PET Imaging of the Dopamine Transporter with ¹⁸F-FECNT: A Polar Radiometabolite Confounds Brain Radioligand Measurements

Materials

FECNT and (2β-carbomethoxy-3β-(4-chlorophenyl)-8-nortropane) (nor-CCT) (7) were purchased from Dr. Mark Goodman (Emory University) and were fully characterized by ¹H nuclear magnetic resonance, melting point, mass spectrometry (MS), and elemental analyses; they were of greater than 99% chemical purity, as assessed by high-performance liquid chromatography (HPLC). All other reagents and solvents were obtained in analytical grade and were used as received unless otherwise stated. Water was obtained from a reverse osmosis–electrodeionization purifier (model Milli-Q Synthesis A 10, RiOs; Millipore).

General Methods

The labeling agent, ¹⁸F-1-fluoro-2-tosyloxyethane, was purified by HPLC with a stainless steel Xterra C₁₈ column (7.8 × 300 mm; Waters Corp.) eluted with an acetonitrile:water gradient (system A) at 6 mL/min. A second gradient system, system B, consisting of CH₃CN:ammonium hydroxide (0.1 mol/L) (pH 10), was used to purify ¹⁸F-FECNT with the same Xterra C₁₈ column eluted at 5 mL/min. A third system, system C, consisting of a Novapak C₁₈ column (100 × 8 mm; Waters Corp.) with radial compression module RCM-100 and a mobile phase of methanol:H₂O:triethylamine (80:20:0.1, v/v) at 1.5 mL/min, was used for radiometabolite analysis. All biologic samples were prefiltered through nylon filters (13 mm × 0.45 μm; Iso-Disk; Supelco). The HPLC system consisted of Beckman Gold (Beckman Coulter, Inc.) analytic pumps equipped with an in-line photodiode-array detector and a flowthrough NaI(Tl) scintillation detector–rate meter (Bioscan). Data from the analysis of radiometabolites were collected and stored with Bio-Chrome Lite software (Bioscan) and analyzed after decay correction of radiochromatograms. Ex vivo measurements of radioactivity were performed by use of a calibrated automatic well-type γ-counter (model 1480 Wizard; Perkin-Elmer) with an electronic window set at between 360 and 1,800 keV (counting efficiency, 51.9%). Decay corrections were performed at a half-life of 109.77 min.

Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (8) and were approved by the National Institute of Mental Health Animal Care and Use Committee.

Radiopharmaceutical Preparation

¹⁸F-FECNT for injection was prepared as previously described (2) but with modifications in the HPLC purification (system A) of the intermediate, ¹⁸F-1-fluoro-2-tosyloxyethane (9). The ¹⁸F-labeled alkylating agent was reacted with nor-CCT (0.75 mg) by evaporating the CH₃CN under helium sweep gas (10 mL/min) in an open vessel heated at 110°C for 10 min. ¹⁸F-FECNT was purified by reverse-phase HPLC (system B).The chemical purity, radiochemical purity, and identity of ¹⁸F-FECNT were assessed by HPLC (system A) at the end of each preparation. The absence of precursor from a preparation also was monitored. Radiochemical purity and identity tests were repeated at the time of radiometabolite analysis (system C). Quantification of the carrier present in each preparation, so as not to exceed 3.5 μg per dose, was performed by LC-MS techniques (10).

