Target occupancy study and whole-body dosimetry with a MAGL PET ligand [¹¹C]PF-06809247 in non-human primates

[¹¹C]PF-06809247 was synthesized as reported previously [8] (Additional file 1). The precursor and reference standard of radioligand were provided from Pfizer Inc.

Seven cynomolgus monkeys (two females and five males, body weight 5850–8000 g) were used. Anesthesia was induced by intramuscular injection of ketamine hydrochloride (10 mg/kg) at Astrid Fagraeus Laboratory and maintained by the administration of a mixture of isoflurane (1.5–2.0%), oxygen and medical air through endotracheal intubation. The subjects’ cranium was immobilized with a fixation device. Body temperature was maintained by a Bair Hugger model 505 warming unit (Arizant Healthcare, MN) and monitored by an esophageal thermometer. Heart rate, blood pressure, respiratory rate and oxygen saturation were continuously monitored throughout the experiments. Fluid balance was maintained by continuous infusion of saline.

PET measurements were conducted using a High Resolution Research Tomograph (HRRT) (Siemens Molecular Imaging). A transmission scan of 6 min using a single ¹³⁷Cs source was performed before the [¹¹C]PF-06809247 injection. List mode data were acquired continuously for 123 min (first two NHPs) or 63 min (remaining five NHPs) immediately after intravenous injection of the radioligand. Images were reconstructed with a series of 34 frames (20 s × 9, 1 min × 3, 3 min × 5, and 6 min × 17) for 123 min data or 33 frames (10 s × 9, 15 s × 2, 20 s × 3, 30 s × 4, 1 min × 4, 3 min × 4, and 6 min × 7) for 63 min data. The ordinary Poisson-3D-ordered subset expectation maximization (OP-3D-OSEM) algorithm was applied with 10 iterations and 16 subsets including modeling of the point spread function (PSF) [9]. Two PET measurements per NHP were performed in 1 day: one baseline and one following pretreatment.

T1-weighted magnetic resonance (MR) images of the individual NHP brains had been obtained using a 1.5 T GE Healthcare Signa system (GE, Milwaukee, Wis, USA). A spoiled gradient recalled (SPGR) sequence had been acquired in the coronal plane with the following parameters: TR = 21 ms; TE = 4 ms; flip angle = 35°; Slice thickness = 1.0 mm; FOV = 12.8 cm; NEX = 2; voxel size = 0.5 × 0.5 × 1 mm³.

An automated blood-sampling system (ABSS) was used to continuously measure the radioactivity for the first 3 min after the radioligand injection. Blood sampling was performed manually for the measurement of radiometabolism and radioactivity at 2 (only for radiometabolism), 4, 10, 20, 30, 60 (also 90 and 120 for first two NHPs) min after the injection.

A reversed-phase radio-HPLC method was used to determine the amount of unchanged [¹¹C]PF-06809247 and its radioactive metabolites in NHP plasma [10]. The plasma obtained after centrifugation of blood at 2000g for 2–4 min was mixed with acetonitrile. The mixture was then centrifuged at 2000g for 2–4 min and the extract was injected into a HPLC system coupled to an on-line radioactivity detector. The radio-HPLC system used consisted of an interface module (D-7000; Hitachi: Tokyo, Japan), a L-7100 pump (Hitachi), an injector (model 7125, with a 5.0-mL loop; Rheodyne: Cotati, USA), and an ultraviolet absorption detector (L-7400, 254 nm; Hitachi) in series with a 150TR; Packard (housed in a shield of 50 mm thick lead) equipped with a 550 μL flow cell. Chromatographic separation was achieved on a XBridge C18 column, (50 mm × 10 mm I.D., 2.5 μm + 10 mm × 10 mm I.D., 5 μm; Waters: New England, USA) by gradient elution. Acetonitrile (A) and 20 mM ammonium phosphate (pH 7) (B) were used as the mobile phase at 6.0 mL/min, according to the following program: 0–3.5 min, (A/B) 20:80 → 55:45 v/v; 3.5–4.0 min, (A/B) 55:45 v/v; 4.0–4.1 min, (A/B) 55:45 → 20:80 v/v; 4.1–5.0 min, (A/B) 20:80 v/v. Peaks for radioactive compounds eluting from the column were integrated and their areas were expressed as a percentage of the sum of the areas of all detected radioactive compounds (decay-corrected to the time of injection on the HPLC).

