Biodistribution and Radiation Dosimetry of ¹⁸F-Fluoro-A-85380 in Healthy Volunteers

Radiosynthesis

Fluoro-A-85380 has been labeled with the positron emitter ¹⁸F by no-carrier-added nucleophilic aromatic trimethylammonium-to-fluoro substitution by K-¹⁸F-F-K₂₂₂ complex with (3-[2(S)-N-(tert-butoxycarbonyl)-2-azetidinylmethoxy]pyridin-2-yl)trimethylammonium trifluoromethanesulfonate as a highly efficient labeling precursor, followed by trifluoroacetic acid removal of the Boc protective group (5). The total synthesis time was 50–53 min from the end of cyclotron ¹⁸F production (end of bombardment [EOB]). Radiochemical yields, with respect to initial ¹⁸F-fluoride ion radioactivity, were 68%–72% (decay corrected) and 49%–52% (nondecay corrected), and the specific radioactivities at EOB were 148–259 GBq/μmol.

Subjects

The Medical Bioethics Committee of the Medical Center at the University of Paris XI approved this study. Three healthy male volunteers, with a mean age of 22 y (range, 21–23 y) and a mean weight of 74.2 kg (range, 69.3–79.8 kg) gave their written informed consent for participation in the study. The subjects were free of illness on the basis of screening by medical history, physical examination, serum chemical analysis, complete blood cell count, and urine analysis. They were nonsmokers as demonstrated by the negative plasma cotinine determination. A standard 12-lead electrocardiogram (ECG) was obtained for screening purposes. Before the start of the experiments, all volunteers were asked to urinate. During the PET experiment, ECG findings and blood pressure were continuously monitored from 10 min before to 10 min after the tracer injection. Serum chemical analysis, complete blood cell count and urine analysis were performed before and within 1 wk after completion of the PET study.

PET Imaging Procedure

During data acquisition, subjects were positioned supine with the arms alongside the body in the ECAT Exact HR+ PET scanner (CTI/Siemens, Knoxville, TN).

For each subject, a whole-body transmission scan was obtained before injection of the radioligand using 3 retractable ⁶⁸Ge rods (each with approximately 74 MBq). Transmission scans lasted 3 min per bed position (field of view, 15 cm), with 8 or 9 steps according to the height of the subject; they were used for subsequent correction of attenuation of emission scans.

Whole-body emission scans were performed (3-dimensional [3D] mode) after an intravenous bolus injection of 191 ± 50.9 MBq (range, 191–238 MBq)—that is, 1.73 ± 0.21 nmol of ¹⁸F-fluoro-A-85380. One-minute step acquisitions, 8 or 9 steps for 1 scan (from the top the head to the upper part of the thigh), were acquired over 2 h.

Urine Collection

All voided urine from the time of injection until 2 h after injection was collected. The urine was collected in a container, and the volume and time of micturition were recorded for all subjects. Three-milliliter urine aliquots were sampled and radioactivity was counted in an automatic γ-counter (model 5000; Packard Instrument Co., Downers Grove, IL). After the counting efficiency of the system had been determined, radioactivity measurements of the samples were corrected for physical decay and multiplied by the urine volume at the micturition time. The amount of radioactivity in the urine at the micturition time was expressed as percentage of the injected radioactivity (%ID). Because the vertebral bodies were clearly seen in all subjects and on all images, the presence of free ¹⁸F-fluoride ion in the urine was checked. Five hundred microliters of urine were added to hydroxyapatite (10 mg) and incubated at 37°C for 30 min. The mixture was centrifuged at 3,000 rpm for 2 min. The supernatant was removed and counted. The pellet was washed with 500 μL distilled water and centrifuged again. The water layer and the pellet were counted.

Blood Collection

Twenty-four venous blood samples were taken at designated time points up to 120 min after injection. For each blood sample and each corresponding plasma sample obtained by centrifugation (5 min at 3,000 rpm), the amount of radioactivity in 100-μL aliquots was counted in an automatic γ-counter as above. The radioactivity measurements of the samples were corrected for physical decay.

⁶⁸Ge-Germanium Cylinder and Determination of Calibration Factor

To obtain an accurate factor for the conversion of ECAT counts to megabecquerels of radioactivity, a ⁶⁸Ge cylinder (radius, 10 cm; length, 20 cm) was imaged with the PET scanner. A volume of 6,283 mL water was mixed with 27.75 MBq ⁶⁸Ge. An emission scan was acquired for 1 h in 3D mode. Cylinder emission data were reconstructed into a coronal image of the cylinder using the same method as for patient data reconstruction. A region of interest (ROI) was drawn over the entire PET image of the cylinder, and the total counts per second in the cylinder were determined. One count per second was found to be equivalent to 11.5 MBq/mL in 3D mode.

PET Data Analysis

The datasets acquired in 3D mode were corrected for scatter using a model-based correction allowing the simulation of the map of single scatter events. This method uses the map of attenuation coefficients and the image of activity concentration to compute the sinogram of single scatter events using the Klein-Nishima formula (6). Raw positron emission data were reconstructed using the ordered-subsets expectation maximization (OSEM) algorithm in the transaxial format with segmented attenuation correction (7). All reconstructions and image analyses used CAPP software, version 7/1 (CTI/Siemens). Two-dimensional or 3D views were assembled with bed position overlap (12 tomographic slices) and smoothed with a gaussian kernel of 5-mm full width at half maximum. All OSEM reconstructions were performed with 4 iterations and 8 subsets. The images were attenuation corrected using the transmission data collected over the same region of emission imaging.

