striatal area peaked at 10 min and displayed improved clearance kinetics compared to the VAChT tracer [18F]VAT, which has been approved by the Food and Drug Administration (FDA) for first-in-man studies. These studies justify further investigation of (-)-[18F]7 and exploration of the structure-activity relationships of these fluoroethoxy and fluoroethylamino analogs.

Chiral resolution of serial potent and selective σ1 ligands and biological evaluation of (-)-[18F]TZ3108 in rodent and the nonhuman primate brain.

Twelve optically pure enantiomers were obtained using either crystallization or chiral high performance liquid chromatography (HPLC) separation methodologies to resolve six racemic sigma-1 (σ1) receptor ligands. The in vitro binding affinities of each enantiomer for σ1, σ2 receptors and vesicular acetylcholine transporter (VAChT) were determined. Out of the 12 optically pure enantiomers, five displayed very high affinities for σ1 (Ki<2nM) and high selectivity for σ1 versus σ2 and VAChT (>100-fold). The minus enantiomer, (-)-14a ((-)-TZ3108) (Ki-σ1=1.8±0.4nM, Ki-σ2=6960±810nM, Ki-VAChT=980±87nM), was chosen for radiolabeling and further in vivo evaluation in rodents and nonhuman primates (NHPs). A biodistribution study in Sprague Dawley rats showed brain uptake (%ID/gram) of (-)-[18F]TZ3108 reached 1.285±0.062 at 5min and 0.802±0.129 at 120min. NHP microPET imaging studies revealed higher brain uptake of (-)-[18F]TZ3108 and more favorable pharmacokinetics compared to its racemic counterpart. Pretreatment of the animal using two structurally different σ1 ligands significantly decreased accumulation of (-)-[18F]TZ3108 in the brain. Together, our in vivo evaluation results suggest that (-)-[18F]TZ3108 is a promising positron emission tomography (PET) tracer for quantifying σ1 receptor in the brain.

Absorbed radiation dosimetry of the D3-specific PET radioligand [18F]FluorTriopride estimated using rodent and nonhuman primate.

[18F]FluorTriopride ([18F]FTP) is a dopamine D3-receptor preferring radioligand with potential for investigation of neuropsychiatric disorders including Parkinson disease, dystonia and schizophrenia. Here we estimate human radiation dosimetry for [18F]FTP based on the ex-vivo biodistribution in rodents and in vivo distribution in nonhuman primates. Biodistribution data were generated using male and female Sprague-Dawley rats injected with ~370 KBq of [18F]FTP and euthanized at 5, 30, 60, 120, and 240 min. Organs of interest were dissected, weighed and assayed for radioactivity content. PET imaging studies were performed in two male and one female macaque fascicularis administered 143-190 MBq of [18F]FTP and scanned whole-body in sequential sections. Organ residence times were calculated based on organ time activity curves (TAC) created from regions of Interest. OLINDA/EXM 1.1 was used to estimate human radiation dosimetry based on scaled organ residence times. In the rodent, the highest absorbed radiation dose was the upper large intestines (0.32-0.49 mGy/MBq), with an effective dose of 0.07 mSv/MBq in males and 0.1 mSv/MBq in females. For the nonhuman primate, however, the gallbladder wall was the critical organ (1.81 mGy/MBq), and the effective dose was 0.02 mSv/MBq. The species discrepancy in dosimetry estimates for [18F]FTP based on rat and primate data can be attributed to the slower transit of tracer through the hepatobiliary track of the primate compared to the rat, which lacks a gallbladder. Out findings demonstrate that the nonhuman primate model is more appropriate model for estimating human absorbed radiation dosimetry when hepatobiliary excretion plays a major role in radiotracer elimination.

Synthesis and biological characterization of a promising F-18 PET tracer for vesicular acetylcholine transporter.

