Voxel-Based Acetylcholinesterase PET Study in Early and Late Onset Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by chronic progressive cognitive decline and displays underlying brain cholinergic dysfunction, providing a rationale for treatment with cholinomimetic medication. The clinical presentations and courses of AD patients may differ by age of onset. OBJECTIVE: The objective of the present study was to illustrate the regional differences of brain acetylcholinesterase (AChE) activity as quantified by N-[11C]methylpiperidinyl-4-acetate ([11C]MP4A) and PET using parametric whole brain analysis and clarify those differences as a function of age. METHODS: 22 early onset AD (EOAD) with age at onset under 65, the remaining 26 as late onset AD (LOAD), and 16 healthy controls (HC) were enrolled. Voxel-based AChE activity estimation of [11C]MP4A PET images was conducted by arterial input and unconstrained nonlinear least-squares method with subsequent parametrical analyses. Statistical threshold was set as Family Wise Error corrected, p-value <0.05 on cluster-level and cluster extent over 30 voxels. RESULTS: Voxel-based group comparison showed that, compared to HC, both EOAD and LOAD showed cortical AChE decrement in parietal, temporal, and occipital cortices, with wider and stringent cortical involvement in the EOAD group, most prominently demonstrated in the temporal region. There was no significant correlation between age and regional cerebral AChE activity except for a small left superior temporal region in the AD group (Brodmann's area 22, Zmax = 5.13, 396 voxels), whereas no significant cluster was found in the HC counterpart. CONCLUSION: Difference in cortical cholinergic dysfunction between EOAD and LOAD may shed some light on the cholinomimetic drug efficacy in AD.

Noninvasive k3 estimation method for slow dissociation PET ligands: application to [11C]Pittsburgh compound B.

BACKGROUND: Recently, we reported an information density theory and an analysis of three-parameter plus shorter scan than conventional method (3P+) for the amyloid-binding ligand [11C]Pittsburgh compound B (PIB) as an example of a non-highly reversible positron emission tomography (PET) ligand. This article describes an extension of 3P + analysis to noninvasive '3P++' analysis (3P + plus use of a reference tissue for input function). METHODS: In 3P++ analysis for [11C]PIB, the cerebellum was used as a reference tissue (negligible specific binding). Fifteen healthy subjects (NC) and fifteen Alzheimer's disease (AD) patients participated. The k3 (index of receptor density) values were estimated with 40-min PET data and three-parameter reference tissue model and were compared with that in 40-min 3P + analysis as well as standard 90-min four-parameter (4P) analysis with arterial input function. Simulation studies were performed to explain k3 biases observed in 3P++ analysis. RESULTS: Good model fits of 40-min PET data were observed in both reference and target regions-of-interest (ROIs). High linear intra-subject (inter-15 ROI) correlations of k3 between 3P++ (Y-axis) and 3P + (X-axis) analyses were shown in one NC (r2 = 0.972 and slope = 0.845) and in one AD (r2 = 0.982, slope = 0.655), whereas inter-subject k3 correlations in a target region (left lateral temporal cortex) from 30 subjects (15 NC + 15 AD) were somewhat lower (r2 = 0.739 and slope = 0.461). Similar results were shown between 3P++ and 4P analyses: r2 = 0.953 for intra-subject k3 in NC, r2 = 0.907 for that in AD and r2 = 0.711 for inter-30 subject k3. Simulation studies showed that such lower inter-subject k3 correlations and significant negative k3 biases were not due to unstableness of 3P++ analysis but rather to inter-subject variation of both k2 (index of brain-to-blood transport) and k3 (not completely negligible) in the reference region. CONCLUSIONS: In [11C]PIB, the applicability of 3P++ analysis may be restricted to intra-subject comparison such as follow-up studies. The 3P++ method itself is thought to be robust and may be more applicable to other non-highly reversible PET ligands with ideal reference tissue.

ß-Amyloid in Lewy body disease is related to Alzheimer's disease-like atrophy.