Because the formalism used to perform absorbed dose calculation is based on the MIRD model (8), all source organs must be first identified from the reconstructed images. ROIs were then drawn over all organs presenting a significant uptake to construct time-activity curves. ROIs were drawn by an experienced investigator on the earliest emission image for liver, abdominal area (including upper and lower large intestine wall as well as small intestine), spleen, kidneys, vertebral bodies, and bone marrow (red); for brain and urinary bladder they were drawn on the emission image obtained 2 h after injection of ¹⁸F-fluoro-A-85380. The shapes and sizes—that is, number of pixels—were kept constant for all individual patients. To determine the biologic clearance of ¹⁸F-fluoro-A-85380, the activity was decay corrected back to the time of injection and time-activity curves were obtained.

Dosimetry

For dosimetry analysis, using the radioactive concentration (in MBq/mL) extracted from the quantitative images, the total activity contained in each source organ was assessed according to the standard-man model defined by Cristy and Eckerman (9). A new set of data, which depicts the behavior of source organ activity according time, was then generated. Data were fitted (least-square method) as follows: a monoexponential decline of radioactivity for upper large intestine and lower large intestine; a biexponential decline for liver, spleen, and kidneys; an uptake phase followed by a monoexponential decline for the brain and the red bone marrow; a monotonic increase for the bladder followed by a complete micturation at 120 min after injection. For all of these organs, the residence times (in h) were calculated by dividing the area under the time-activity curves by the injected dose. MIRDOSE3 software (10) was then used to estimate the mean absorbed dose by all organs included in the phantom due to the activity contained in the source.

Because healthy volunteers have been used in this study, the concept of effective dose must be used to express the risk encountered. Preliminary calculations demonstrated that kidneys were receiving an absorbed dose as high as the most exposed organ (the liver). Therefore, according the recommendations of International Commission on Radiological Protection (IRCP) Publication 60 (11), a weighting factor, W_T, equal to 0.025 was applied to the kidneys (equal to that of the remainder of the body).

Biosafety

After injection of an average of 191 MBq ¹⁸F-fluoro-A-85380 (range, 137–238 MBq; 1.7 nmol; range, 1.5–1.9 nmol), no adverse or subjective effects were noticed in any of the subjects. Their vital signs remained stable throughout the experiment. No significant changes in heart rate, blood pressure, or ECG findings were observed during the 10 min after injection of ¹⁸F-fluoro-A-85380. Moreover, no meaningful changes were observed in any of the clinical laboratory assays performed on the blood and urine specimens obtained before and after administration of the radio1igand (data not shown).

Biodistribution and Dosimetry

A whole-body image of 1 subject showing the biodistribution of radioactivity on injection of ¹⁸F-fluoro-A-85380 at 92–104 min after injection is presented in Figure 1. Ten minutes after injection, high activity was clearly seen in the kidneys, intestines, and liver, and low activity was observed in the brain as well as in the lungs and the heart (Table 1). The images obtained between 1 and 2 h after injection showed that most of the radioactivity was distributed over the kidneys, bladder, liver, and intestines, whereas the uptake in the thalamus (a nAChR-rich region) became more visible at later times. A substantial and rather constant amount (0.4 %ID/100 g tissue from the beginning to the end of the PET acquisition) of radioactivity was seen on the vertebral bodies in all subjects. The radioactivity was rapidly increasing in the urinary bladder, indicating prompt excretion through the renal system. The mean measured urinary excretion at 2 h after injection was 22% ± 8% of the administered activity. No free ¹⁸F-fluoride ion was detected in the urine. No clear evidence of biliary excretion of ¹⁸F-fluoro-A-85380 was seen on the images.

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FIGURE 1.

Whole-body coronal image of ¹⁸F-fluoro-A-85380 distribution in healthy volunteer (subject 1, 1-min acquisition per step) obtained 92–104 min after intravenous injection of 200 MBq ¹⁸F-fluoro-A-85380. Activity clears from other organs to kidneys and bladder over time.

¹⁸F-Fluoro-A-85380 was rapidly cleared from blood, and most of the activity is in the plasma portion. The average %ID remaining in the blood at 60 min after injection was 0.055 %ID/100 g tissue (range, 0.05–0.06 %ID/100 g tissue).

Penetration of ¹⁸F-fluoro-A-85380 across the blood-brain barrier peaked at 25 min after injection and was followed by a slow clearance (Fig. 2). The average %ID was 0.182 %ID/100 g tissue (range, 0.16–0.196 %ID/100 g tissue). The thalamus showed the highest radioactivity (Fig. 3).

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FIGURE 2.

Thalamic (Thal), cerebellar (Cereb), and venous plasma (plasma) time-activity curves (decay corrected) after injection of ¹⁸F-fluoro-A-85380 (200 MBq; subject 1). At 120 min after injection, thalamus-to-cerebellum ratio of radioactivity is 1.8.

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FIGURE 3.

Late (one 5-min image, 120 min after injection) distribution of ¹⁸F-fluoro-A-85380 in brain. Thalamus has highest uptake; in other structures, distribution is rather uniform.

The residence times were derived from the experimental organ distribution data and urine measurements of the 3 subjects (Table 2). The dataset of each subject was fitted independently. The residence time was highest for the remainder of the body, followed by the liver and the urinary bladder. The mean absorbed dose was then estimated using the MIRDOSE3 software (Table 3). Taking into account the weighting factor from ICRP Publication 60 (11), including the kidneys as previously described, the mean effective dose for the healthy adults was estimated to be 0.0194 mSv/MBq (range, 0.0178–0.0218 mSv/MBq).