Evaluation of 11C-NR2B-Sme and Its Enantiomers As PET Radioligands for Imaging the NR2B Subunit Within the NMDA Receptor Complex

Journal of Nuclear Medicine, published on January 10, 2020 as doi:10.2967/jnumed.119.235143

Evaluation of 11C-NR2B-SMe and its Enantiomers as PET Radioligands for Imaging the NR2B Subunit within the NMDA Receptor Complex in Rats  Lisheng Cai,1 Jeih-San Liow,1 Cheryl L. Morse,1 Sanjay Telu,1 Riley Davies,1 Michael P. Frankland,1 Sami S. Zoghbi,1 Ken Cheng,2 Matthew D. Hall,2 Robert B. Innis,1 Victor W. Pike1

1Molecular Imaging Branch, National Institute of Mental Health (NIMH), National Institutes of Health, Bethesda, MD 20892, USA. 2NCATS Chemical Genomics Center (NCGC), National Institutes of Health, Rockville, MD 20850, USA.

Running title: Radioligands for Imaging NR2B Subunit

Correspondence to: Dr. Lisheng Cai, Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, 10 Center Drive, Building 10, Room B3 C346, Bethesda, MD 20892, USA. Tel. (301) 451-3905; Fax (301) 480- 5112; E-mail: [email protected].

1 ABSTRACT [S-Methyl-11C](±)-7-methoxy-3-(4-(4-(methylthio)phenyl)butyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin- 1-ol (11C-NR2B-SMe) and its enantiomers were synthesized as candidates for imaging the NR2B subunit within the N-methyl-D-aspartate receptor with positron emission tomography (PET). Methods: Brains were scanned with PET for 90 min after intravenous injection of one of the candidate radioligands into rats. To detect any NR2B specific binding of radioligand in brain, various pre-blocking or displacing agents were evaluated for their impact on the PET brain imaging data. Radiometabolites from brain and other tissues were measured ex vivo and in vitro. Results: Each radioligand gave high early whole brain uptake of radioactivity, followed by a brief fast decline and then a slow final decline. 11C-(S)-NR2B-SMe was studied extensively. Ex vivo measurements showed that radioactivity in rat brain at 30 min after radioligand injection was virtually unchanged radioligand. Only less lipophilic radiometabolites appeared in plasma. High-affinity NR2B ligands, Ro- 25-6981, ifenprodil, and CO10124, showed increasing preblock of whole brain radioactivity retention with increasing dose (0.01 to 1.25 mg/kg, i.v.). Five 1 antagonists (FTC146, BD1407, F3, F4, and NE100) and four 1 agonists ((+)-pentazocine, (±)-PPCC, PRE-084, (+)-SKF10047) were ineffective preblocking agents, except FTC146 and F4 at high dose. Two potent σ1 receptor agonists, TC1 and SA4503, showed dose-dependent preblocking effects in the presence or absence of pharmacological 1 receptor blockade with FTC146. Conclusions: 11C-(S)-NR2B-SMe has adequate NR2B-specific PET signal in rat brain to warrant further evaluation in higher species. TC1 and SA4503 likely have off-target binding to NR2B in vivo.

Keywords: NR2B subunit; NMDA receptor; GluN2B; Sigma 1; NR2B-SMe.

2 INTRODUCTION N-Methyl-D-aspartate (NMDA) receptors are ligand and voltage-gated ion channels that mediate influx of Ca2+, Na+, and K+ into the synapse (1). These receptors are expressed throughout the central nervous system (CNS) and play key physiological roles in synaptic plasticity, learning, and memory. NMDA receptors are also implicated in the pathophysiology of several CNS disorders (2-4) and more recently have been identified as a target for the treatment of disease-associated genomic variation (5). NMDA receptors exist as diverse subtypes as a result of variation in the assembly of seven subunits [NR1, NR2 subunits (NR2A−NR2D), and NR3 (A or B)] into tetrameric receptor complexes. Unique structural features of the NMDA receptor subtypes account for the tuning of their physiological roles and their distinct pharmacological properties. NMDA receptors, especially those enriched with NR2B subunits, endow the prefrontal cortex not only with important functionality but also with a major vulnerability to environmental insults and to risk factors for psychiatric disorders (6). NMDA receptors have distinct binding sites for L-glutamate, glycine, D-serine, polyamines, Mg2+, phencyclidine, and Zn2+. Ligands for NMDA are of four types, namely glutamate binding site ligands, glycine binding site ligands, channel blockers, and N-terminal domain binding ligands. Some NMDA ligands have been developed into drugs, such as memantine for the treatment of Alzheimer’s disease (7,8). Attempts to produce radioligands for imaging NMDA receptors in living subjects with positron emission tomography (PET) is an active area of research that has nonetheless met with limited success (9,10). NR2B is the most studied subunit within the NMDA receptor complex. This subunit is expressed throughout the CNS, with highest concentration in forebrain and the dorsal horn of the spinal cord (11). The NR2B subunit is a therapeutic target for schizophrenia, stroke, and neurodegenerative diseases, especially neuro-pain. Therapeutics targeting NR2B rather than the NMDA channel may have fewer side- effects. The quantification of NMDA NR2B subunits receptors in vivo with PET could help to elucidate the contribution of this receptor to neuropsychiatric disorders and also assist in drug development (10). A promising PET radioligand for NR2B, named 11C-(R)-Me-NB1, has been reported very recently and shows modest displaceable signal for NR2B in rats in vivo (12,13). Here, within a broader medicinal chemistry campaign to develop PET radioligands with high specific signal and low background noise, we discovered (±)-7-methoxy-3-(4-(4- (methylthio)phenyl)butyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-ol (NR2B-SMe) (for chemical structure, see Supplemental Fig. 1). This compound shows high affinity for NR2B in the nanomolar range,

