Biodistribution and Radiation Dosimetry of the Integrin Marker ¹⁸F-RGD-K5 Determined from Whole-Body PET/CT in Monkeys and Humans

Radiopharmaceutical Preparation

¹⁸F-RGD-K5 is a triazole-containing RGD peptide useful for detecting α_vβ₃ integrin expression in vivo.

Production of ¹⁸F-RGD-K5 suitable for human injection was accomplished in 2 steps on an automated synthesis module. First, pentyne tosylate was reacted with K¹⁸F in the presence of Kryptofix 222 (Sigma Aldrich), K₂CO₃, and acetonitrile (MeCN) at 110°C for 10 min. After fluorination, the ¹⁸F-pentyne was distilled into a vial containing RGD-K5-N₃, CuSO₄, sodium ascorbate, and a copper ligand tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine in the presence of alcohol (EtOH) and water (Fig. 2). After reacting for 10 min, the crude reaction mixture was purified by reversed-phase high-performance liquid chromatography (HPLC) (MeCN:water with 0.05% trifluoroacetic acid), and the collected fraction was reconstituted as an injectable solution of 10% EtOH:water via C18 trap and release. The final solution was passed through a sterile filter (Pall Corp.). This process produced ¹⁸F-RGD-K5 that was free of Kryptofix 222, pyrogens, and residual solvents (MeCN ≤ 0.04%); was sterile; and possessed an acceptable pH profile (pH 5–7.5).

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FIGURE 2.

Synthesis of ¹⁸F-RGD-K5 from precursor.

The production of ¹⁸F-RGD-K5 investigational product was conducted according to our Investigational New Drug application (no. 102,075), submitted to the U.S. Food and Drug Administration. Individual doses of ¹⁸F-RGD-K5 contained a maximum of 740 MBq. Each manufactured batch of ¹⁸F-RGD-K5 was formulated to yield a minimum specific activity of more than 14.8 GBq/μmol. Therefore, a typical individual patient dose contained, by weight, a microdose ranging up to a maximum of 50 μg of RGD-K5 per dose.

Human Subjects

The study was approved by the Research Review Committee, Institutional Review Board, and Radiation Safety Committee of Fox Chase Cancer Center. Four healthy female volunteers (mean age ± SD, 53 ± 18 y; age range, 32–75 y) were included in the study. Written informed consent was obtained from each subject. The subjects’ weights were 64 ± 5 kg (range, 57–68 kg). All subjects were healthy based on history, physical examination, electrocardiogram, urinalysis, and standard blood tests.

Human PET/CT Acquisition and Image Analysis

After the intravenous injection of ¹⁸F-RGD-K5 (583 ± 78 MBq; range, 474–659 MBq) to the volunteers, 5 successive whole-body (WB) PET/CT scans were obtained on a Discovery LS (GE Healthcare) PET/CT scanner. The lowest possible milliampere setting on the scanner was used to acquire the CT scans for attenuation correction. The helical CT scan acquisition parameters were 140 kVp, 10 mA, 0.8-s rotation, 5-mm slice thickness, and 4.25-mm interval.

The WB PET scans were acquired in 2-dimensional mode and ranged from the top of the head to mid thigh. The 5 WB scans were acquired at approximately 15, 60, 90, 120, and 140 min after injection. The scan time was 1.5 min per bed position, and each scan covered 7 bed positions, with single-slice overlap between the bed positions. In addition, after the first WB scan, a PET/CT scan of mid thigh to toes was obtained. Blood pressure, body temperature, pulse, and electrocardiogram were monitored before the administration of ¹⁸F-RGD-K5, after the first and second PET/CT scans, and at 24 h after dose administration.

Blood and urine were collected before the drug injection and in the breaks between the PET scans. Urine was also collected at the end of the last PET scan. Samples of urine were assayed in a well counter to estimate the excreted activity in urine. HPLC was performed to determine the amounts of intact ¹⁸F-RGD-K5 and its radioactive metabolites in the plasma. Briefly, the radioactivity of the whole-blood samples was measured in a well counter in counts per minute. The whole blood was centrifuged at 3,500 rpm for 5 min to separate plasma from whole cells. The plasma fraction was removed and placed in a separate, empty, preweighed tube to record the weight of plasma. An aliquot of plasma (400 μL) was removed, followed by spiking of the aliquot with nonradioactive RGD-K5 standard (80 μL of 2.5 mg/mL) and acetonitrile (400 μL). After the sample was stirred in a vortex mixer for 30 s and centrifuged at 13,000 rpm for 8 min to separate proteins from plasma, the plasma supernatant was removed and weighed and the counts per minute of pellet and extract were measured. An aliquot (50–100 μL) of the processed plasma sample was injected onto the radio-HPLC column. The radio-HPLC conditions were B = 0.1% trifluoroacetic acid in water; D = 0.1% trifluoroacetic acid in MeCN; t = 0 min 95% B, 5% D; t = 19 min 70%B, 30%D; t = 21 min 5%B, 95% D; flow, 2 mL/min; and wavelength, 206 nm. The radio-HPLC eluent was collected in 1-min fractions and individually counted. No attempts were made to identify the radioactive metabolites in the plasma or urine.

The PET scans were reconstructed to a 50-cm display field of view using an ordered-subsets expectation maximization algorithm with 28 subsets and 2 iterations. The reconstruction included corrections for random coincidences and scatter. Attenuation correction was applied on the basis of the low-dose CT. The accuracy of the activity in the PET images was verified by summing the activity in the first WB PET scan and the PET scan from mid thigh to toes and comparing it with the decayed injected activity.