Nine fluorine-containing vesicular acetylcholine transporter (VAChT) inhibitors were synthesized and screened as potential PET tracers for imaging the VAChT. Compound 18a was one of the most promising carbonyl-containing benzovesamicol analogs; the minus enantiomer, (-)-18a displayed high potency (VAChT Ki=0.59 ± 0.06 nM) and high selectivity for VAChT versus σ receptors (>10,000-fold). The radiosynthesis of (-)-[(18)F]18a was accomplished by a two-step procedure with 30-40% radiochemical yield. Preliminary biodistribution studies of (-)-[(18)F]18a in adult male Sprague-Dawley rats at 5, 30, 60 and 120 min post-injection (p.i.) were promising. The total brain uptake of (-)-[(18)F]18a was 0.684%ID/g at 5 min p.i. and by 120 min p.i. slowly washed out to 0.409 %ID/g; evaluation of regional brain uptake showed stable levels of ∼0.800 %ID/g from 5 to 120 min p.i in the VAChT-enriched striatal tissue of rats, indicating the tracer had crossed the blood brain barrier and was retained in the striatum. Subsequent microPET brain imaging studies of (-)-[(18)F]18a in nonhuman primates (NHPs) showed high striatal accumulation in the NHP brain; the standardized uptake value (SUV) for striatum reached a maximum value of 5.1 at 15 min p.i. The time-activity curve for the target striatal region displayed a slow and gradual decreasing trend 15 min after injection, while clearance of the radioactivity from the cerebellar reference region was much more rapid. Pretreatment of NHPs with 0.25mg/kg of the VAChT inhibitor (-)-vesamicol resulted in a ∼90% decrease of striatal uptake compared to baseline studies. HPLC metabolite analysis of NHP plasma revealed that (-)-[(18)F]18a had a good in vivo stability. Together, these preliminary results suggest (-)-[(18)F]18a is a promising PET tracer candidate for imaging VAChT in the brain of living subjects.

Neuroimaging Analysis of the Dopamine Basis for Apathetic Behaviors in an MPTP-Lesioned Primate Model.

Apathy commonly occurs in Parkinson disease (PD) patients; however, the role of dopamine in the pathophysiology of apathy remains elusive. We previously demonstrated that dopaminergic dysfunction within the ventral tegmental area (VTA)-nucleus accumbens (NAcc) pathway contributes to the manifestation of apathetic behaviors in monkeys treated with the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We now extend these studies to identify dopaminergic dysfunction in cortical regions that correlate with development of apathetic behaviors. Specifically, we measured the effects of MPTP on monkeys' willingness to attempt goal directed behaviors, which is distinct from their ability to perform tasks. A total of 16 monkeys had baseline magnetic resonance imaging (MRI) and positron emission tomography (PET), using 6-[18F]fluorodopa (FD), [11C]dihydrotetrabenazine (DTBZ), and 2ß-[11C]carbomethoxy-3ß-(4-fluorophenyl)tropane (CFT). The monkeys received unilateral infusion of different doses of MPTP (0 - 0.31mg/kg) to produce a wide range of severity of motor parkinsonism. Eight weeks after MPTP, PET scans were repeated and animals were euthanized. Apathetic behavior and motor impairments were assessed blindly both pre- and post-MPTP infusion. Apathy scores were compared to in vitro and in vivo dopaminergic measures. Apathy scores increased following MPTP and correlated with PET measures of dopaminergic terminals (DTBZ or CFT) in dorsal lateral prefrontal cortex (DLPFC), ventromedial prefrontal cortex (VMPFC), and insular cortex (IC). Among all the cortical regions assessed, forward step-wise regression analyses indicated that only stereologic cell counts in VTA, and not counts in the substantia nigra (SN), predict dopamine transporter changes in IC. Our findings suggest that dopaminergic dysfunction within the VTA-IC pathway plays a role in the manifestation of apathetic behaviors in MPTP-lesioned primates.

In vitro and in vivo characterization of two C-11-labeled pet tracers for vesicular acetylcholine transporter.