The aim of this study was to investigate whether amyloid deposition is associated with Alzheimer's disease (AD)-like cortical atrophy in Lewy body (LB) disease (LBD). Participants included 15 LBD with dementia patients (8 with dementia with Lewy bodies [DLB] and 7 with Parkinson's disease [PD] with dementia [PDD]), 13 AD patients, and 17 healthy controls. Age, gender, and Mini-Mental State Examination scores were matched between patient groups. All subjects underwent PET scans with [(11)C]Pittsburgh Compound B to measure brain amyloid deposition as well as three-dimensional T1-weighted MRI. Gray-matter volumes (GMVs) were estimated by voxel-based morphometry. Volumes-of-interest analyses were also performed. Forty percent of the 15 DLB/PDD patients were amyloid positive, whereas all AD patients and none of the healthy controls were amyloid positive. Amyloid-positive DLB/PDD and AD patients showed very similar patterns of cortical atrophy in the parahippocampal area and lateral temporal and parietal cortices, with 95.2% of cortical atrophy distribution being overlapped. In contrast, amyloid-negative DLB/PDD patients had no significant cortical atrophy. Compared to healthy controls, parahippocampal GMVs were reduced by 26% in both the amyloid-positive DLB/PDD and AD groups and by 10% in the amyloid-negative DLB/PDD group. The results suggest that amyloid deposition is associated with AD-like atrophy in DLB/PDD patients. Early intervention against amyloid may prevent or delay AD-like atrophy in DLB/PDD patients with amyloid deposition.

A method to predict the ratio of the tracer conversion rate to the tracer back-diffusion rate of an irreversible-type radiotracer in humans by preclinical evaluations.

OBJECTIVE: The aim of this study was to develop a method to predict a tracer's α-value in the human brain on the basis of animal data. The α-value is the ratio of the conversion rate and the back-diffusion rate (k3/k2) and is one of the critical kinetic features of the detection sensitivity of target molecule activity, such as enzyme activity, in the measurement of PET and single-photon emission computed tomography using an irreversible-type radiotracer. METHOD: The α-value in the rat brain was estimated by a simultaneous assay of the tracer uptake and the target biochemical activity using N-[C]-methylpiperidin-4-yl acetate ([C]MP4A) and N-[C]-methylpiperidin-4-yl propionate ([C]MP4P) as test tracers, both of which are metabolic trapping tracers for measurement of brain acetylcholinesterase. The α-value in humans was then extrapolated from the α-value in rats by considering the differences between the species. The predicted human α-values were compared with those obtained from the kinetic analyses of human PET studies using [C]MP4A and [C]MP4P. RESULT: The α-values in the human brain cortex were predicted to be 0.51±0.1 for MP4A and 0.25±0.05 for MP4P. These results were close to values reported in other PET studies: 0.48±0.1 to 0.73±0.2 for MP4A and 0.15±0.04 to 0.18±0.04 for MP4P. CONCLUSION: The α-value predicted by this method would be used for practical selection or development of irreversible-type radiotracers for human use.

Effects of halogenation on tyrosine phosphorylation and peptide binding to the SRC homology 2 domain of lymphocyte-specific protein tyrosine kinase.

Phosphorylation of tyrosine residues by protein tyrosine kinases (PTK) and phosphotyrosine/Src homology 2 (SH2) domain interactions are crucial not only for signal transduction but also for regulation of PTK activity. Tyrosine residues also receive nitration and halogenation under oxidative conditions. It has been reported that nitration of tyrosine residue caused peptides to be a poor substrate for PTK and that nitrotyrosine residues could bind to SH2 domains as a phosphotyrosine mimic to activate Src family kinase. However, the effect of halogenation on tyrosine phosphorylation or SH2 domain binding is not well understood. We examined the phosphorylation of model peptides containing 3-halotyrosine or 3-nitrotyrosine using typical receptor tyrosine kinase, epidermal growth factor receptor (EGFR), and nonreceptor tyrosine kinase, lymphocyte-specific protein tyrosine kinase (Lck). The EGFR- and Lck-mediated phosphorylation was markedly inhibited by tyrosine halogenation. Iodination showed the strongest inhibition of the phosphorylation among four types of halogenation, and its inhibitory effect was stronger than that of nitration. We also examined the effect of iodination and nitration of tyrosine residues on binding to the SH2 domain of Lck, using a model peptide containing the phosphoTyr-Glu-Glu-Ile motif, which has a high affinity for the SH2 domain. The relative affinities of the modified peptides whose phosphotyrosine was substituted with unphosphorylated tyrosine, 3-nitrotyrosine, and 3-iodotyrosine, and of the model peptide were 0.024, 0.26, 1, and 16, respectively. These results suggest that tyrosine iodination may have an effect on the phosphorylation or binding to the SH2 domain similar to nitration. Tyrosine iodination possibly modulates signal transduction, with the potential impairment of cell function.

