Measuring glucocorticoid receptor expression in vivo with PET

The glucocorticoid receptor (GR) is an emerging drug target for several common and deadly solid tumors like breast and prostate cancer, and clinical trials studying the antitumor effects of GR antagonists are beginning. Since GR expression can be variable in tumor cells, and virtually all normal mammalian tissues express some GR, we hypothesized that an imaging tool capable of detecting GR positive tumors and/or measuring GR occupancy by drug in tumor and normal tissues could improve the precision application of anti-GR therapies in the clinic. To this end, we developed a fluorine-18 labeled corticosteroid termed GR02 that potently binds the endogenous ligand binding pocket on full length GR. Binding of 18F-GR02 was suppressed in many normal tissues by co-treatment with mifepristone, a GR antagonist in human use, and was elevated in many normal tissues among mice lacking circulating corticosteroids due to adrenalectomy. 18F-GR02 also accumulated in GR positive subcutaneous and subrenal capsule prostate cancer models, and uptake in tumors was competed by mifepristone. Combined with a straightforward and high yielding radiosynthesis, these data establish the foundation for near-term clinical translation of 18F-GR02.

All chemicals, including anhydrous solvents, were purchased from Sigma Aldrich and used without further purification, unless otherwise noted. Deuterated solvents for NMR spectroscopy were purchased from Cambridge Isotopes Laboratories. NMR spectra were recorded on a Bruker Advance III 400 MHz spectrometer at the UCSF NMR lab, and spectra were analyzed using Mestrelab software. High resolution mass spectra were recorded on a Bruker microTOF II at the University of Notre Dame's Mass Spectrometry and Proteomics core facility. Fluorine-18 was obtained as a NaF salt from the cyclotron core at UCSF.
The C21 primary alcohol on prednisolone was converted to a methanesulfonyl ester following a previously described protocol [2]. To a solution of prednisolone (1.5 g, 4.16 mmol, 1 eq.) in pyridine (10 mL) at 0° C was added methanesulfonyl chloride (1.5 mL, 19.4 mmol, 4.7 eq.). The reaction was stirred for 2 hours at 0° C. The reaction was added dropwise to a rapidly stirring solution of ice water (100 mL) and stirring was continued until the ice completely melted. The solution was filtered under reduced pressure and the collected precipitate was washed with cold water and dried overnight under high vacuum.

radiosynthesis of 18 F-Gr01
[ 18 F]-fluoride anion (3.7-18.5 GBq) was eluted off a QMA Sep-Pak cartridge using 0.5 mL of a K 2.2.2 /KHCO 3 solution (11 mg/mL of K 2.2.2 , 1 mg/mL of K 2 CO 3 , 9:1 v:v acetonitrile:H 2 O). The resulting solution was subjected to 3 drying cycles at 115°C under a gentle stream of nitrogen. Compound 4 (~5 mg) was dissolved in anhydrous DMSO (800 mL) and added to [ 18 F]-fluoride, and the resulting solution was sealed and heated at 130° C for 20 min. 18 F-GR01 was isolated via semi-preparative reverse phase-HPLC coupled to a radiation detector (5%-95% gradient of acetonitrile in H 2 O with 0.1% formic acid.) The product fraction isolated off of the HPLC was diluted to ~30 mL in H 2 O and loaded onto a C18-light Sep-Pak cartridge. The product fraction isolated off of the HPLC was diluted to ∼30 mL in H 2 O and loaded onto a C18-light Sep-Pak cartridge preconditioned with ethanol (5 mL) and H 2 O (5 mL). The cartridge was washed with 10 mL H 2 O and the activity was eluted with ethanol (500 µL). The resulting solution was then concentrated for 1 h at 50° C under reduced pressure, and 18 F-GR02 was then dissolved in a solution of 15% ethanol (aq.) for animal studies. The radiotracer was confirmed to be >99% radiochemical purity prior to administration in animals.

radiosythesis of 18 F-Gr02
[ 18 F]-fluoride anion (3.7-18.5 GBq) was eluted off a QMA Sep-Pak cartridge using 0.5 mL of a K 2.2.2 / KHCO 3 solution (11 mg/mL of K 2.2.2 , 1 mg/mL of K 2 CO 3 , 9:1 v:v acetonitrile:H 2 O). The resulting solution was subjected to 3 drying cycles at 115° C under a gentle stream of nitrogen. Ethyl 4-(tri-methylammonium triflate) benzoate (∼5 mg) was dissolved in anhydrous acetonitrile (800 μL) and added to [ 18 F]-fluoride, and the resulting solution was sealed and heated at 90° C for 10 min. A 25% solution of tetra-propylammonium hydroxide (20 µL) in 0.5 mL acetonitrile was added in the reaction vial, and the solution was sealed and heated at 90° C for 8 minutes. The solution was dried down and washed with acetonitrile three times. A solution of N,N,N′,N′-tetra-methyl-O-(N-succinimidyl) uronium tetrafluoroborate (30 mg) in acetonitrile (1 mL) was added to the reaction vial and heated for 8 minutes at 90° C. N-succinimidyl 4-[ 18 F]-fluorobenzoate was then eluted off a HLB plus Sep-Pak cartridge in acetonitrile.
N-succinimidyl 4-[ 18 F]-fluorobenzoate was reacted with 8 (2.5 mg) in a solution of 1:1 dimethyl sulfoxide:acetonitrile (500 µL total volume) with triethylamine (25 µL) at 90° C for 10 min. 18 F-GR02 was isolated via semi-preparative reverse phase-HPLC coupled to a radiation detector (20-60% gradient of acetonitrile in H 2 O with 0.1% formic acid). The product fraction isolated off of the HPLC was diluted to ∼30 mL in H 2 O and loaded onto a C18-light Sep-Pak cartridge. The Sep-Pak cartridge was washed with ethanol (5 mL) and H 2 O (5 mL), and then the collected activity was added onto the Sep-Pak cartridge and was washed with 10 mL H 2 O and the activity was eluted with ethanol (0.5 µL). The resulting solution was then concentrated for 1 h at 50° C under reduced pressure, and 18 F-GR02 was then dissolved in a solution of 15% ethanol (aq.) for animal studies. The radiotracer was >99% pure prior to administration in animals.t