The Synthesis and Structural Requirements for Measuring Glucocorticoid Receptor Expression In Vivo with (±)-¹¹C-YJH08 PET

In vivo blocking studies reveal that (±)-¹¹C-YJH08 specifically binds GR in vivo. (A) Biodistribution data collected 20 min after injection showing suppression of (±)-¹¹C-YJH08 uptake in several mouse tissues by dexamethasone (Dex., n = 5/arm). Dexamethasone was administered via 2 routes involving oral gavage of 50 mg/kg for 3 d before radiotracer injection or administration of water-soluble dexamethasone–cyclodextrin complex via intraperitoneal injection at 10 mg/kg 1 h before radiotracer injection. *P < 0.05. At right are shown blocking data observed in brain. *P < 0.01. (B) Percentage changes in radiotracer uptake per tissue among mice exposed to dexamethasone or dexamethasone–cyclodextrin versus vehicle. (C) Representative CT and PET/CT images showing suppression of (±)-¹¹C-YJH08 binding in selected organs in vivo by dexamethasone treatment. Dex. = dexamethasone; Dex-CD = dexamethasone–cyclodextrin complex; ID = injected dose.

Resolution and analysis of (R)/(S) YJH08 enantiomers reveals similar affinity for GR in vitro and biodistribution patterns in vivo. (A) HPLC trace over chiral stationary phase shows separation of racemic YJH08 to provide (R) and (S) isomers. Starred peak at 29.1 min was isolated in and recrystallized in hexanes to give single crystal. (B) Oak Ridge thermal ellipsoid plot showing absolute conformation of crystal, which was (R) enantiomer of YJH08. Thermal ellipsoids are drawn at 40% probability. (C) ³H-dexamethasone displacement curves on DU145 cells showing relative affinities of (R)-YJH08, (S)-YJH08, and dexamethasone. Each ligand has affinity of less than 5 nM, and all are equipotent. R ² values for curve fitting were 0.99 (R), 0.94 (S), and 0.97 (dexamethasone). (D) Mouse biodistribution data showing tissue uptake of (R)- and (S)-¹¹C-YJH08. Data were acquired 20 min after injection in male C57BL6/J mice. (E) Structures of (S) and (R) enantiomers of YJH08. Bonds are colored-coded to enable distinction of small-molecule structure in ligand-binding domain of GR. Below each enantiomer are results of molecular dynamics simulation to identify lowest-energy interaction between ligand binding domain of GR and respective enantiomer. Receptor side chains within 5 Å of respective YJH08 enantiomer are shown. For clarity, 2 docked poses are shown for each ligand–receptor complex. At bottom is shown schematic representation of docked pose for each enantiomer within GR ligand binding domain, to highlight amino acids within 5 Å of respective enantiomer. ID = injected dose.

Summary of K_i, 95% CIs, and coefficient of determination for YJH08. Data were calculated from ³H-dexamethasone displacement assay on cells. K_i was calculated using 1-site K_i fit nonlinear regression with Prism. Data are representative of 2 independent assays.