[11C]MePPEP was prepared with 100 ± 0%, radiochemical purity and 99.9 ± 0%, chemical purity. The specific activity at time of radioligand injection was 112.0 ± 54.7 GBq/μmol (n = 34 batches). The mean injected activity was 13.1 ± 5.5 MBq (0.12 ± 0.07 nmol) for mouse studies and 49.3 ± 23 MBq (0.44 ± 0.30 nmol) for rat studies.
Receptor Occupancy by [11C]MePPEP
Due to the relatively small body mass of rodents, PET ligands can cause significant (> 5–10%) receptor occupancy, which violates the assumptions of tracer methodologies. We estimated the receptor occupancy in rodents caused by the injected mass dose of MePPEP based on reports of receptor density and on our measurements of brain uptake. The density (Bmax
) of CB1
receptors in cerebellum is 1750 fmol per mg protein in rat (Hirst et al., 1996
) and 1810 fmol per mg protein in mouse (Abood et al., 1997
). Assuming that 10% of brain weight is protein, these densities correspond to 175 nM in rats and 181 nM in mice. The maximal brain uptake was ~190% SUV (2.4 nM) in rats and ~260% SUV (11.2 nM) in mice. Assuming that 80% of the ligand was specifically bound, the maximal occupancy of CB1
receptors was 1.1% (= 80% * 2.4 nM/175 nM) in rats and 5.0% (= 80%* 11.2 nM/181 nM) in mice.
[11C]MePPEP Was Not a P-gp Substrate in Mouse
[11C]MePPEP was not a substrate for P-gp, an efflux transporter expressed at the blood-brain barrier. P-gp knockout (n = 4) and wild type (n = 4) mice were injected with [11C]MePPEP. Brain uptake peaked at ~20 min with similar concentrations of 200 and 182% SUV in the knockout and wild type mice, respectively (). In addition, the area under the curve of activity versus time from 0 to 90 min in knockout mice was only 1.1 times that of wild type mice with no significant difference between the two groups (P = 0.58, two-tailed student t-test).
Figure 1 Forebrain radioactivity after injecting [11C]MePPEP in mice. Symbols represent the mean ±SD for wild type (▲; n = 4) and P-gp knockout (□; n = 4) mice. The peak uptake was 199% SUV at 18 min in the knockout mice and 181% SUV at (more ...)
To determine whether greater brain uptake in P-gp knockout than in wild type mice was merely the result of higher plasma concentration of the radioligand, we euthanized animals at 30 min and measured radioactivity in brain and plasma. The concentrations of [11C]MePPEP in plasma were 5.5 and 8.2% SUV for wild type and P-gp knockout mice, respectively, with corresponding forebrain uptake of 191 and 383% SUV (). The brain to plasma ratio at this single time point was actually lower in P-gp knockout (45 = 383%/8.2%) compared to wild type (65 = 191%/5.5%) mice. Thus, [11C]MePPEP was not a substrate for P-gp in mice as assessed with brain uptake, either corrected or uncorrected for the plasma concentration of radioligand.
Effect of P-gp Gene Knockout on Plasma and Brain Concentrations of [11C]MePPEP in Mice
Specificity of [11C]MePPEP for CB1 Receptors in Mice
[11C]MePPEP was selective for the CB1 receptor in mice when assessed by pharmacological displacement of a highly selective CB1 inverse agonist. Baseline and displacement scans were acquired with a 3 h interval in the same animal. Rimonabant (3 mg/kg IV) administered at 25 min after [11C]MePPEP displaced more than half of the brain activity by 90 min compared to the baseline scan. After displacement by rimonabant, brain uptake decreased from peak of 260% at 25 min to about 100% SUV at 90 min. The residual activity after displacement was similar to that of a pre-block study in a different animal (). In the preblock study, the same dose of rimonabant was given at 30 min before [11C]MePPEP. A ratio of areas under the curve of preblocked vs. baseline animals from 0 to 90 min was 0.32, indicating that 68% of brain uptake was specifically bound to CB1 receptors.
Figure 2 Displacement of brain activity in P-gp knock out and CB1 receptor knockout mice. (A) P-gp knockout mice were examined under baseline (□), displacement (▲), and preblocked (○) conditions. Rimonabant (3 mg/kg, IV) was administered (more ...)
Comparing CB1 knockout to wild type mice with [11C]MePPEP yielded results of receptor selectivity consistent with the displacement by rimonabant. Brain activity in CB1 receptor knockout mice was 100% SUV at 3 min and quickly declined to 30% SUV at 90 min. In contrast, maximal brain uptake in wild type animals was 170% SUV at 20 min and slowly declined to 100% SUV at 90 min (). Thus, compared to knockout animals, the CB1 receptors in the wild type mice bound more radioligand and therefore required greater time to achieve peak activity in brain. The ratio of areas under the curve from 0 to 90 min in knockout compared to wild type mice was 0.35, indicating that 65% of the uptake was specifically bound to CB1 receptors.