A blood sample was taken at 3 min before injection for measurement of protein binding and determination of free fraction of [¹¹C]PF-06809247 in the plasma. The free fraction, f_p, of [¹¹C]PF-06809247 in plasma was estimated using an ultrafiltration method [11]. Plasma (400 µL) or phosphate buffered saline solution (400 µL) as a control were mixed with [¹¹C]PF-06809247 (40 µL, ~ 1 MBq) and incubated at room temperature for 10 min. After the incubation, 200 µL portions of the incubation mixtures were pipetted into ultrafiltration tubes (Centrifree YM-30, molecular weight cutoff, 30,000; Millipore: Billerica, USA) and centrifuged at 1500g for 15 min. Equal aliquots (20 µL) of the ultrafiltrate (C_free) and of the plasma (C_total) were counted for their radioactivity with a 2480 Wizard2 Automatic Gamma Counter (Perkin Elmer: Massachusetts, USA). Each determination was performed in duplicate. The free fraction was then calculated as f_p = C_free/C_total, and the results were corrected for the membrane binding measured with the control samples.

PF-06818883 was administered intravenously as a bolus infusion (15 s, volume; 0.5 mL/kg) of seven different doses (0.01–1.27 mg/kg) approximately 1 h before PET scanning [12] (Table 1). PF-06818883 was formulated by dissolving in PBS with a final pH of 7.5. A single dose was administered to four NHPs while three NHPs received two different doses.

Venous blood samples (1 mL each) were taken at − 63, − 30, − 1, 30, 60 (also 90 and 120 for first two NHPs) min after the radioligand injection of PET measurements to measure the plasma concentration of PF-06807893. PF-06818883 is a pro-drug and it converts to PF-06807893, an irreversible MAGL inhibitor (IC₅₀ = 7 nM). The blood samples were collected in a plasma-tube containing K2 EDTA as an anticoagulant. The plasma samples were harvested by centrifuging the blood sample at a speed of 1200×g for 10 min at 4 °C. The harvested plasma samples were immediately stored in − 80 °C freezer. The plasma concentration of PF-06807893 was measured at an analysis laboratory (Unilabs York Bioanalytical Solutions, UK).

The regions of interest (ROIs) were delineated manually on MRI images of each NHP for the whole brain, cerebellum, caudate, putamen, thalamus, frontal cortex, temporal cortex, and hippocampus. The summed PET images of the whole duration were co-registered to the MRI image of the individual NHP. After applying the co-registration parameters to the dynamic PET data, the time-activity curves of brain regions were generated for each PET measurement.

Kinetic parameters as K₁, k₂ and k₃ were estimated by two tissue compartment (2TC) using metabolite corrected plasma radioactivity as the input function [13]. K₁ and k₂ are uptake and clearance rate constants between arterial plasma and non-displaceable compartment. k₃ is a rate constant from non-displaceable to specific binding compartment. k₄, a rate constant from specific binding to non-displaceable compartment, was set as 0 according to the irreversible binding of [¹¹C]PF-06809247. As the main outcome measures, K_i defined as (K₁ × k₃)/(k₂ + k₃) and Patlak slope were calculated [14]. K_i represents an uptake rate constant that incorporates both net inward transport and trapping in the tissue. Due to low reliable data for parent fraction of [¹¹C]PF-06809247 at later phase, only up to 30 min data was used for the quantification. The relation between K_i by 2TC and Patlak slope was evaluated by linear correlation.

The target occupancy was calculated by the following equation: Occupancy (%) = (K_{i_baseline}− K_{i_pretreatment})/K_{i_baseline} × 100, as K_{i_baseline} is K_i at baseline condition and K_{i_pretreatment} is K_i at pretreatment condition. The K_i values calculated by 2TC and Patlak slope were used to determine the occupancy. The average occupancy by all ROIs was used for further evaluation.

The relationship between average plasma concentration (C_ave) of − 1 and 30 min of PF-06807893 (active metabolite) and occupancy using Patlak slope was estimated by an E_max model with the following equation: Occupancy (%) = C/(EC₅₀ + C) × E_max, as C is the plasma concentration of PF-06807893, EC₅₀ is the plasma concentration required to achieve 50% of the maximum occupancy, and E_max is maximum occupancy. In this analysis, E_max was set as 100%.