moderate lipophilicity, and amenability to labeling with carbon-11 (t1/2 = 20.4 min). We therefore

3 prepared 11C-NR2B-SMe and its enantiomers from S-methyl propionyl precursors for evaluation as NR2B PET radioligands in rat.

MATERIALS AND METHODS General Methods Animal experiment ethics and approval. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (https://grants.nih.gov/grants/olaw/guide-for-the- care-and-use-of-laboratory-animals.pdf) and were approved by the National Institute of Mental Health (NIMH) Animal Care and Use Committee. Statistics. Subsequent numerical data are expressed as mean  S.D. for n > 2, or as mean and range for n = 2. Absolute Configuration and In Vitro Properties. The absolute configurations of (–)-NR2B-SMe and (+)-NR2B-SMe were determined by vibrational circular dichroism (see Supplemental Information for more information). Radiosynthesis Preparation of 11C-NR2B-SMe. (±)-Methyl 3-((4-(4-(1-hydroxy-7-methoxy-1,2,4,5-tetrahydro- 3H-benzo[d]azepin-3-yl)butyl)phenyl)thio)propanoate (NR2B-ester; 0.5 mg; 1.1 μmol) in DMF (0.40 mL) and tetra-n-butylammonium hydroxide in methanol (5 L of 1 M stock solution in MeOH; 5 μmol) were loaded into a septum-sealed reaction vial (1-mL neck vial, Waters Corp.) of a Synthia apparatus (14). 11C-Iodomethane (> 37 GBq) was then swept with a stream of helium (15 mL/min) into the vial from a Microlab module and left at RT for 5 min. 11C-NR2B-SMe was separated out with HPLC on a Luna C18 column (10 μm, 4.6 × 250 mm; Phenomenex; Torrance, CA) eluted at 6 mL/min for 20 min with TFA (0.1% w/v) in MeCN-water (35:65 v/v). Eluate was monitored for absorbance at 255 nm and radioactivity. 11 The fraction containing C-NR2B-SMe (tR = 10.0 min) was collected in ascorbic acid solution (10 mg/mL; 0.1 mL) and then rotary evaporated to dryness (80 C, water bath). The residue was dissolved in

sterile saline for injection USP (4 mL) containing ethanol USP (1 mL) and aq. NaHCO3 solution (8.4% w/v; 40 µL). The solution (pH ~ 5.5) was sterile-filtered into a sterile and pyrogen-free dose vial that had been preloaded with sterile saline for injection USP (5 mL). This product was analyzed with HPLC on an X-Bridge C18 column (10 μm, 4.6 × 250 mm; Waters Corp.) eluted for 15 min with TFA (0.1% w/v) in MeCN-water (40:60 v/v) at 1.5 mL/min. Eluate was monitored for absorbance at 255 nm and for radioactivity. The retention time of 11C-NR2B-SMe was 9.5

4 min. The response of the analytical system had been calibrated for mass of NR2B-SMe to allow molar activity to be calculated. The identity of the radioligand was confirmed by its co-mobility in analytical HPLC with added reference compound and by LC-MS of residual carrier after decay of the formulated radioligand overnight. The formulated radioligand had greater than 98% radiochemical purity. The stability of formulated 11C- NR2B-SMe was assessed with radio-HPLC at 1 and 2 h after radiosynthesis. Radiometabolites of 11C-NR2B-SMe in rat tissues ex vivo Rats were anesthetized with 1.5% isoflurane in oxygen. Formulated 11C-NR2B-SMe (200 µL; 37 MBq) was then injected intravenously through the penile vein of each rat. Other experimental parameters related to this study are listed in Supplemental Table 1. Thirty minutes after injection of 11C-NR2B-SMe, a large anticoagulated (heparin) blood sample was drawn from each rat. The rats were then immediately sacrificed by decapitation, and their brains and myocardial tissues were excised. For Brain and Myocardium. The radioactive tissue was weighed, placed in acetonitrile (1 mL), measured for radioactivity, and homogenized along with carrier NR2B-SMe (50 µg) using a hand-held tissue Tearor (model 985-370; BioSpec Products Inc.; Bartlesville, OK). Water (500 µL) was then added and the tissue was further homogenized before measurement of radioactivity. The homogenate was centrifuged at 10,000 g for 1 min. The supernatant was then analyzed with general HPLC method A