A short-scan method for k(3) estimation with moderately reversible PET ligands: application of irreversible model to early-phase PET data.

Long dynamic scans (60-120 min) are often required for estimating the k(3) value, an index of receptor density, by positron emission tomography (PET). However, the precision of k(3) is usually low in kinetic analyses for reversible PET ligands compared with irreversible ligands. That is largely due to unstable estimation of the dissociation rate constant, k(4). We propose a novel '3P+' method for estimating k(3) of moderately reversible ligands, where a 3-parameter model without k(4) is applied to early-phase PET data to obtain a good model-fit of k(3) estimation. By using [(11)C] Pittsburgh compound B (PIB) (k(4) = 0.018/min) as an example of a moderately reversible ligand, the 3P+ method simulation with a 28 min PET scan yielded less than 3% k(3) relative bias with a +100% k(3) change. In [(11)C]PIB PET scans of 15 normal controls (NC) and nine patients with Alzheimer's disease (AD), the 3P+ method provided a precise k(3) estimate (mean SE of 13.6% in parietal cortex; covariance matrix method). The results revealed linear correlations (r = 0.964) of parietal k(3) values in 24 subjects between 28minute 3P+ method and conventional 90 minute 4-parameter method. A good separation of k(3) between NC and AD groups (P < 0.001; t-test) was replicated in 28 minute 3P+ method. The short-scan 3P+ method may be a practical alternative method for analyzing reversible ligands.

Effect of radiolabeled metabolite elimination from the brain on the accuracy of cerebral enzyme activity estimation using positron emission tomography with substrate tracers.

Cerebral enzyme activity can be quantified using positron emission tomography (PET) in conjunction with a radiolabeled enzyme substrate. We investigated the relationship between the elimination rate (k(el)) of tracer metabolites from the brain and the precision of target enzyme activity estimation (k(3)). An initial simulation study indicated that the precision of k(3) estimates was highly dependent on k(el), and was characterized by several kinetic parameters including the ratio of k(el) and the efflux rate (k(2)) of authentic tracer (ß≡k(el)/k(2)). The optimal tracer condition for high sensitivity was found to be ß<0.1. To verify the simulation results, we performed a PET study with a single monkey using two PET tracers, N-[(18)F]fluoroethylpiperidin-4-ylmethyl acetate ([(18)F]FEP-4MA) and N-[(11)C]methylpiperidin-4-yl acetate ([(11)C]MP4A). Both of these substrate type tracers were developed for measuring cerebral acetylcholinesterase activity. There was good retention of the radioactive metabolite of [(11)C]MP4A in the brain (k(el)=0.0036±0.0013 min(-1), ß=0.028), whereas that of [(18)F]FEP-4MA was eliminated from the brain (k(el)=0.012±0.0010 min(-1), ß=0.085). A non-linear least square analysis for simultaneous estimation of all parameters showed that the precision of the k(3) estimate for [(18)F]FEP-4MA was as high (7.4%) as that for [(11)C]MP4A (10%). These results indicate that tracers with metabolites that are eliminated from the brain at a slow rate (ß<0.1) may be useful for the quantitative measurement of target enzyme activity.

Cholinergic imaging in corticobasal syndrome, progressive supranuclear palsy and frontotemporal dementia.

