Reduced density of the α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) in the cortex and hippocampus of the human brain has been reported in aging and patients with neurodegenerative disease. This study assessed the pharmacokinetic behavior of 18F-(−)-JHU86428 (18F-XTRA), a new radiotracer for in vivo PET imaging of the α4β2-nAChR, particularly in extrathalamic regions of interest in which the α4β2-nAChR is less densely expressed than in thalamus. 18F-XTRA was also used to evaluate the α4β2-nAChR in the hippocampus in human aging. Methods: Seventeen healthy nonsmoker adults (11 men, 6 women; age, 30–82 y) underwent PET neuroimaging over 90 or 180 min in a high-resolution research tomograph after bolus injection of 18F-XTRA. Methods to quantify binding of 18F-XTRA to the α4β2-nAChR in the human brain were compared, and the relationship between age and binding in the hippocampus was tested. Results: 18F-XTRA rapidly entered the brain, and time–activity curves peaked within 10 min after injection for extrathalamic regions and at approximately 70 min in the thalamus. The 2-tissue-compartment model (2TCM) predicted the regional time–activity curves better than the 1-tissue-compartment model, and total distribution volume (VT) was well identified by the 2TCM in all ROIs. VT values estimated using Logan analysis with metabolite-corrected arterial input were highly correlated with those from the 2TCM in all regions, and values from 90-min scan duration were on average within 5% of those values from 180 min of data. Parametric images of VT were consistent with the known distribution of the α4β2-nAChR across the brain. Finally, an inverse correlation between VT in the hippocampus and age was observed. Conclusion: Our results extend support for use of 18F-XTRA with 90 min of emission scanning in quantitative human neuroimaging of the extrathalamic α4β2-nAChR, including in studies of aging.
Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand–gated ion channels, of which the α4β2 and α7 are the most abundant subtypes in the human brain. The loss of activity of even a small quantity of neuronal nAChRs can have wide-ranging effects on neurotransmission across neural circuits (1). Altered density of the α4β2-nAChR is linked to several neurodegenerative disorders (2–5). Additionally, postmortem work using 3H-epibatidine (6) and some in vivo human imaging, including that using 2-18F-fluoro-A-85380 (2-18F-FA) with PET (5,7), suggest diminished availability of the α4β2-nAChR in human aging. The binding of 2-18F-FA in the hippocampus and thalamus inversely correlated with performance on a cognitive task of processing speed in a cohort of elderly healthy participants (8).
There is need for α4β2-nAChR–targeting radiotracers with faster pharmacokinetics and high specific uptake in brain tissue outside the thalamus (extrathalamic regions such as the cortex and striatum) (9,10), in which the α4β2-nAChR is less densely expressed (11). 18F-(−)-JHU86428 (18F-XTRA) (12,13) is among such recently developed radioligands (13–15) and has promising in vitro binding characteristics, including subnanomolar binding affinity (Ki = 0.06 nM) and improved lipophilicity (LogD7.4 = 0.67) over that of 2-18F-FA (13). 18F-XTRA also showed stable, high binding estimates in extrathalamic regions of the baboon brain in vivo (12).
This study assessed use of 18F-XTRA with PET imaging in the human brain, particularly in extrathalamic regions of interest (ROIs). Estimates of total distribution volume (VT) generated using kinetic modeling methods with arterial input function and using alternative scan durations were compared. Finally, we investigated the correlation between age and VT in the hippocampus, a region in which low availability of the α4β2-nAChR may be linked to subtle deficits in cognition even in otherwise healthy older individuals (8).
MATERIALS AND METHODS
This prospective study was approved by a Johns Hopkins Institutional Review Board, and all subjects provided written informed consent. Seventeen healthy adult (≥18 y) participants were recruited through local advertising. Each subject completed a screening interview and laboratory testing (blood counts, metabolic panel, coagulation studies), electrocardiogram, and urine toxicology. Eligible participants had stable health with no clinical abnormality on the screening assessment and structural MRI. Exclusion criteria included nicotine use in the past year, past psychiatric or neurologic illness, history of substance abuse including marijuana (assessed by self-report and urine toxicology), medication known to affect acetylcholine signaling, current psychotropic medication use, contraindication to MRI, or contraindication to PET imaging with arterial line.
All older (≥50 y) participants were also assessed with neuropsychologic testing that included the Clinical Dementia Rating scale (16) to ensure a global Clinical Dementia Rating of 0, consistent with normal cognition. Since the apolipoprotein ε4 (APOE ε4) allele may play a role in aberrant cholinergic signaling (17) that may be linked to altered α4β2-nAChR availability (18), older participants were assessed for APOE ε4 carrier status using methods described previously (19).