Displacement by Agonists vs. Inverse Agonists
Direct and indirect acting agonists caused no discernable changes in brain uptake (), and all agonist curves at their terminal points were within one standard deviation of baseline scans (n = 8). The direct acting agonists anandamide (10 mg/kg IV, n = 2), methanandamide (10 mg/kg IV, n = 1), and CP 55,940 (1 mg/kg IV, n = 2) were administered at 40 min. Inhibition of anandamide reuptake by inhibiting both FAAH, the enzyme that metabolizes anandamide, and the anandamide transporter has an indirect agonist effect by increasing the concentration of this endocannabinoid. The anandamide reuptake process blocker, URB597, was administered at 40 min after radioligand injection in two animals (0.3 mg/kg and 0.6 mg/kg IV), and as a pretreatment at 1 h before radioligand injection in a third animal (0.3 mg/kg IV). Two additional animals were pretreated with URB597 (0.6 mg/kg IV), and anandamide (10 mg/kg IV) was administered 40 min after radioligand injection.
The direct agonist agents were administered at doses that had pharmacological effects. Anandamide transiently decreased respiration rate, with shallow and irregular breathing. Methanandamide caused the death of one animal after 35 min, possibly by respiratory depression. CP 55,940 caused decreased respiration rate for the duration of the scan, and later the death of one animal after 50 min. In contrast, URB597 had no noticeable pharmacological effect.
In contrast to the agonists, rimonabant rapidly displaced brain activity when administered at 3 mg/kg IV at 40 min after the radioligand (n = 2 rats). Compared to baseline studies (n = 8 rats), rimonabant decreased brain activity by 63% at the end of the scan (2 h) (). Rimonabant had no noticeable effects on temperature or respiration.
Ex Vivo Mass Spectrometry
Our results of agonist state assessment using PET were reliably replicated using ex vivo mass spectrometry. Rimonabant (an inverse agonist) caused >90% receptor blockade, whereas CP 55,940 (an agonist) caused no receptor blockade. More specifically, rimonabant (10 mg/kg) pretreatment decreased MePPEP in rat frontal cortex at 60 min from a baseline, vehicle-treated concentration of 34.4 ± 5.3 to 2.3 ± 0.6 ng/g. In contrast, CP 55,940 (1 mg/kg) caused no significant change in the concentration of MePPEP against vehicle-treated rats from 40.0 ± 4.4 to 40.3 ± 1.5 ng/g. Rimonabant appeared to displace or block more MePPEP binding measured by ex vivo mass spectrometry (>90%) than by in vivo PET (~65%).
Compartmental Modeling of [11C]MePPEP in Rat Brain
Pharmacokinetic modeling of brain and plasma time-activity data showed that estimates of total distribution volume (VT) achieved stable, well identified values during a 2-hour scan. We used brain time-activity data and serial plasma concentrations of [11C]MePPEP to estimate VT, which is proportional to receptor density (Bmax). Due to the limited resolution of PET, the measured concentrations of brain activity were similar in all regions. For this reason, we sampled activity from a large area that included the entire forebrain.
In three out of four rodents the two-tissue compartment model was superior to the one-tissue compartmental model, and we used both to assess the minimal imaging time required for stable identification of VT. The brain time-activity data from 0–120 min (n = 4 rats) as well as the measured serial concentration of [11C]MePPEP were analyzed with compartmental modeling. The two-tissue compartment model gave better statistical fit of brain data than the one-tissue compartment in three of the four rats with P < 0.01, with the other animal achieving P = 0.08 (F-test; ). To assess the stability of distribution volume over time, we increasingly truncated the brain data, from 0–120 to 0–30 min, at terminal times of 120, 100, 80, 60, 50, 40, and 30 min. The calculated distribution volume increased asymptotically from 30 to 120 min and reached 90% of the terminal value within ~70 and 55 min for one-tissue and two-tissue compartmental models, respectively ().
Figure 4 Brain and plasma concentrations of radioactivity after injecting [11C]MePPEP in rats. (A) The brain time-activity curve of this rat was visually and statistically better fit by the two- (solid line) than the one- (dashed line) tissue compartment model. (more ...)
Figure 5 Time instability of distribution volume (VT) in rat. Increasingly truncated intervals of brain time-activity data were analyzed from time 0 to that indicated on the x-axis. Time points represent the mid-point of the acquisition time, therefore data captured (more ...)
The concentration of unchanged [11C]MePPEP rapidly declined in plasma. From a peak of ~1,100% SUV at about 45 s, its concentration declined to 50% SUV at 5 min and further to 12% SUV at 120 min (). Metabolism was also rapid, as shown by the accumulation of radiometabolites in plasma. [11C]MePPEP as a percentage of total radioactivity declined from 100% in the injected sample to 50 ± 9.1% at 5 min, 27 ± 5.8% at 26 min, and 16 ± 1.5% at 60 min (). Rat plasma samples contained at least three radiometabolites, which eluted earlier than [11C]MePPEP on HPLC and were, therefore, less lipophilic than the parent radioligand ().
Figure 6 Metabolism of MePPEP in rat. (A) Metabolism of MePPEP (■) was rapid with most of the radioactivity comprised of the most polar metabolite (×) by the end of the scan. The curves represent the mean of 4 animals. (B) At 90 min three metabolites (more ...)
Ex Vivo Measurements in Rat
To assess the chemical composition of radioactivity in brain over time, we euthanized three animals at 30, 60, and 90 min, and two animals at 120 min. Radiometabolites of [11C]MePPEP were present in rat brain but at relatively stable percentage (~13%) of total radioactivity. The percentages of radiometabolites in rat brain were 12.9 ± 0.4% at 30 min, 13.2 ± 6.0% at 60 min, 16.4 ± 9.3% at 90 min, and 12.3 ± 4.8% at 120 min.