Rat Brain Biodistribution Study

For ex vivo brain analyses, female Sprague–Dawley rats (weight, 250–350 g) were anesthetized by inhalation of 1.5% isoflurane in O₂. The rats were infused (through the tail vein) with no-carrier-added ¹⁸F-FECNT (31.5 MBq/h, at a rate of 1.5 mL/h for 1.0 h). At the end of the infusion, a blood sample (2 mL) was removed by cardiac puncture, and the rat was sacrificed with an intravenous administration of potassium chloride (0.5 mL, 20 mEq/10 mL; Abbott Laboratories). Plasma samples (200 μL) were isolated from whole blood by centrifugation at 1,800g for 3 min and placed in CH₃CN (700 μL) spiked with standard FECNT (20 μg). The whole cerebellum, striatum, and cortex each were resected, weighed, and placed in CH₃CN (1–2 mL) that had been spiked with FECNT (50 μg), and activity was determined with the γ-counter. The brain tissues were homogenized by use of a Tissue Tearor (model 985-370; BioSpec Products Inc.) with sham washings between homogenizations in 3 consecutive vessels, each containing pure CH₃CN; the homogenizer then was wiped dry to prevent cross-contamination. Samples were centrifuged at 9,400g for 2 min (Allegra 21R; Beckman Coulter). The supernatants were subjected to HPLC analysis, and the activities in the resulting precipitates were used to calculate the percent recovery of activity in the CH₃CN supernatants. The composition of the activity in each tissue sample was determined by HPLC.

In vitro rat brain and blood stability studies were performed with 2 rats. The first rat was anesthetized by inhalation of 1.5% isoflurane in O₂; the second was anesthetized with a mixture of ketamine (75 mg per kilogram of body weight; Ketaset III; Wyeth) and xylazine hydrochloride (13 mg per kilogram of body weight; Xyla-Ject; Phoenix Scientific Inc.) (0.2 mL intraperitoneally). Blood (2 mL) was removed by cardiac puncture from each rat, which then was sacrificed. The first rat was perfused with cold (4°C) 0.9% NaCl (10 mL) containing heparin at 100 U/mL (heparin sodium, 1,000 U/mL; Elkins Sinn Inc.). The brain was placed in cold heparinized 0.9% NaCl (5 mL). The second rat was decapitated. The brains were homogenized in an ice bath at the highest speed with three 30-s pulses and 5-min pauses and then cooled over ice. ¹⁸F-FECNT (∼740 kBq) was added to each brain homogenate, and 74 kBq was added to each 1 mL of whole blood; these samples then were incubated at 37°C in a reciprocating shaker water bath (model 25; Precision Scientific) at 60 oscillations per minute. Brain homogenate samples (50 μL; n = 11) were periodically removed and placed in CH₃CN (300 μL) for up to 90 min for HPLC analysis. At the end of the 90-min period, ¹⁸F-FECNT (10 kBq) was added to each brain homogenate, and the mixture was incubated for an additional 2–4 h. Whole blood was incubated with ¹⁸F-FECNT for 10 min, and then plasma was isolated. Each sample of brain homogenate (50 μL) or plasma (200 μL) was placed in CH₃CN (300 or 700 μL, respectively) containing FECNT (20 μg) and mixed well. Next, H₂O (100 or 200 μL) was added to the brain-CH₃CN or plasma-CH₃CN mixture, respectively, and mixed well. The activity in the samples was measured with the γ-counter, the samples were centrifuged at 9,400g for 2 min, and the supernatants were injected into the HPLC column. The recovery of radioactivity from the HPLC column was monitored either by γ-counter measurements of the radioactivity in collected eluates for comparison with the radioactivity injected or by injection of 2 mL of absolute methanol at the end of radiochromatography with continued monitoring for radioactive peak elution.

The distribution of ¹⁸F-FECNT between the plasma and the cellular component of the blood was calculated with the following formula: (radioactivity in plasma or cellular blood fraction/total radioactivity) × 100.

