Positron emission tomography imaging using an inverse agonist radioligand to assess cannabinoid CB1 receptors in rodents

Abstract

[11C]MePPEP is an inverse agonist and a radioligand developed to image cannabinoid CB1 receptors with positron emission tomography (PET). It provides reversible, high specific signal in monkey brain. We assessed [11C]MePPEP in rodent brain with regard to receptor selectivity, susceptibility to transport by P-glycoprotein (P-gp), sensitivity to displacement by agonists, and accumulation of radiometabolites. We used CB1 receptor knockout mice and P-gp knockout mice to assess receptor selectivity and sensitivity to efflux transport, respectively. Using serial measurements of PET brain activity and plasma concentrations of [11C]MePPEP, we estimated CB1 receptor density in rat brain as distribution volume. CB1 knockout mice showed only nonspecific brain uptake, and [11C]MePPEP was not a substrate for P-gp. Direct acting agonists anandamide (10 mg/kg), methanandamide (10 mg/kg), CP 55,940 (1 mg/kg), and indirect agonist URB597 (0.3 and 0.6 mg/kg) failed to displace [11C]MePPEP, while the inverse agonist rimonabant (3 and 10 mg/kg) displaced >65% of [11C]MePPEP. Radiometabolites represented ~13% of total radioactivity in brain between 30 and 120 min. [11C]MePPEP was selective for the CB1 receptor, was not a substrate for P-gp, and was more potently displaced by inverse agonists than agonists. The low potency of agonists suggests either a large receptor reserve or non-overlapping binding sites for agonists and inverse agonists. Radiometabolites of [11C]MePPEP in brain caused distribution volume to be overestimated by approximately 13%.

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 18 min in the wild type mice.

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 30 min before or 25 min (↑) after [¹¹C]MePPEP. (B) Forebrain time activity curves in a wild type (□) and CB₁ receptor knockout (▲) mouse.

Figure 3

Displacement of [¹¹C]MePPEP by agonists and inverse agonist in rats. Drugs were administered IV 40 min after [¹¹C]MePPEP. Anandamide (△) 10 mg/kg; methanandamide (▽) 10 mg/kg; CP 55,940 (◇) 1 mg/kg; URB597 (anandamide reuptake inhibitor, ×) 0.6 mg/kg; URB597 with anandamide (+) 0.6 mg/kg and 10 mg/kg, respectively; and rimonabant (○) 3 mg/kg. The curves are representative of two studies with anadamide, CP 55,940, URB597 both alone and with anandamide, and rimonabant, and of one study with methanandamide. The baseline curve (■) is the mean of 8 animals.

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. (P = 0.007 by F-test). (B) The plasma concentration of [¹¹C]MePPEP separated from radiometabolites from same animal as panel A peaked at 45 s and rapidly declined thereafter. The range of the concentration was large, and the insert has an overlapping value at 5 min.

Figure 5

Time instability of distribution volume (V_T) 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 in a time frame from 100 to 120 min is plotted at 110min. V_T was calculated for one-(■) and two-(●) tissue compartment models and expressed as a ratio to that determined with the complete scanning data from 0 to 120 min. The terminal V_T for forebrain was 9.4 ± 3.7 mL•cm^-3 for one-tissue and 10.0 ± 4.2 mL•cm^-3 for two-tissue compartment models. The error bar represents SD of the ratios, n = 4 animals.

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 less lipophilic than MePPEP were eluted by HPLC. The most polar metabolite (1) was followed by two more lipophilic metabolites (2 and 3), separated from MePPEP (4). This representative chromatograph comes from a single animal.