Animals
Male P-gp knock-out (KO) (Mdr1a (-/-)) mice were purchased from Taconic (Hudson, NY, USA) and male wild-type (WT) mice (FVB) were purchased from Charles River Laboratories (Brussels, Belgium) or Elevage Janvier (Le Genest Saint Isle, France). The study was approved by the Ghent University local ethical committee, and all procedures were performed in accordance with the regulations of the Belgian law. All mice had access to food and water ad libitum before the start of the study.
During the entire scan procedure, the animals were kept under anesthesia with 1.5% isoflurane (Medini N.V., Oostkamp, Belgium) administered through a mask and were placed on a heating pad (37°C).
Radiosynthesis
The synthesis of 11C-dLop was performed by the methylation of the precursor didesmethylloperamide with 11C-iodomethane (Figure 1) as reported earlier by our institution [8]. Didesmethylloperamide was kindly provided by Janssen Pharmaceutica (Beerse, Belgium), while tetrabutylammoniumhydroxide, N,N-dimethylformamide and dimethylsulfoxide were purchased from Sigma-Aldrich (Bornem, Belgium).
Comparison of 11C-dLop left heart ventricle time-activity curve and blood counter measurement time-activity curve
WT mice (n = 3) were anesthetized with isoflurane (1.5%) and cannulated with a polyethylene catheter (60 cm, PE10), filled with heparinised saline (0.9%). One end of the catheter was inserted in the carotid artery of the mice by a precise operation, and at the other end, a syringe needle was inserted. The animals were fixed on the μPET scanner, the catheter was inserted inside the detector and the withdrawing syringe was placed on the main pumping unit as described by Convert et al.
[9]. Both the μPET scanner (LabPet8; resolution, 1.5 mm) and microvolumetric blood counter (Gamma Medica-Ideas, Quebec, Canada) acquisitions were started in synchronization and subsequent 20-MBq 11C-dLop, dissolved in 100 to 150 μl saline/ethanol mixture (9/1, v/v) was injected intravenously (i.v.). Blood was collected at a constant rate of 10 μl/min for the entire 30-min acquisition time, and the blood time-activity curve was displayed in real time by the software of the microvolumetric blood counter. Immediately after the end of the 11C-dLop scan, the mice were injected with 18.5 MBq of 18F-FDG in a tail vein. Twenty minutes after 18F-FDG injection, a static μPET scan was started for 20 min.
Dynamic 11C-dLop PET data were sorted into frame sequences of 5 s (n = 12), 10 s (n = 6), 1 min (n = 4), 2 min (n = 2), 5 min (n = 2), 10 min (n = 1). A region of interest (ROI) was drawn manually around the left ventricle of the heart (Figure 2A) on the 18F-FDG scan images. Since the position of the mice was unaffected between the 11C-dLop and the 18F-FDG scan, the ROI of the left heart ventricle on the 18F-FDG scan could be pasted on the 11C-scan images (Figure 2B) to derive an arterial blood input function. Data from the blood counter were corrected for dispersion with the following formula: C
a(t) = g(t) + τ
disp × (dg/dt), where C
a(t) is the real whole blood activity curve in mice, g(t) the measured data and dg/dt the derivative of g. τ
disp, the dispersion factor was calculated according to Convert et al.
[9].
The estimated input function (18F-FDG-derived) and the measured input function (blood counter) were compared by a direct and indirect method. The direct method, as described by Fang and Muzic [10], evaluated the input functions by calculating the area under the curve (AUC) difference. Indirect comparison examined the impact of the estimated 18F-FDG-derived input function on an estimated kinetic parameter from the kinetic model, like the K
1/k
2 ratio, as described later on (see PET data analysis and kinetic modeling of 11C-dLop). The AUC difference was calculated as absolute values of (AUCPET - AUCbloodcounter)/AUCbloodcounter × 100 and the error percentage of K
1/k
2 ratio as absolute values of (K
1/k
2PET - K
1/k
2bloodcounter)/(K
1/k
2bloodcounter) × 100.