Characterization of Rat Brain Radiometabolite

Two male Sprague–Dawley rats (280 and 443 g) that had been given ketamine:xylazine anesthesia each were infused through the penile vein with ¹⁸F-FECNT for 60 min (53 and 34 MBq/h plus 120 and 150 μg of carrier FECNT at rates of 1.6 and 1.8 mL/h for 1.0 h, respectively). Body temperature was maintained at between 36.5 and 37.0°C. At the end of the infusion time, urine was collected. The brain was excised, homogenized in 1.5 times its weight of CH₃CN, diluted with H₂O (500 μL), and rehomogenized. These homogenates were divided into 4 samples, activity was counted with the γ-counter, and the samples were centrifuged at 9,400g for 2 min. The supernatants then were injected into the HPLC column. The eluates corresponding to the polar radiometabolite, the parent radioligand, and the intermediate region (between these peaks) were collected, and ∼2-mL samples of each fraction were evaporated with a SpeedVac concentrator (Thermo Electron Corp.). The residue was reconstituted in CH₃CN:H₂O (50:50, v/v; 100 μL), and the resulting solution (5 μL) was injected into the LC-MS instrument, which was equipped with an electrospray ionization interface (model LCQ Deca; Thermo Electron Corp.). Urine was centrifuged at 9,400g for 5 min, and aliquots (10 μL) were injected directly into the LC instrument. LC separation was performed with a reverse-phase C₁₈ column (2 × 150 mm, 5 μm; Luna; Phenomenex). The LC mobile phase consisted of a mixture of A (aqueous ammonium formate at 10 mmol/L) and B (CH₃CN) run as a linear gradient at 150 μL/min from 80% of A to 20% of A over 8 min and then isocratically at 20% of A for 4 min (system D). The source voltage for the electrospray was 4.5 kV, and the capillary voltage was 16 V. The capillary temperature was 260°C. In the MS mode, the LC-MS instrument was set up to acquire ions between m/z 150 and m/z 500. For MS-MS acquisition, the molecular ions (including the ³⁷Cl isotope) of FECNT and the metabolite were isolated (isolation width of 5 amu) and energized at 38% collision energy.

A similar analysis was performed with synthetic standards of FECNT and the metabolite (nor-CCT). The excised brain from a control rat (466 g) was processed in a manner similar to the brains treated with the carrier and was used to provide a baseline pattern. Urine analysis was performed after samples were centrifuged (5,000g, 5 min), and the supernatants (2 μL) were injected into the LC-MS instrument. After LC separation, full-scan mass spectral analysis revealed m/z 280 and m/z 282 ions for the metabolite nor-CCT and m/z 312 and m/z 314 ions for the metabolite FECNT carboxylic acid; these mass spectra were identical to the corresponding mass spectra of the synthetic standards.

Nonhuman Primate Studies

Six rhesus monkeys (Macaca mulatta) that had fasted and that weighed 13.3 ± 1.7 kg (mean ± SD) were used for plasma analysis studies of ¹⁸F-FECNT. Three of them (13.8 ± 0.8 kg) were imaged for 240 min. Animals were immobilized with ketamine and maintained under anesthesia with 1.6% isoflurane in O₂. A blood sample (2 mL) was withdrawn before radiopharmaceutical administration for determination of plasma protein binding and for construction of standards A and B for plasma analysis. Standard A was whole blood incubated at room temperature with ¹⁸F-FECNT for the duration of the study to confirm its stability. Standard B was plasma mixed with a sample of the radiopharmaceutical and processed in a manner similar to that used for the rest of the sample to determine the efficiency of the analysis method. One sample was used immediately for the protein binding assay. The radiopharmaceutical was injected intravenously at 115 ± 22 MBq (4.5 mL), and PET serial dynamic image data were acquired with an Advance PET scanner (GE Healthcare) for 300 min. Acquisition was done for 57 frames with a scan duration ranging from 30 s to 5 min. A second, intra-arterial line in the contralateral limb was infused with 0.9% NaCl solution (11 mL/kg/h) to obtain 21 blood samples. For analysis of plasma ¹⁸F-FECNT, 8 samples (0.5 mL each) were drawn at 15-s intervals until 2 min, and then samples (1 mL) were collected at 2.5, 3, 6, 10, 20, 40, 60, 90, 120, 150, 180, 210, 240, and 300 min in heparin-treated syringes. PET images were coregistered to magnetic resonance images, and time–activity curves were obtained for irregular regions of interest for the striatum and the cerebellum.