Corticobasal syndrome, progressive supranuclear palsy and frontotemporal dementia are all part of a disease spectrum that includes common cognitive impairment and movement disorders. The aim of this study was to characterize brain cholinergic deficits in these disorders. We measured brain acetylcholinesterase activity by [11C] N-methylpiperidin-4-yl acetate and positron emission tomography in seven patients with corticobasal syndrome (67.6+/-5.9 years), 12 with progressive supranuclear palsy (68.5+/-4.1 years), eight with frontotemporal dementia (59.8+/-6.9 years) and 16 healthy controls (61.2+/-8.5 years). Two-tissue compartment three-parameter model and non-linear least squares analysis with arterial input function were performed. k3 value, an index of acetylcholinesterase activity, was calculated voxel-by-voxel in the brain of each subject. The k3 images in each disease group were compared with the control group by using Statistical Parametric Mapping 2. Volume of interest analysis was performed on spatially normalized k3 images. The corticobasal syndrome group showed decreased acetylcholinesterase activity (k3 values) in the paracentral region, frontal, parietal and occipital cortices (P<0.05, cluster corrected). The group with progressive supranuclear palsy had reduced acetylcholinesterase activity in the paracentral region and thalamus (P<0.05, cluster corrected). The frontotemporal dementia group showed no significant differences in acetylcholinesterase activity. Volume of interest analysis showed mean cortical acetylcholinesterase activity to be reduced by 17.5% in corticobasal syndrome (P<0.001), 9.4% in progressive supranuclear palsy (P<0.05) and 4.4% in frontotemporal dementia (non-significant), when compared with the control group. Thalamic acetylcholinesterase activity was reduced by 6.4% in corticobasal syndrome (non-significant), 24.0% in progressive supranuclear palsy (P<0.03) and increased by 3.3% in frontotemporal dementia (non-significant). Both corticobasal syndrome and progressive supranuclear palsy showed brain cholinergic deficits, but their distribution differed somewhat. Significant brain cholinergic deficits were not seen in frontotemporal dementia, which may explain the unresponsiveness of this condition to cholinergic modulation therapy.

In vivo detection of neuropathologic changes in presymptomatic MAPT mutation carriers: a PET and MRI study.

BACKGROUND: Microglial activation and disrupted neurotransmissions may herald symptomatic manifestations in neurodegenerative tauopathies. METHODS: We investigated microglial activation with [(11)C]DAA1106 positron emission tomography (PET), striatal dopaminergic function with l-[beta-(11)C]dopa PET, acetylcholinesterase (AChE) activity with [(11)C]N-methylpiperidin-4-yl acetate PET, and morphologic brain changes with MRI in three persons (aged 38-41 years) with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), who were presymptomatic gene carriers (PGCs) from an American kindred with pallidopontonigral degeneration. The results from these 3 PGCs were compared with [(11)C]DAA1106 PET results from age-matched 9 healthy volunteers (HV), and with l-[beta-(11)C]dopa and [(11)C]MP4A PET results from 10 HV. Values considered significant were more than 2 SDs greater or less than the normal control mean, as the number of subjects was small for group comparisons. RESULTS: Glial activities were increased in the frontal cortex of one PGC, the occipital cortex of two PGCs, and the posterior cingulate cortex of one PGC, although none of the PGCs showed overt glial activation in the brain. Only one of the PGCs showed reduced AChE activity in the temporo-parietal cortex. Three PGCs showed low dopamine synthesis rates in the putamen. Hippocampal atrophy was observed in two PGCs. CONCLUSIONS: Hippocampal atrophy and striatal dopaminergic dysfunction may be early disease processes in the pathogenesis of FTDP-17. Neuroinflammation may also be an in vivo signature of tau pathology at a prodromal stage, although current PET techniques may not constantly reveal it as the earliest neuroimaging abnormality.

Use of a novel radiometric method to assess the inhibitory effect of donepezil on acetylcholinesterase activity in minimally diluted tissue samples.

BACKGROUND AND PURPOSE: Cholinesterase inhibitors have been widely used for the treatment of patients with dementia. Monitoring of the cholinesterase activity in the blood is used as an indicator of the effect of the cholinesterase inhibitors in the brain. The selective measurement of cholinesterase with low tissue dilution is preferred for accurate monitoring; however, the methods have not been established. Here, we investigated the effect of tissue dilution on the action of cholinesterase inhibitors using a novel radiometric method with selective substrates, N-[(14)C]methylpiperidin-4-yl acetate ([(14)C]MP4A) and (R)-N- [(14)C]methylpiperidin-3-yl butyrate ([(14)C]MP3B_R), for AChE and butyrylcholinesterase (BChE) respectively. EXPERIMENTAL APPROACH: We investigated the kinetics of hydrolysis of [(14)C]-MP4A and [(14)C]-MP3B_R by cholinesterases, and evaluated the selectivity of [(14)C]MP4A and [(14)C]MP3B_R for human AChE and BChE, respectively, compared with traditional substrates. Then, IC(50) values of cholinesterase inhibitors in minimally diluted and highly diluted tissues were measured with [(14)C]MP4A and [(14)C]MP3B_R. KEY RESULTS: AChE and BChE activities were selectively measured as the first-order hydrolysis rates of [(14)C]-MP4A and [(14)C]MP3B_R respectively. The AChE selectivity of [(14)C]MP4A was an order of magnitude higher than traditional substrates used for the AChE assay. The IC(50) values of specific AChE and BChE inhibitors, donepezil and ethopropazine, in 1.2-fold diluted human whole blood were much higher than those in 120-fold diluted blood. In addition, the IC(50) values of donepezil in monkey brain were dramatically decreased as the tissue was diluted. CONCLUSIONS AND IMPLICATIONS: This method would effectively monitor the activity of cholinesterase inhibitors used for therapeutics, pesticides and chemical warfare agents.