Human Brain Imaging
Synthesis of 18F-XTRA
18F-XTRA was synthesized as previously described (12). Radiochemical purity was greater than 99%, with high specific radioactivity (1,586 ± 937 GBq/μmol) at the time of injection. The mean administered mass and radioactivity of 18F-XTRA were 0.08 ± 0.04 μg (range, 0.03–0.17 μg) and 335 ± 38.3 MBq (range, 235–387 MBq), respectively. There were no adverse or clinically detectable pharmacologic effects, and no significant changes in vital signs, laboratory results, or electrocardiograms were observed.
Brain PET Image Acquisition
All participants wore a thermoplastic facemask to minimize head movement and underwent both radial arterial line and antecubital venous catheter insertion. PET scans were acquired using a High-Resolution Research Tomograph (Siemens Healthcare) with 2.5-mm reconstructed image resolution (20). Each emission scan started at the time of bolus intravenous injection of 18F-XTRA, with continuous list-mode data collection for 90 (n = 10) or 180 (n = 7) min. Imaging data were reconstructed using methods described in the supplemental materials (available at http://jnm.snmjournals.org).
Measurement of the arterial plasma input function was conducted through collection of 35–50 blood samples (1 mL), obtained after injection using the previously published protocol (19). Samples from 120 to 180 min after injection were collected every 10 min. Plasma was immediately isolated from whole blood using centrifugation. Radioactivity was counted in a cross-calibrated γ-well-counter. The fraction of parent radioligand in plasma was determined by high-performance liquid chromatography (HPLC) with blood sampling as previously described (19), with additional blood sampling at 105, 120, 150, and 180 min after injection for 180-min scans.
The modified column-switching HPLC method (21) used a Waters reverse phase XBridge BEH C18 5 μM 4.6 × 150 mm analytic column, with an analytic mobile phase (45% acetonitrile and 55% aqueous solution of 0.1% ammonium hydroxide) at 2 mL/min. The HPLC system was standardized using nonradioactive XTRA and 18F-XTRA before analysis of plasma samples, which were spiked with 10 μL of XTRA (1 mg/mL) for each run. Metabolite-corrected plasma time–activity curves were obtained by applying percentage parent ligand time profiles, generated by HPLC analysis, to the total plasma time–activity curves after linear interpolation in PMOD (version 3.7; PMOD Technologies Ltd.).
T1-weighted brain MRI at 3 T was acquired for each participant using methods identical to those as previously described (19), to obtain a 0.8 × 0.8 × 0.8 mm 3-dimensional image with a magnetization-prepared rapid gradient-echo sequence.
PET Image Analysis and Volumes of Interest
PET image processing, including motion correction and kinetic analysis, was conducted using PMOD as previously described (19). PET time–activity curves were generated for 10 ROIs that were segmented from each MR image using the FreeSurfer image analysis suite (http://surfer.nmr.mgh.harvard.edu/). ROIs included the thalamus, striatum, hippocampus, corpus callosum, as well as cerebellar, temporal, occipital, cingulate, frontal, and parietal cortices. Total intracranial volume was also defined using FreeSurfer for generating regional volume ratio values (ROI volume normalized to total intracranial volume).
Derivation of Rate Constants and VTs
VT (22) for each ROI was derived using the metabolite-corrected arterial input function and compartmental modeling (1-tissue-compartment model with 3 parameters [1TCM]; 2-tissue-compartment model with 5 parameters [2TCM]) or Logan graphical analysis (23). In compartmental modeling, nonlinear least-squares analysis was performed, with the Marquardt algorithm for parameter estimation (24). Logan-derived VT values were determined using ordinary least squares after transformation of the PET data with t* = 45 min. The contribution of cerebral blood volume was set at 5% of brain volume. As in other recent PET imaging of this target (12,25), reference-tissue models were not applied because there is no clearly identified human brain region devoid of the α4β2-nAChR.
Compartmental model fitting was assessed by visual inspection of the model fit to the time–activity curves and by relative goodness of fit using the Akaike information criterion (26). The standard errors of nonlinear least-square estimates of rate constants and VT from modeling were computed from the covariance matrix in PMOD and expressed as the coefficient of variation (% COV) (27). Regional VT estimates from variable scan durations were evaluated using the 180-min acquisition as the reference for comparison of VT values from data shortening (shortened to 90 min). For each duration, denoted X, relative bias values were expressed as |VTX – VT 180 min|/VT 180 min.