PURPOSE: The vesicular acetylcholine transporter (VAChT) is a specific biomarker for imaging presynaptic cholinergic neurons. Herein, two potent and selective (11)C-labeled VAChT inhibitors were evaluated in rodents and nonhuman primates for imaging VAChT in vivo. PROCEDURES: For both (-)-[(11)C]2 and (-)-[(11)C]6, biodistribution, autoradiography, and metabolism studies were performed in male Sprague Dawley rats. Positron emission tomography (PET) brain studies with (-)-[(11)C]2 were performed in adult male cynomolgus macaques; 2 h dynamic data was acquired, and the regions of interest were drawn by co-registration of the PET images with the MRI. RESULTS: The resolved enantiomers (-)-2 and (-)-6 were very potent and selective for VAChT in vitro (K i < 5 nM for VAChT with >35-fold selectivity for VAChT vs. σ receptors); both radioligands, (-)-[(11)C]2 and (-)-[(11)C]6, demonstrated high accumulation in the VAChT-enriched striatum of rats. (-)-[(11)C]2 had a higher striatum to cerebellum ratio of 2.4-fold at 60 min; at 30 min, striatal uptake reached 0.550 ± 0.086 %ID/g. Uptake was also specific and selective; following pretreatment with (±)-2, striatal uptake of (-)-[(11)C]2 in rats at 30 min decreased by 50 %, while pretreatment with a potent sigma ligand had no significant effect on striatal uptake in rats. In addition, (-)-[(11)C]2 displayed favorable in vivo stability in rat blood and brain. PET studies of (-)-[(11)C]2 in nonhuman primates indicate that it readily crosses the blood-brain barrier (BBB) and provides clear visualization of the striatum; striatal uptake reaches the maximum at 60 min, at which time the target to nontarget ratio reached ~2-fold. CONCLUSIONS: The radioligand (-)-[(11)C]2 has high potential to be a suitable PET radioligand for imaging VAChT in the brain of living subjects.

Radiosyntheses and in vivo evaluation of carbon-11 PET tracers for PDE10A in the brain of rodent and nonhuman primate.

The radiosyntheses and in vivo evaluation of four carbon-11 labeled quinoline group-containing radioligands are reported here. Radiolabeling of [(11)C]1-4 was achieved by alkylation of their corresponding desmethyl precursors with [(11)C]CH3I. Preliminary biodistribution evaluation in Sprague-Dawley rats demonstrated that [(11)C]1 and [(11)C]2 had high striatal accumulation (at peak time) for [(11)C]1 and [(11)C]2 were 6.0-fold and 4.5-fold at 60 min, respectively. Following MP-10 pretreatment, striatal uptake in rats of [(11)C]1 and [(11)C]2 was reduced, suggesting that the tracers bind specifically to PDE10A. MicroPET studies of [(11)C]1 and [(11)C]2 in nonhuman primates (NHP) also showed good tracer retention in the striatum with rapid clearance from non-target brain regions. Striatal uptake (SUV) of [(11)C]1 reached 1.8 at 30 min with a 3.5-fold striatum:cerebellum ratio. In addition, HPLC analysis of solvent extracts from NHP plasma samples suggested that [(11)C]1 had a very favorable metabolic stability. Our preclinical investigations suggest that [(11)C]1 is a promising candidate for quantification of PDE10A in vivo using PET.

Radiosynthesis and in vivo evaluation of a novel σ1 selective PET ligand.

The σ1 receptor is an important target for CNS disorders. We previously identified a σ1 ligand TZ3108 having highly potent (Ki-σ1 = 0.48 nM) and selective affinity for σ1 versus σ2 receptors. TZ3108 was 18F-labeled with F-18 for in vivo evaluation. Biodistribution and blocking studies of [18F]TZ3108 in male Sprague-Dawley rats demonstrated high brain uptake, which was σ1-specific with no in vivo defluorination. MicroPET studies in cynomolgus macaques showed high brain penetration of [18F]TZ3108; the regional brain distribution was consistent with that of the σ1 receptor. Pseudo-equilibrium in the brain was reached ~ 45 min post-injection. Metabolite analysis of [18F]TZ3108 in NHP blood and rodent blood and brain revealed that ~ 70% parent remained in the plasma of NHPs 60 min post-injection and the major radiometabolite did not cross the blood-brain barrier in rats. In summary, the potent, selective and metabolically stable σ1 specific radioligand [18F]TZ3108 represents a potentially useful PET radioligand for quantifying the σ1 receptor in the brain.

Striatal dopamine D1-like receptor binding is unchanged in primary focal dystonia.