Piperidine-4-methanthiol ester derivatives for a selective acetylcholinesterase assay.

The activity of acetylcholinesterase (AChE) is measured to obtain pathological information about the cholinergic system in various disease states and to assess the effect of AChE inhibitors. Using Ellman's method that is commonly used in such examinations, butyrylcholinesterase inhibitors must be added to measure AChE-specific activity because of low selectivity of AChE toward traditional substrates; however, such inhibitors also inhibit AChE. Therefore, it is desirable to obtain an AChE selective substrate that can be used with the Ellman's method. Here, we synthesized novel AChE substrates, 1-methyl-4-acetylthiomethylpiperidine and 1,1-dimethyl-4-acetylthiomethylpiperidine, and evaluated the hydrolysis rate and AChE selectivity by comparison with the results obtained when traditional substrates were used. The hydrolysis rate of the novel compounds by human AChE was one order of magnitude lower than that of the traditional substrates, acetylthiocholine and acetyl-beta-methylthiocholine, whereas the hydrolysis rate using human butyrylcholinesterase was two orders of magnitude lower than that of the traditional substrates. This indicated that AChE showed selectivity towards the novel substrates which was one order of magnitude higher than that of the traditional substrates. The hydrolysis of the novel compounds in a rat cerebral cortical homogenate and a monkey whole blood was completely inhibited by 1 muM of the specific AChE inhibitor, 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one, indicating the high specificity of AChE towards the novel substrates in a crude tissue sample. From these results, we conclude that the novel compounds developed would be suitable AChE-selective substrates for Ellman's method.

In vivo evaluation of N-[18F]fluoroethylpiperidin-4ylmethyl acetate in rats compared with MP4A as a probe for measuring cerebral acetylcholinesterase activity.

[(11)C]MP4A is an established radioprobe for quantification of cerebral acetylcholinesterase (AChE) activity by positron emission tomography (PET) based on the kinetics of AChE-mediated metabolism and metabolite trapping. It has been used to assess the deficiency in cholinergic innervation in the brain of patients with dementia. Because (18)F has a longer half-life than (11)C, (18)F-labeled derivatives of [(11)C]MP4A allow delivery of the probe to other PET centers, making AChE measurement more widely applicable. Previously, N-[(18)F]fluoroethylpiperidin-4ylmethyl acetate ([(18)F]FEP-4MA) showed that the (18)F-labeled analog of MP4A possessed desirable properties for the quantification of cerebral AChE activity by PET. Here, we evaluated the in vivo kinetics of [(18)F]FEP-4MA and validated the responsiveness of brain uptake to AChE activity based on a mathematical model derived from the AChE-mediated trapping rationale and compared it with MP4A in rats. Almost all radioactivity in the brain was composed of [(18)F]FEP-4MA and the hydrolyzed metabolite at 0-60-min postinjection. When the authentic radioprobe was not observed in the brain, the regional (18)F uptake in the brain correlated well with regional MP4A uptake, and the elimination rate of (18)F from the brain was higher than that of the metabolite of MP4A. The responsiveness of regional (18)F uptake in the brain was examined by simultaneous assay of (18)F concentration, relative blood flow, and AChE activity. Regional (18)F uptake correlated with regional AChE activity as well as that of MP4A. Therefore, we concluded that [(18)F]FEP-4MA would be applicable to clinical PET study for quantifying cerebral AChE activity.

Estimation of plasma IC50 of donepezil for cerebral acetylcholinesterase inhibition in patients with Alzheimer disease using positron emission tomography.