Human Studies

Two healthy humans (men; 24 and 33 y; 93 and 99 kg) participated in this study. One venous antecubital catheter was placed for radioligand injection, and an arterial catheter was placed in the contralateral radial artery for blood sampling. Just before radioligand injection, blood samples (4 mL) were drawn for protein binding determinations and for construction of standards A and B for plasma radiochromatographic analysis. This step was followed by an 8-min transmission scan, performed with a ⁶⁸Ge rotating pin source, to provide a measured correction of photon attenuation of the head. ¹⁸F-FECNT doses of 360 and 396 MBq were administered over 60 s. Simultaneously with the radioligand administration, a series of 2-h emission scans were started: 6 for 30 s, 3 for 1 min, 2 for 2 min, and 22 for 5 min. After a 1-h interval, another set of emission scans (24 for 5 min) were obtained from 3 to 5 h. To measure the plasma radiochromatographic analysis. of ¹⁸F-FECNT, 10 samples were drawn at 15-s intervals until 2.5 min, and then samples (1.5 mL) were collected at 3, 4, 6, 8, 10, 15, 20, 30, 40, 50, 60, 75, 90, 115, and 150 min in heparin-treated syringes.

Plasma ¹⁸F-FECNT was used as the input function for compartmental nonlinear least squares analysis. For time points before the plasma ¹⁸F-FECNT peak, the measured values were used. After the peak, the measured values were fitted biexponentially, and their estimates were used in subsequent calculations. Brain data were weighted on the basis of noise-equivalent counts. An unconstrained 2-tissue compartment model was applied to calculate the total distribution; the rate constants k₁, k₂, k₃, and k₄ were used as previously described (11) using PMOD2.55. Throughout this article, brain and blood volumes are reported in cubic centimeters and milliliters, respectively, to distinguish between data sources. Tomographic images were reconstructed by 3-dimensional filtered backprojection with a Hanning filter. Volumes of interest were drawn on coregistered magnetic resonance images.

Plasma Analysis

Arterial blood samples collected from the 2 humans and the 6 nonhuman primates were centrifuged for 2 min at 1,800g. Each plasma sample was added to 1.5 times its volume of CH₃CN. The mixture was agitated, H₂O was added at 9% the final volume, and mixing was done again. Activity measurements for these samples were decay corrected to time of dose administration and expressed as disintegrations per minute per milliliter. Plasma samples were centrifuged at 9,400g for 2 min, and the supernatants were analyzed by HPLC (system B). Recounting was done for the precipitates to monitor the extraction efficiency. The parent activity concentration in the plasma was calculated as the product of the HPLC fractional parent activity and the total plasma disintegrations per minute per milliliter. Activities in the brain and plasma also were expressed as percent standardized uptake values, which were calculated with the following formula: [(activity per gram of tissue)/injected activity] × grams of body weight × 100.

The plasma free fraction of ¹⁸F-FECNT (the non–protein-bound fraction) was measured in duplicate for each study by ultrafiltration with Amicon Centrifree units (Millipore; molecular weight cutoff, 30,000) at 3,000g for 20 min as described previously (12).

Radiopharmaceutical

¹⁸F-FECNT was prepared from the ¹⁸F-fluoride ion with an overall decay-corrected radiochemical yield of 18.8% ± 6.7% (mean ± SD; n = 11). The radiochemical purity was 98.7% ± 1.4% (n = 11). The specific activity, decay corrected to the time of injection, was 98.5 ± 46.5 GBq/μmol (n = 10).

Rat Brain Biodistribution

The composition of the activity in the rat brain and plasma was measured after 60 and 120 min of constant infusion (CI) of ¹⁸F-FECNT. As expected, all tissues showed a higher level of accumulation of the radiometabolite at 120 min than at 60 min. However, the percentage of activity that represented the parent radioligand decreased in that time interval. ¹⁸F-FECNT decreased from 90.2% at 60 min after injection to 71.0% at 120 min in the rat striatum and from 59.3% to 12.6% in the cerebellum (Table 1). The recovery of radioactivity from the various tissues and fluids into the CH₃CN that was injected into the HPLC column was more than 90%. The recovery of radioactivity from the HPLC column was complete. Thus, the efficiency of the radiometabolite analysis was not less than 90%.