The relationship between VT in the hippocampus and age was tested using Spearman rank-order correlation analysis because age was not normally distributed across the study population. Secondary analyses testing the relationship between age and VT in the other 9 ROIs were also explored.
Statistical analyses were performed using SPSS Statistics (version 23.0; IBM Corp.). Data were checked for outliers (28), and descriptive statistics were obtained. Normality of the data was assessed using the Shapiro–Wilk test. Data are presented as mean ± SD, and significance was set to a P value less than 0.05 unless otherwise noted.
Seventeen healthy nonsmokers (11 men, 6 women; age, 30–82 y; median age, 60 y; interquartile range, 37 y) underwent PET neuroimaging with 18F-XTRA (Table 1). All older participants (n = 10) had a global Clinical Dementia Rating of 0, and none of the participants was an APOE ε4 carrier. ROI volumes and volume ratios from the study population are presented in Supplemental Table 1.
Plasma activity peaked within 90 s after injection and decreased to less than 5% of the peak by 20 min (Fig. 1A). HPLC easily isolated 18F-XTRA (retention time, 7.5 min) from its radiolabeled metabolites, which were more polar and well resolved from the parent compound. 18F-XTRA represented 21.8% ± 10.7% of total plasma activity by 90 min (Supplemental Fig. 1) and 15.2% ± 10.5% by 180 min.
Determination of VT
18F-XTRA readily entered the brain and, for extrathalamic ROIs, activity peaked within 10 min after injection and then declined over the remaining 90- or 180-min scan duration (Fig. 1B). Highest peak uptake occurred in the thalamus at approximately 70 min after injection except in 1 individual (a 76-y-old Caucasian man) whose thalamic activity peaked just before the end of the 90-min emission scan. Activity in the thalamus washed out gradually after the peak. The lowest uptake was observed in the corpus callosum.
Across the entire population, the kinetic behavior of 18F-XTRA over the 90-min scan in each ROI yielded a visually better fit using the 2TCM compared with the 1TCM (Fig. 1C for representative data) except for within the thalamus of the aforementioned individual who had unusually late, 90-min peak thalamic activity that did not converge for either compartmental model. Those outlier data were excluded from further analyses, and when all other 90 min of continuous data were used, the Akaike information criterion favored the 2TCM in all 10 ROIs (Supplemental Table 2). The 2TCM identified VT well (COV < 5%) for all ROIs except for the thalamus, which had a COV of 5.4% (Table 2). K1 was also identified well (COV < 5%) using the 2TCM. The other rate constants from the 2TCM were identified with COV values of approximately 8%–22% for k2 and 9%–24% for k3/k4 across all 10 ROIs. All VT estimates (compartmental modeling, Logan) using 90-min emission data were highest in the thalamus and were more homogeneous across regions of the striatum, hippocampus, and cortical ROIs. VT was lowest in the corpus callosum.
When 90-min data were used, values of regional VT from Logan analysis correlated well with those of the 2TCM (Fig. 2). Regional VT values generated using Logan analysis from the 90-min continuous scans were also within 5% of the VT values obtained using 180 min of continuous data from the same 7 individuals (Fig. 3; Supplemental Table 3). Parametric images of VT derived using Logan analysis from 90-min emission scans demonstrated binding of 18F-XTRA throughout the brain (Fig. 4).
Correlation Between Age and VT in Hippocampus
An inverse correlation between age and 18F-XTRA VT in hippocampus (rho = −0.589, P = 0.014) was found (Supplemental Fig. 2). Secondary analyses revealed no significant correlation between age and VT in other ROIs after applying correction for multiple comparisons (P < 0.005 after Bonferroni adjustment for all 10 ROIs). There was also no correlation between body mass index and VT in any of the ROIs. One-way ANOVA analysis revealed no effect of sex or race on VT. There was no correlation between age and volume or volume ratio for any of the 10 ROIs.
PET imaging using newly developed radiotracers that have superior specificity for the α4β2-nAChR and faster brain kinetics over previously used radioligands (29) may further our understanding of changes in cholinergic activity over the course of cognitive decline (30). Here we present the first human neuroimaging data using PET and 18F-XTRA, a radiotracer with promising physical (13) and in vivo (12) characteristics.