The PET images for the middle time point for each patient were displayed on an imaging workstation. For each organ—including the brain, kidneys, bladder, liver, gallbladder, and large intestine (upper and lower)—volume regions of interest (ROIs) were drawn. For the small intestine, which was not visualized above background, a volume ROI was determined by marking the boundaries of visualized adjacent organs. The PET images for other time points were displayed and registered with this PET scan to transfer the volume ROI and determine the total activity in the organs. Adjustments to the individual ROIs were made to ensure the inclusion of visualized organs (e.g., bladder, gallbladder). The percentage injected activity was determined for the organs for each of the time points. The decay-corrected percentage administered activity for the top 3 organs are shown in Figure 3.

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FIGURE 3.

Mean percentage administered activity and SD for top 3 organs determined on basis of four ¹⁸F-RGD-K5 PET emission scans in human volunteers, as function of time after injection. Rapid clearance of activity was observed in organs.

Normalized Number of Disintegrations

The percentage administered activity for each organ for each time point was fitted to an exponential or sum-of-exponentials function in OLINDA/EXM software (8) to determine the total number of disintegrations per unit administered activity, hereafter referred to as normalized number of disintegrations. Activity in the remainder of the body was calculated for each time point as the injected activity minus the activity in all the source organs and in collected urine. The half-times for biologic excretion were computed by exponential fitting of injected activity minus accumulated urine activity as a function of time. The 1- and 4.8-h bladder-voiding models in OLINDA/EXM were used to determine the normalized number of disintegrations in the bladder. Absorbed doses in the various organs were calculated by entering the normalized number of disintegrations of all source organs for each subject into OLINDA/EXM, using the standardized adult male and female models.

Animal Subjects

The rhesus monkey studies were approved by the West Point Institutional Animal Care and Use Committee at Merck Research Laboratories. Three male rhesus monkeys (ages, 7, 9, and 11 y) were initially anesthetized with ketamine (10 mg/kg intramuscularly), then induced with propofol (5 mg/kg intravenously), intubated, and ventilated with medical-grade air. Anesthesia was maintained with propofol (0.4 mg/kg/min) for the duration of the study, and the vital signs (electrocardiogram, expired tidal CO₂, SPO₂, and temperature) were monitored and maintained in the reference range for the duration of the study. Animals were administered an intravenous bolus of 167 ± 19 MBq of ¹⁸F-RGD-K5 in a 3 mL-volume injected with a 10- to 15-s bolus duration.

PET/CT Acquisition

After the injection of 167 ± 19 MBq of ¹⁸F-RGD-K5 to the monkeys, low-dose unenhanced CT (120 kVp, 60 mA, 0.5-s rotation, and 3.75-mm slice thickness) was performed, followed by dynamic whole-body PET on a Discovery ST (GE Healthcare) PET/CT scanner. Twenty-four WB PET images were acquired in 2-dimensional mode for 5 bed positions covering 72.9 cm axially for a total duration of 200 min (time per bed range, 15 s to 4 min).

After the completion of WB PET, contrast-enhanced CT (200 mA, 120 kVp, 0.5-s rotation, and 3.75-mm slice thickness) using iohexol (Omnipaque 300; GE Healthcare) (2.5 mL/kg) was performed to assist with organ identification.

Data Analysis

The PET scans were reconstructed using an ordered-subsets expectation maximization algorithm with 30 subsets and 2 iterations and a gaussian postprocessing filter of 3 mm in full width at half maximum. The reconstruction included corrections for scatter and random coincidences. Attenuation correction was applied on the basis of the low-dose CT.

For each study, ROIs were delineated using both the enhanced CT and the summed PET. ROIs included the brain (CT), heart content (first PET), liver (summed PET), spleen (CT), kidneys (CT), testes (CT), and urinary bladder (last PET). The gut ROI was drawn around the abdomen, excluding all other delineated organs. As such, the gut ROI included the gallbladder. ROIs were projected onto the PET images at each time point. Time–activity curves were obtained by calculating the total activity in the ROIs and expressing them as percentage of the total injected activity.

Absorbed Dose Calculations

The percentage administered activity in each source organ was iteratively fitted to a biexponential function using a nonlinear least-squares regression algorithm (SAAM II, version 1.2, software) (9) to obtain the normalized number of disintegrations for important source organs.

Rhesus monkeys accumulated radioactivity in the urinary bladder over the duration of the study. The percentage administered activity in the bladder, obtained from the monkey images, was used as input, and the number of disintegrations from the urinary bladder's contents was calculated using the dynamic bladder model of Cloutier et al. (10). Bladder-voiding intervals of 1 and 4.8 h were used.

In this analysis, the gut ROI comprised the gallbladder, small bowel, upper large intestine, and lower large intestine. The value for fecal excretion fraction (f_gut) was taken as the value at the last image time (≅200 min). The gut residence times were calculated using the model for gastrointestinal tract kinetics from the International Commission on Radiation Protection publication 30 (11), assuming the radioactivity entered via the small bowel.

To obtain an estimate of the corrected total-body time–activity curve (which excluded the activity in the gut and bladder), the total-body time–activity curve was first constructed as decay-corrected injected activity minus the urinary excreted activity and was fit to a biexponential function. Second, to correct the total-body time–activity curve for fecal excretion, the total-body curve was also multiplied by a factor of 1 – f_gut, where f_gut is the fecal excretion fraction. The corrected total-body number of disintegrations was calculated by integrating the corrected total-body time–activity curve fit. The remainder of the body number of disintegrations was calculated as the corrected total-body number of disintegrations minus all other disintegrations, except the bladder and gut.

Human radiation doses were calculated from these rhesus monkey normalized number of disintegrations using OLINDA/EXM for the adult male and female phantoms. No scaling of the rhesus biodistribution data was done to estimate human absorbed radiation doses. Thus, it was assumed that the biodistribution in these monkeys was the same as in humans.