The absolute concentrations of ¹⁸F-FECNT and the radiometabolite across brain regions and in plasma after 60 min of CI were compared (Fig. 1). The concentrations of ¹⁸F-FECNT varied across brain regions and were highest in the striatum, being about 7-fold higher than those in the cerebellum. In contrast, the concentrations of the radiometabolite were uniform across brain regions, consistent with it being an inactive metabolite, that is, one that does not accumulate in DAT-rich brain regions. The concentrations of ¹⁸F-FECNT were higher in all brain regions than in plasma. Ratios of ¹⁸F-FECNT concentrations in brain tissues to those in plasma ranged from 5 (cerebellum to plasma) to as high as 34 (striatum to plasma). Conversely, the concentrations of the radiometabolite were higher in plasma than in all brain regions. The ratio of radiometabolite concentrations in all brain regions to those in plasma was 0.5. These data are consistent with the generation of the metabolite in the periphery, with crossing of the blood–brain barrier.

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FIGURE 1. 

Characteristic distribution of ¹⁸F-FECNT and its radiometabolite, presented as their specific concentrations in rat brain regions and plasma after 1 h of intravenous CI of ¹⁸F-FECNT.

¹⁸F-FECNT was shown to be metabolically stable in both rat whole blood and rat brain homogenates in vitro. In rat whole blood, ¹⁸F-FECNT was distributed 59% to cellular blood elements and 41% to plasma. The radioligand was stable for up to 4 h (>99%) in rat brain homogenates maintained at 37°C. The in vitro homogenate incubations were performed with brains obtained from 2 rats given 2 different forms of anesthesia (i.e., isoflurane and ketamine:xylazine) to determine whether anesthesia had any effect on the in vitro metabolism of ¹⁸F-FECNT. Both brains showed no degradation of the parent radioligand, as analyzed with multiple and periodic monitoring of aliquots by reverse-phase HPLC. Similarly, perfusion of the rat brain to remove residual blood did not affect the in vitro metabolism of the radioligand.

Characterization of Rat Brain Radiometabolite

The brains from 2 rats that were infused with carrier-added ¹⁸F-FECNT as well as the brain from a control rat were used to identify the metabolite(s) in the brain. Brain extracts from each carrier-treated rat were divided into 3 samples and analyzed sequentially. HPLC (system C) showed that 67.3% ± 0.7% (n = 3) of the activity was associated with the polar radiometabolite and that the remaining 32% ± 0.1% of the activity was associated with the parent. These data constituted a ratio of 2.1 ± 0.01 (n = 3) for polar radiometabolite activity to parent activity in the carrier-treated rat brains. Samples from the carrier-treated and control rat brains were subjected to LC-MS and MS-MS analyses; this step was followed by comparison of ion chromatograms. Figure 2A is an ion chromatogram (encompassing m/z 275 through m/z 330 ions; system D) for a carrier-treated sample (obtained with HPLC system C; the fraction containing the radioactive parent radioligand) showing parent FECNT eluting at a retention time of 8.5 min and the major metabolite eluting at a retention time of 7.4 min. The mass spectrum for the metabolite peak is shown in Figure 2B, and that for FECNT is shown in Figure 2C. The electrospray ionization of the metabolite generated m/z 280 and m/z 282 ions (³⁷Cl isotope) whereas, as expected, FECNT generated the m/z 326 molecular ion (and the m/z 328 ion for the ³⁷Cl isotope). The relative abundances of the ³⁷Cl isotope in FECNT and nor-CCT in brain extracts were 31.7% and 32.3%, respectively. These measured values are consistent with a 32.5% natural abundance of ³⁷Cl. Authentic FECNT and nor-CCT had similar elution times of 6.2 and 6.6 min, respectively, with system C.

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FIGURE 2. 

(A) Partial-ion chromatogram (between m/z 275 and m/z 330) from LC-MS analysis of brain extracts showing peaks for metabolite nor-CCT (retention time, 7.4 min) and for FECNT (retention time, 8.5 min). (B and C) Mass spectra for nor-CCT peak (B) and for FECNT peak (C).