BACKGROUND: Multiple studies have demonstrated decreases in striatal D2-like (D2, D3) radioligand binding in primary focal dystonias. Although most investigations have focused on D2-specific receptors (D2R), a recent study suggests that the decreased D2-like binding may be due to a D3-specific (D3R) abnormality. However, only limited data exist on the role of D1-specific receptors (D1R) and the D1R-mediated pathways within basal ganglia in dystonia. Metabolic positron emission tomography (PET) data in primary generalized dystonia suggest resting state over activity in the D1R-mediated direct pathway, leading to excessive disinhibition of motor cortical areas. This work investigated whether striatal D1-like receptors are affected in primary focal dystonias. METHODS: Striatal-specific (caudate and putamen) binding of the D1-like radioligand [(11)C]NNC 112 was measured using PET in 19 patients with primary focal dystonia (cranial, cervical, or arm) and 18 controls. RESULTS: No statistically significant difference was detected in striatal D1-like binding between the two groups. The study had 91% power to detect a 20% difference, indicating that false-negative results were unlikely. CONCLUSIONS: Because [(11)C]NNC 112 has high affinity for D1-like receptors, very low affinity for D2-like receptors, and minimal sensitivity to endogenous dopamine levels, we conclude that D1-like receptor binding is not impaired in these primary focal dystonias.

Validation of nigrostriatal positron emission tomography measures: critical limits.

OBJECTIVE: Molecular imaging and clinical endpoints are frequently discordant in Parkinson disease clinical trials, raising questions about validity of these imaging measures to reflect disease severity. We compared striatal uptake for 3 positron emission tomography (PET) tracers with in vitro measures of nigral cell counts and striatal dopamine in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys. METHODS: Sixteen macaques had magnetic resonance imaging and baseline PETs using 6-[18F]fluorodopa (FD), [11C]dihydrotetrabenazine (DTBZ), and 2beta-[11 C]carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT). MPTP (0-0.31 mg/kg) infused unilaterally via the internal carotid artery produced stable hemiparkinsonism by 3 weeks. After 8 weeks, PETs were repeated and animals were euthanized for striatal dopamine measurements and unbiased counts of tyrosine hydroxylase-stained nigral cells. RESULTS: Striatal uptake for each radiotracer (FD, DTBZ, CFT) correlated with stereologic nigral cell counts only for nigral loss<50% (r2=0.84, r2=0.86, r2=0.87, p<0.001 respectively; n=10). In contrast, striatal uptake correlated with striatal dopamine over the full range of dopamine depletion (r2=0.95, r2=0.94, r2=0.94, p<0.001; n=16). Interestingly, indices of striatal uptake of FD, DTBZ, and CFT correlated strongly with each other (r2=0.98, p<0.001). INTERPRETATION: Tracer uptake correlated with nigral neurons only when nigral loss was <50%. This along with previous work demonstrating that nigral cell counts correlate strongly with parkinsonism ratings may explain discordant results between neuroimaging and clinical endpoints. Furthermore, strong correlations among striatal uptake for these tracers support lack of differential regulation of decarboxylase activity (FD), vesicular monoamine transporter type 2 (DTBZ), and dopamine transporter (CFT) within 2 months after nigrostriatal injury.

Basal ganglia intensity indices and diffusion weighted imaging in manganese-exposed welders.