Kinetic model for 11C-dLop
PET experiments
Before positioning the anesthetized mice on the scanner, WT mice (n = 3) were injected i.v. 30 min before the tracer injection with saline (100 μl, controls, n = 3) or 50 mg cyclosporine/kilogram body weight (n = 3) (Novartis, Vilvoorde, Belgium). Approximately 20 MBq of 11C-dLop, dissolved in 100 to 250 μl saline/ethanol mixture (9/1, v/v) was administered via a tail vein, and the dynamic μPET scan was initiated. After the 11C-dLop scan, the mice were injected with approximately 18.5 MBq of 18F-FDG in a tail vein (100 μl). Twenty minutes after the 18F-FDG injection, a static μPET scan was started for 20 min. KO mice (n = 3) were handled in the same way as the WT mice, with exception of the pretreatment procedure.
Determination of percent parent compound in plasma and plasma-whole blood ratio of 11C-dLop
The determination of percent parent compound (11C-dLop) in plasma over time was performed in WT (pretreated with saline or 50 mg cyclosporine/kilogram body weight, n = 3 per group and per time point) and KO mice (n = 3 per time point) using a high-performance liquid chromatography (HPLC) assay. Thirty minutes after pretreatment, the mice were injected with 22.2 to 30 MBq of 11C-dLop (300 μl) and were killed at 1, 10, and 30 min postinjection (p.i.). Blood was collected by cardiac puncture, and the brain was excised. Plasma (200 μl) was obtained after centrifugation (3,000 g, 6 min). Subsequently, 800 μl and 1 ml of acetonitrile (Chem-Lab N.V., Zedelgem, Belgium) were added to the brain and plasma, respectively. Both samples were vortexed (1 min), centrifuged (3,000 g, 3 min), and counted for radioactivity. A supernatant was isolated and analyzed with an HPLC system (Grace Econosphere C18, 10 μm, 10 × 250 mm, eluted with acetonitrile/20 mM sodium acetate (70/30, v/v) as mobile phase at 7 ml/min). Elution fractions of 30 s were collected and counted for radioactivity. Percent parent compound was calculated as the sum of the counts determined in the fractions containing 11C-desmethylloperamide (determined by co-injection with cold desmethylloperamide and UV detection at 220 nm) divided by the total counts of all collected fractions.
To determine the plasma-whole blood ratio, the mice (n = 3) were injected with 4.80 to 5.55 MBq of 11C-dLop (300 μl) and were killed at 0.5, 1, 2, 3, 5, and 10 min p.i.. Blood was collected from the heart by cardiac puncture, counted for radioactivity, and centrifuged for 10 min (3,000 g). Plasma and blood pellet were separated, weighted, and counted for radioactivity. To obtain the plasma-to-whole blood ratio, counts from plasma and blood pellet were averaged for weight.
PET data analysis and kinetic modeling of 11C-dLop
Dynamic 11C-dLop PET data were sorted into frame sequences as mentioned above. The arterial blood input curve obtained from the μPET was corrected for plasma-whole blood ratio and metabolites. An ROI was signed around the whole brain on the 18F-FDG scan images and was used to determine the 11C-dLop brain time-activity curve (Figure 3). All data were loaded and analyzed with the PMOD software package (version 3.1., PMOD Technologies Ltd., Zurich, Switzerland).
Standardized uptake values (SUVs) were calculated using the following equation: A/(ID/BW), where A is the decay-corrected radioactivity concentration in the brain (measured in kilobecquerels per cubic centimeter), ID is the injected dose of 11C-dLop (measured in kilobecquerels), and BW is the mice body weight (measured in grams), resulting in SUVs expressed as grams per milliliter. To account for mice differences in the blood concentrations, which are the driving force for the brain concentrations, the brain-to-blood ratio was calculated using the SUVs in the blood and in the brain. A one-tissue compartment model was investigated, in which the rate constants K
1 and k
2 represent, respectively, the rate of transport from plasma to brain and the rate of outflow from the brain to the plasma. A two-tissue compartment model (with or without k4 fixed to 0) was also considered, since interaction of 11C-dLop in the brain might occur. The volume of vasculature was set as a variable in the compartment model.
Statistical analysis
All calculated outcome parameters, differences between WT mice with and without cyclosporine, and KO mice were investigated with ANOVA and Bonferroni post hoc testing. The level of statistical significance was set to 5%.