OBJECTIVES: Estimate the value of in vivo plasma IC50 of donepezil, the concentration of donepezil in plasma that inhibits brain acetylcholinesterase (AChE) activity by 50% at the steady-state conditions of donepezil between the plasma and the brain. METHODS: N-[C] methylpiperidin-4-yl acetate ([C]MP4A) positron emission tomography was performed in 16 patients with probable Alzheimer disease (AD) before and during the treatment of donepezil (5 mg/day) with a mean interval of 5.3 months. The plasma IC50 value of donepezil was estimated from plasma donepezil concentrations and cerebral cortical mean AChE inhibition rates measured by positron emission tomography, using one-parameter model. RESULTS: Donepezil reduced AChE activity uniformly in the cerebral cortex compared with the baseline in each AD patient, and the mean reduction rate in the cerebral cortex was 34.6%. The donepezil concentrations in the plasma ranged from 18.5 to 43.9 ng/mL with a mean of 28.9 +/- 7.3 ng/mL. The plasma IC50 value was estimated to be 53.6 +/- 4.0 ng/mL. CONCLUSIONS: Once the plasma IC50 of donepezil is determined, the brain AChE inhibition rate could be estimated from the plasma concentration of donepezil in each subject based on the plasma IC50. Now that the mean donepezil concentrations in the plasma, when the patients took 5 mg/day, remained 28.9 ng/mL, approximately half of the plasma IC50, higher dose of donepezil might provide further benefits for patients with AD. This technique can be also applied to measure the in vivo plasma IC50 of other cholinesterase inhibitors such as rivastigmine and galantamine.

Reactivity of 6-halopurine analogs with glutathione as a radiotracer for assessing function of multidrug resistance-associated protein 1.

6-Bromo-7-[(11)C]methylpurine is reported to react with glutathione via glutathione S-transferases in the brain and to be converted into a substrate for multidrug resistance-associated protein 1 (MRP1), an efflux pump. The compound with a rapid conversion rate allows quantitative assessment of MRP1 function, but this rate is probably susceptible to interspecies differences. Hence, for application to different species, including humans, it is necessary to adjust the conversion rate by modifying the chemical structure. We therefore designed 6-halo-9-(or 7)-[(14)C]methylpurine (halogen: F, Cl, Br, or I), and evaluated them in vitro with respect to enzymatic reactivity with glutathione using brain homogenates from the mouse, rat, or monkey. There was a marked difference in reactivity between these species. Changes in the position of the methyl group and halogen on N-methyl-6-halopurine provided various compounds possessing wide-ranging reactivity with glutathione. In conclusion, the adjustment of reactivity of 6-bromo-7-[(11)C]methylpurine may allow assessment of MRP1 function in the brain in various species.

A radiotracer method to study efflux transport of iodide liberated from thyroid hormones via deiodination metabolism in the brain.

AIMS: Thyroid hormones (TH) play an important role in the development and functional maintenance of the central nervous system. The purpose of this study was to develop a radiotracer method for studying the in vivo efflux transport of iodide liberated by the TH metabolism in the brain. The rationale of our method is as follows: a radioiodinated compound can enter the brain and rapidly release iodide in situ; the iodide efflux rate can be estimated from the clearance of brain radioactivity after disappearance of the iodinated compound. MAIN METHODS: 6-[(125)I]Iodo-9-pentylpurine ([(125)I]9Pe6IP) was designed to enter the brain and release (125)I(-) by the reaction with glutathione and synthesized from the corresponding bromo derivative in a Br/(125)I exchange reaction. The brain kinetics of radioactivity and radioactive metabolites were investigated after intravenous injection of [(125)I]9Pe6IP into mice. The iodide efflux rate was estimated in mice pretreated with perchlorate, an inhibitor of iodide transport from the brain. KEY FINDINGS: High brain uptake (5.3% injected dose/g) was observed at 1 min, and almost complete conversion of [(125)I]9Pe6IP to (125)I(-) occurred 10 min after injection. The (125)I(-) uptake from the blood was negligible. (125)I(-) was eliminated from the brain along a single-exponential curve with a half-life of 6.0 min. Furthermore, dose-dependent inhibition of (125)I(-) efflux was observed in mice pretreated with perchlorate. SIGNIFICANCE: We conclude that 9Pe6IP labeled with (124)I (positron emitter) or (123)I (single-photon emitter) may be useful for studying the in vivo efflux transport of iodide in the brain using nuclear medicine imaging devices.

Noninvasive and quantitative assessment of the function of multidrug resistance-associated protein 1 in the living brain.