The identities of both the metabolite nor-CCT (N-dealkylated) and FECNT in rat brains were confirmed by the finding that their LC retention times (Fig. 2A), mass spectra (Figs. 2B and 2C), and MS-MS product ion spectra were identical to those of synthetic standards (data not shown). However, the fluoroalkyl moiety itself was not detectable by existing LC-MS techniques. A different method of chromatographic separation and chemical modification (not available at the time) would have been needed to detect and identify this molecule. The 2.1 molar ratio of nor-CCT to FECNT, as measured by LC-MS (system D), was similar to that of the radiometabolite to ¹⁸F-FECNT (2.1 ± 0.01; n = 3), as measured by the HPLC (system C). Two major molecules were detected in rat urine, parent FECNT and nor-CCT, and traces of the β-carboxylic acid of FECNT also were present.

Primate Studies

Analysis of arterial plasma samples from both humans and nonhuman primates showed composition profiles similar to those of the rats. Two main radioactivity peaks were detected in each plasma sample. One peak eluted in the void volume, and the other eluted at a retention time corresponding to that of parent ¹⁸F-FECNT. The plasma free fractions of ¹⁸F-FECNT were 9.4% ± 2.7% (n = 6) in monkeys and 4.7% ± 1.3% (n = 4) in humans. The relative compositions of the activity in plasma over time were similar in the 6 monkeys and the 2 humans (Fig. 3), although the appearance of radiometabolites was somewhat delayed in the humans. At about 6 min after the administration of ¹⁸F-FECNT, monkey plasma was composed of 50% radiometabolite and 50% parent radioligand, with little variability, as indicated by the small SD error bars (Fig. 3). The 50% radiometabolite:50% parent composition occurred at 18 and 27 min after injection in humans, as shown in Figure 3.

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FIGURE 3. 

Arterial plasma compositions of ¹⁸F-FECNT and its polar radiometabolite after intravenous administration. Data are shown individually for 2 human participants, human 1 (♦) and human 2 (•) for ¹⁸F-FECNT and human 1 (⋄) and human 2 (○) for radiometabolite, and collectively (mean ± SD) for 6 rhesus monkeys for ¹⁸F-FECNT (▾) and radiometabolite (▵).

The uptake and washout of brain activity were similar in monkeys and humans, with peak uptake in the striatum at about 100 min and then relatively slow washout (Fig. 4). Although activity in the striatum showed a gradual decline after about 100 min, the activity in the cerebellum was virtually stable. Such differential washout rates in target and background regions can be attributable to the effect of the accumulation of a radiolabeled metabolite(s) in the brain. Although this differential washout can be demonstrated by a time-dependent increase in the ratio of the activity in the striatum to that in the cerebellum, a better parameter for evaluating this effect is distribution volume assessed relative to that of the parent compound in plasma. Compartmental analysis with an arterial input function provides the ratio for each region to plasma (rather than the ratio for region to region) and uses an integrated area under the curve (rather than a single time point measurement). The cerebellar distribution volume for both monkey and human studies increased as a function of the duration of image analysis. In monkeys, the distribution volume increased 2.0-fold ± 0.2 (n = 3) between 60 and 240 min (Fig. 5A), whereas in humans, the increases were 1.7-fold between 60 and 300 min and 1.5-fold between 60 and 260 min (Fig. 5B). However, the distribution volume in the striatum did not increase during the same periods, indicating a smaller contribution of the radiometabolite to the total activity (specific binding plus nondisplaceable activity). The distribution volume in the striatum became stable after 180 min, with negligible changes after 160 and 200 min in the 2 participants.

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FIGURE 4. 

Time–activity curves for striatum and cerebellum for rhesus monkeys (A) and humans (B). Data are reported as percent standardized uptake values (%SUV). Arterial plasma ¹⁸F-FECNT input function was corrected for radiometabolites.

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FIGURE 5. 

(A) Average cerebellar and striatal distribution volumes (V) for 3 rhesus monkeys and corresponding percent coefficients of variation, determined by use of increasing amounts of available data from each study, after intravenous administration of ¹⁸F-FECNT at 114.7 ± 22.3 MBq. (B) Cerebellar and striatal distribution volumes (V) for 2 humans and percent coefficients of variation, determined by use of variable portions of data from time 0 to time shown on x-axis, after administration of 359.3 and 396.1 MBq of ¹⁸F-FECNT. Error bars indicate percent coefficient of variation.