Whole-body PET measurements were made in two cynomolgus monkeys (two females, body weight 5350 and 5600 g). Anesthesia was administered by intramuscular injection of ketamine hydrochloride (approximately 10 mg/kg) at AFL and maintained by intravenous infusion of ketamine (4 mg/kg/h) and xylazine (0.4 mg/kg/h). The body of the NHP was immobilized using a vacuum pad. Body temperature was maintained by a Bair Hugger model 505 and monitored by an esophageal thermometer. Heart rate, blood pressure, and oxygen saturation were continuously monitored throughout the experiments. Fluid balance was maintained by continuous infusion of saline.

Whole-body PET scans were conducted using a GE Discovery PET/CT 710 (GE healthcare, Waukesha, WI, USA) with around 5 mm full width half maximum (FWHM). One low-dose CT scan was performed before intravenous administration of [¹¹C]PF-06809247 for attenuation correction. Then four series of PET acquisitions, each covering four axial fields of view (AFOV), were conducted. The four PET series consisted of two 20 s × 4 AFOV scans, three 40 s × 4 AFOV scans, four 80 s × 4 AFOV scans, and six 160 s × 4 AFOV scans respectively. PET images were reconstructed with a 3D ordered-subset expectation maximization (OSEM) algorithm with three iterations and eighteen subsets, including the time of flight information (VUE Point FX) and the point spread function correction (Sharp IR). A 2D Gaussian filter with 5.5 mm cut-off was used. The time for the bed to return to the original position was approximately 20 s, and the total duration of the whole-body scan was 100 min.

Regions of interest (ROIs) were drawn on the brain, heart, liver, kidney, lung, stomach, spleen, bone (lumbar vertebrae), gall bladder, urinary bladder and small intestine with the help of the CT images for anatomic landmarks. Radioactivity concentration in each PET scan was decay-corrected to the time of injection. Time activity curve was expressed as percentage of the injected dose (%ID) calculated as follows: radioactivity (Bq/cc) × ROI volume (cc)/injected dose (Bq) × 100.

Estimates of the absorbed radiation dose for humans was calculated with OLINDA/EXM 1.1 (Organ Level INternal Dose Assessment code) software, using the adult male (70 kg) reference model [15]. The fractional uptake in NHP organs was assumed to be equal to the uptake in human organs.

The injected radioactivity (n = 20) of [¹¹C]PF-06809247 was 146 ± 10 (mean ± SD) (range; 120–162) MBq. The molar radioactivity at the time of injection was 1510 ± 963 (392–4388) GBq/µmol, and the injected mass was 0.06 ± 0.04 (0.01–0.15) µg. Retention of brain uptake was clearly decreased after pretreatment of PF-06818883 (NHP4; 0.42 mg/kg, Fig. 1). TACs for several brain regions demonstrated decreased radioactivity, which corresponded to PET images (NHP4; 0.42 mg/kg, Fig. 2a, b, Additional file 1: Figure S1).

All radiometabolites showed shorter retention times than the parent (Fig. 3). The percent values of plasma parent fraction became less than 10% after 30 min. The percent values of plasma protein binding were 86.1 ± 2.8%.

K_i by 2TC and Patlak slope were well correlated (Table 2 and Fig. 4). The occupancy range was calculated as 22.8–94.1% and 25.4–100.5% by 2TC K_i and Patlak slope, respectively (Table 1). The average plasma concentration at − 1 and 30 min of PF-06807893 was 0.7–370.0 ng/mL. (Additional file 1: Figure S2) One plasma concentration (dose of 0.01 mg/kg for NHP5) showed below lower limit of detection. The pro-drug PF-06818883 was not detected during the PET measurement in most cases (data not shown). EC₅₀ of PF-06807893 was estimated to be 1.3 ng/mL (Fig. 5).

The injected radioactivity of [¹¹C]PF-06809247 was 272 and 302 MBq for the two measurements, respectively. The molar radioactivity at the time of injection was 186 and 494 GBq/µmol, and the injected mass was 0.6 and 0.3 µg for the two measurements, respectively. High uptakes were observed in the liver, small intestine, kidney, and brain (Figs. 6, 7a, b). The effective dose is 4.3 μSv/MBq (Table 3).