Multidrug resistance-associated protein 1 (MRP1) acts as a defense mechanism by pumping xenobiotics and endogenous metabolites out of the brain. The currently available techniques for studying brain-to-blood efflux have significant limitations related to either their invasiveness or the qualitative assessment. Here, we describe an in vivo method, which overcomes these limitations for assessing MRP1 function, using positron emission tomography (PET) and a PET probe. 6-Bromo-7-[(11)C]methylpurine was designed to readily enter the brain after intravenous administration and to be efficiently converted to its glutathione conjugate (MRP1 substrate) in situ. Dynamic PET scan provided the brain time-activity curve after injection of 6-bromo-7-[(11)C]methylpurine into mice. The efflux rate of the substrate was kinetically estimated to be 1.4 h(-1) with high precision. Moreover, knockout of Mrp1 gene caused approximately a 90% reduction of the efflux rate, compared with wild-type mice. In conclusion, our method allows noninvasive and quantitative assessment for MRP1 function in the living brain.

PET study of brain acetylcholinesterase in cerebellar degenerative disorders.

To elucidate characteristic changes of brain acetylcholinesterase (AChE) in cerebellar degenerative disorders. Eight patients with the cerebellar variant of multiple system atrophy (MSA-C), 7 patients with spinocerebellar ataxia type-3 (SCA-3), 3 patients with SCA-6, and 13 healthy age-matched volunteers participated in this study. Brain AChE activity was measured by [(11)C] N-methylpiperidin-4-yl propionate PET in all subjects. Brain AChE activities were significantly decreased in the thalamus (-27%) and the posterior lobe of cerebellar cortex (-36%) in patients with MSA-C and in the thalamus (-23%) in patients with SCA-3 compared with healthy controls (P < 0.01). Thalamic AChE activities of SCA-3 patients were negatively correlated with the unified Parkinson's disease rating scale motor subscore (P < 0.001). AChE activities were not significantly altered in the cerebral cortex in any disease group. Reduction of AChE activities in the thalamus and cerebellum in MSA and in the thalamus in SCA-3 suggest that cholinergic modulating drugs may have a role in the treatment of ataxia and other symptoms in these disorders.

Cerebral acetylcholinesterase imaging: development of the radioprobes.

Cerebral acetylcholinesterase (AChE) imaging is not only useful for diagnosis of dementia disorders but also for therapeutic monitoring of the effects of cholinesterase (ChE) inhibitors and for decision of the appropriate clinical dosage of newly developed ChE inhibitors. Several ChE inhibitors or the derivatives such as 1,2,3,4-tetrahydro-9-methylaminoacridine (MTHA), donepezil, physostigmine, CP126,998 and 2-fluoro-CP118,954 have been labeled with positron emitters for mapping cerebral AChE by positron emission tomography (PET). When [(11)C]MTHA or [(11)C]donepezil was injected in animals, the uptake poorly reflect the regional distribution of AChE in the brain because of high non-specific binding and/or less specific to AChE in vivo in the brain tissue. [(11)C]physostigmine, [(11)C]CP126,998 and 2-[(18)F]fluoro-CP118,954 were distributed corresponding well to the regional AChE activity in animals, and also former two probes were successfully applied to clinical PET trial. The other approach is measuring cerebral AChE activity with radiolabeled acetylcholine analogue substrates. We have developed the principle for measuring cerebral enzyme activity by PET and radiolabeled N-methylpiperidinyl esters for quantitative measurement of cerebral AChE activity. N-[(11)C]methylipiperidin-4-yl acetate (MP4A) and N-[(11)C]methylpiperidin-4-yl propionate (MP4P) have been used for clinical studies of other demented disorders including Alzheimer's disease (AD), and the probes have demonstrated not only the reduction of AChE activity in the cerebral cortex of patients with AD but also the inhibitory effects of donepezil and rivastigmine on AChE activity in the brain of AD patients. Following this succession, widely available [(18)F]-labeled derivatives of MP4A and MP4P have been developed based on the structure-activity relationships between AChE and piperidinol esters.

A novel noninvasive method for assessing glutathione-conjugate efflux systems in the brain.