18F-XTRA readily accessed the brain in 17 healthy participants, all of whom underwent PET for 90 or 180 min. The highest uptake was in the thalamus, with relatively lower uptake in the striatum, hippocampus, and cortex and lowest uptake in the corpus callosum, consistent with direct assessment in postmortem tissue (2). After exclusion of thalamic data from 1 individual in whom thalamic activity peaked toward the end of the 90-min scan, VT values were well estimated using the 2TCM and 90-min acquisition, especially in extrathalamic regions. The 2TCM was favored over the 1TCM by goodness of fit and Akaike information criterion. The 2TCM VT and K1 values were well identified (COV < 5%) in all extrathalamic regions and reasonably identified (COV = 5.4%) in the thalamus. K1 was also high (K1 > 0.48 mL cm−3 min−1 in all cortical and subcortical ROIs; K1 = 0.30 mL cm−3 min−1 in corpus callosum), consistent with high radiotracer delivery. Overall, the observed high uptake into the brain, fast pharmacokinetics, and ability to estimate VT in extrathalamic regions with a 90-min scan supports further use of 18F-XTRA in clinical research.
This initial evaluation of 18F-XTRA in healthy humans revealed a negative correlation between age and VT in the hippocampus. Since amyloid plaque may negatively influence expression of this receptor (18), all elderly (≥50-y-old) participants were evaluated for APOE ε4 carrier status. Those older individuals lacked even 1 APOE ε4 allele and were therefore at relatively low risk for having high amyloid burden. There was also no correlation between age and hippocampal volume or volume ratio among these participants. Together, our results suggest that 18F-XTRA PET may be sufficiently sensitive to measure the hypothesized loss of α4β2-nAChR availability over aging in extrathalamic regions (5–7), particularly the hippocampus, in which reduced expression of the β2 subunit may account for the lower α4β2-nAChR binding in the elderly (31). This aging effect was not found using 18F-nifene with PET, but this study population consisted of only 8 subjects (age, 21–69 y) (32).
18F-XTRA VT estimates were higher in most human extrathalamic brain regions than VT values from bolus injection of other recently developed radiotracers with fast pharmacokinetics, such as (−)-18F-flubatine (25,33) and 18F-AZAN (34). Since VT represents the sum of both specific binding and nondisplaceable uptake (22), we note the limitation that a displacement study, such as using nicotine, is needed to compare specific binding patterns between recently developed radiotracers. Limited blocking studies in baboons using 18F-XTRA PET after subcutaneous administration of cytisine, a selective partial agonist at the α4β2-nAChR, support the specificity of this radiotracer for its target (12). Although 18F-XTRA also has high affinity for the α6 nicotinic receptor subunit (13), central receptors containing the α6 subunit are relatively limited in distribution (retina, catecholaminergic nuclei) compared with the widespread, higher density of the α4β2-nAChR (35). We also note that thalamic data from 1 individual among the 17 participants did not peak until close to the end of the 90-min scan, rather than peaking at approximately 70 min. Since we saw a similar, late peak in thalamic data in 1 of 5 baboons (12), a conservative approach for studying the α4β2-nAChR in the human thalamus may use longer 18F-XTRA scan duration (180 min) or use an alternative radiotracer that has not shown outlier thalamic pharmacokinetics, such as 18F-AZAN (34).
18F-XTRA is a promising new radiotracer for measuring the human cerebral α4β2-nAChR in vivo. Analysis by the 2TCM using 18F-XTRA data from 90-min scan duration is sufficient to estimate VT in extrathalamic ROIs, such as the cerebral cortex, hippocampus, and striatum. 18F-XTRA PET is also a promising tool for further study of the effect of aging on the availability of the α4β2-nAChR, particularly in the hippocampus of the human brain in vivo.
This work was supported by the Henry N. Wagner, Jr. Endowment (Martin G. Pomper), a Johns Hopkins Doris Duke Early Clinician Investigator Award (Jennifer M. Coughlin), the Alexander Wilson Schweizer Fellowship (Jennifer M. Coughlin), and the National Institutes of Health (R33AG037298 [Andrew G. Horti and Martin G. Pomper], AG038893 [Gwenn S. Smith], AG041633 [Gwenn S. Smith], Shared Instrument Grants S10RR023623 [Dean F. Wong], S10RR017219 [Dean F. Wong]). No other potential conflict of interest relevant to this article was reported.
We thank the Johns Hopkins PET Center for expert provision of 18F-XTRA (Daniel P. Holt, Robert C. Smoot, Jack C. Brown), with special thanks to Alimamy Kargbo for aid in the PET metabolite analyses.
- © 2018 by the Society of Nuclear Medicine and Molecular Imaging.
- Received for publication November 19, 2017.
- Accepted for publication February 3, 2018.