OBJECTIVES: Manganese exposure leads to diffuse cerebral metal deposition with the highest concentration in the globus pallidus associated with increased T1-weighted MRI signal. T1 signal intensity in extra-pallidal basal ganglia (caudate and putamen) has not been studied in occupationally exposed workers. Diffusion weighted imaging is a non-invasive measure of neuronal damage and may provide a quantification of neurotoxicity associated with welding and manganese exposure. This study investigated extra-pallidal T1 basal ganglia signal intensity as a marker of manganese exposure and basal ganglia diffusion weighted imaging abnormalities as a potential marker of neurotoxicity. METHODS: A 3T MR case:control imaging study was performed on 18 welders and 18 age- and gender-matched controls. Basal ganglia regions of interest were identified for each subject. T1-weighted intensity indices and apparent diffusion coefficients were generated for each region. RESULTS: All regional indices were higher in welders than controls (p ≤ 0.05). Combined basal ganglia (ρ = 0.610), caudate (ρ = 0.645), anterior (ρ = 0.595) and posterior putamen (ρ = 0.511) indices were more correlated with exposure than pallidal (ρ = 0.484) index. Welder apparent diffusion coefficient values were lower than controls for globus pallidus (p = 0.03) and anterior putamen (p = 0.004). CONCLUSIONS: Welders demonstrated elevated T1 indices throughout the basal ganglia. Combined basal ganglia, caudate and putamen indices were more correlated with exposure than pallidal index suggesting more inclusive basal ganglia sampling results in better exposure markers. Elevated indices were associated with diffusion weighted abnormalities in the pallidum and anterior putamen suggesting neurotoxicity in these regions.

Reduced uptake of FDOPA PET in end-stage liver disease with elevated manganese levels.

OBJECTIVE: To investigate whether manganese toxicity secondary to end-state liver disease is associated with nigrastriatal dysfunction as measured by 6-[(18)F]fluoro-L-DOPA (FDOPA) positron emission tomographic (PET) imaging. DESIGN: Observational case report. SETTING: The Movement Disorder Center at Washington University, St Louis, Missouri. PATIENT: An individual with manganese toxicity secondary to end-stage liver disease. His FDOPA PET was compared with those of 10 idiopathic Parkinson disease patients and 10 age- and sex-matched healthy controls. MAIN OUTCOME MEASURE: The average estimated net FDOPA uptake by Patlak graphical analysis for caudate, anterior putamen, and posterior putamen. RESULTS: The FDOPA uptake for the patient with secondary manganese toxicity was reduced across all regions by more than 2 SDs compared with healthy controls: caudate (reduced 24.7%), anterior putamen (28.0%), and posterior putamen (29.3%). The ratio of uptake between the caudate/posterior putamen was 0.99 and was different from that of idiopathic Parkinson disease patients, in whom the greatest reduction of FDOPA was in the posterior putamen (mean [SD] ratio, 1.65 [0.41]). CONCLUSIONS: Reduce striatal uptake of FDOPA uptake indicates dysfunction of the nigrostriatal pathways in manganese toxicity secondary to end-stage liver disease. The pattern of striatal involvement with equal reduction of FDOPA uptake in the caudate compared with posterior putamen appears different from those previously reported in individuals with occupational manganese toxicity and idiopathic Parkinson disease and may be specific to manganese toxicity secondary to end-stage liver disease.

Amyloid imaging of Lewy body-associated disorders.

Clinicopathologic studies of Parkinson disease dementia (PDD) and dementia with Lewy bodies (DLB) commonly reveal abnormal ß-amyloid deposition in addition to diffuse Lewy bodies (α-synuclein aggregates), but the relationship among these neuropathologic features and the development of dementia in these disorders remains uncertain. The purpose of this study was to determine whether amyloid-ß deposition detected by PET imaging with Pittsburgh Compound B (PIB) distinguishes clinical subtypes of Lewy body-associated disorders. Nine healthy controls, 8 PD with no cognitive impairment, 9 PD with mild cognitive impairment, 6 DLB, and 15 PDD patients underwent [(11)C]-PIB positron emission tomography imaging, clinical examination, and cognitive testing. The binding potential (BP) of PIB for predefined regions and the mean cortical BP (MCBP) were calculated for each participant. Annual longitudinal follow-up and postmortem examinations were performed on a subset of participants. Regional PIB BPs and the proportion of individuals with abnormally elevated MCBP were not significantly different across participant groups. Elevated PIB binding was associated with worse global cognitive impairment in participants with Lewy body disorders but was not associated with any other clinical or neuropsychological features, including earlier onset or faster rate of progression of cognitive impairment. These results suggest that the presence of fibrillar amyloid-ß does not distinguish between clinical subtypes of Lewy body-associated disorders, although larger numbers are needed to more definitively rule out this association. Amyloid-ß may modify the severity of global cognitive impairment in individuals with Lewy body-associated dementia.