Brain efflux systems export such conjugated metabolites as glutathione (GSH) and glucuronate conjugates, generated by the detoxification process, from the brain and serve to protect the brain from harmful metabolites. The intracerebral injection of a radiolabeled conjugate is a useful technique to assess brain efflux systems; however, this technique is not applicable to humans. Hence, we devised a novel noninvasive approach for assessing GSH-conjugate efflux systems using positron emission tomography. Here, we investigated whether or not a designed proprobe can deliver its GSH conjugate into the brain. Radiolabeled 6-chloro-7-methylpurine (7m6CP) was designed as the proprobe, and [(14)C]7m6CP was prepared by the reaction of 6-chloropurine with [(14)C]CH(3)I as a model of [(11)C]CH(3)I. The radiochemical yield and purity of [(14)C]7m6CP were 10-20% and greater than 99%, respectively. High brain uptake (0.8% ID/g) at 1 min was observed, followed by gradual radioactivity clearance from the brain for 5-60 min after the injection of [(14)C]7m6CP into rats. Analysis of metabolites confirmed that the presence of [(14)C]7m6CP was hardly observed, and 80% of the radioactivity was identical to its GSH conjugate for 15-60 min. The brain radioactivity was single-exponentially decreased during the period of 15-60 min post-injection of [(14)C]7m6CP, and the first-order efflux rate constant of the conjugate, estimated from the slope, was 0.0253 min(-1). These results showed that (1) [(14)C]7m6CP readily entered the brain, (2) it efficiently and specifically transformed to the GSH conjugate within the brain, and (3) after [(14)C]7m6CP disappearance, the clearance of radioactivity represented the only efflux of GSH conjugate. We conclude that 7m6CP can deliver the GSH conjugate into the brain and would be useful for assessing GSH-conjugate efflux systems noninvasively.

Evaluation of 4'-[methyl-14C]thiothymidine for in vivo DNA synthesis imaging.

UNLABELLED: We evaluated 4'-[methyl-11C]thiothymidine ([methyl-11C]S-dThd) to obtain a thymidine analog that might prove simpler to use for imaging DNA synthesis and that might follow the same biochemistry as its surrogate. METHODS: [Methyl-14C]S-dThd was synthesized by rapid methylation of 5-trimethylstannyl-4'-thio-2'-deoxyuridine via a palladium-mediated Stille coupling reaction with 14C-methyl iodide. Degradation of [methyl-14C]S-dThd, when incubated in human blood, was analyzed by high-performance liquid chromatography (HPLC). The in vivo potential of [methyl-14C]S-dThd was evaluated by studying its distribution in EMT-6 mammary carcinoma-bearing mice. 2-Fluoro-2'-deoxycytidine, a potent inhibitor of DNA synthesis, was used to modulate cell proliferation. Tissue extraction was also performed to investigate the incorporation of [methyl-14C]S-dThd into DNA. RESULTS: [Methyl-14C]S-dThd was obtained in a 31%-41% radiochemical yield (calculated from 14C-methyl iodide) at 130 degrees C, 5-min reaction in N,N-dimethylformamide followed by semipreparative HPLC purification. The radiochemical purity of [methyl-14C]S-dThd was >99% and the specific activity was 2.04 GBq/mmol (according to the specific activity of 14C-methyl iodide). [2-14C]Thymidine, when incubated with human blood, demonstrated rapid degradation. In contrast, [methyl-14C]S-dThd was stable with <3% degradation at 60 min. An in vivo distribution study showed the accumulation of radioactivity in proliferating tissues (spleen, thymus, duodenum, and tumor). On the other hand, the radioactivity of nonproliferating tissues (lung, liver, kidney, and muscle) was rapidly cleared in parallel with the clearance of blood radioactivity. The tumor uptake of [methyl-14C]S-dThd was high (8.8 percentage injected dose per gram at 60 min) and selective (tumor-to-blood ratio, 12.2 at 60 min). 2-Fluoro-2'-deoxycytidine pretreatment significantly reduced the tumor uptake of [methyl-14C]S-dThd. Relative blood flow as measured by the uptake of 4-[N-methyl-14C]iodoantipyrine was similar between the treated and untreated groups. Tissue extraction studies showed that most of the total tissue radioactivity of rapidly proliferating tissues was recovered in the DNA fraction at 60 min after [methyl-14C]S-dThd injection. CONCLUSION: The labeling procedure is rapid and suitable for 11C labeling. Positron-labeled 4'-thiothymidine should be useful for imaging DNA synthesis by PET.