(NR2B-SMe, tR ~ 6.4 min. See supplemental material). The precipitate was measured for radioactivity to determine the recovery of radioactivity in the supernatant that had been injected onto the HPLC. For Plasma. Plasma was separated from blood cells and a sample (50 µL) was measured for radioactivity. An aliquot (450 µL) was placed in acetonitrile (720 µL) along with carrier NR2B-SMe (5 µg) and mixed well. Water (100 µL) was added, mixed well, measured for radioactivity, and centrifuged at 10,000 g for 1 min. The supernatant was analyzed with radio-HPLC and the precipitate measured for radioactivity, as described for brain. The stability of 11C-NR2B-SMe in rat tissues ex vivo was obtained by dividing the percentage of radioactivity present as radioligand in the tissue sample measured with HPLC by the fractional radiochemical purity of the radioligand (Table 1). The SUV due to radioligand or radiometabolite only was calculated by multiplying the total SUV of the tissue by the fraction of the radioligand or radiometabolite measured with HPLC (Table 2). PET Imaging in Rats

5 Selection of Agents for Preblocking and Displacement for PET Experiments in Rats. The aims of preblocking and displacement experiments were to assess radioligand target engagement and to determine radioligand selectivity in vivo. Many ligands do not show selectivity for binding to NR2B over 1 receptors (15-19). In addition, 1 receptors exist in close proximity on cell membranes and may have direct interactions with NMDA receptors, modulating the behavior of NMDA receptor complexes (20). NR2B-SMe showed high binding affinity to the NR2B receptor, but also showed weak binding affinity to 1 receptors in vitro (Table 3). To address NR2B-SMe selectivity in PET imaging, we chose various NR2B, σ1, and σ2 ligands for preblocking and displacement experiments in normal rats in vivo with PET. The studied NR2B ligands are summarized in Table 4, 1 antagonists and ligands of undetermined 1 intrinsic efficacy in Table 5, and putative 1 agonists in Table 6. 11 Estimation of ED50 Values In Vivo from Dose-response Data. After administering C-NR2B-SMe or one of its enantiomers to rat at baseline, whole brain radioactivity concentration (SUV) was seen to almost stabilize by 20 min. The SUV unit normalizes radioactivity concentration for rat weight and injected dose. We used the SUV values of time-activity curves (TACs) of the deployed radioligand between 20 and 90 min to calculate areas-under the curve (AUCs). AUCs obtained from the same production of radioligand were scaled to the value (or mean value) for the baseline experiment to give relative values (dubbed: relative AUC 20–90 min). These data were used to estimate the dose of blocking

agent that was effective for 50% reduction of the AUC 20–90 min at baseline, here termed ED50 and reported as moles of administered blocker per kg body weight. Where data permitted, the dose-response

curves were fitted with GraphPad Prism software (version 8.1.1; San Diego, CA) to estimate the ED50 values. A dummy value of 100 pmol per kg bodyweight was used for zero concentration of the challenge agent in the construction of these dose-response curves.

RESULTS NR2B-SMe and Its Chemical Properties Absolute Configuration. (–)-NR2B-SMe and (+)-NR2B-SMe were found to have R and S absolute configuration, respectively, as determined by comparison of VCD and IR spectra measured with the calculated Boltzmann-averaged spectra of the calculated conformations (see Supplemental Fig. 2 and 3 and Supplemental Table 2), after separation of the racemic mixture by chiral HPLC columns (see Supplemental Fig. 4 and 5).

6 Binding Affinities In Vitro and Other Physical Properties. The Ki value of NR2B-SMe in the in vitro assay in transiently transfected mouse fibroblast cells expressing NMDA was 2.2 nM (Table 3). The

pKa was determined to be 5.04 (Supplemental Fig. 6), and the logD7.4 was 3.41. Pharmacological Screen. NR2B-SMe was found to be selective for binding to the NR2B subunit because 10 M concentration only weakly inhibited the binding of reference radioligands to numerous binding sites and receptors, as listed in Supplemental Information. At 10 m concentration, inhibition was greater than 10% for only a few binding sites and receptors. These were the calcium channel (10.6%), hERG channels (66.3%), guinea pig 1 (83.7%), and PC12 cell2 (90.0%). Experiments in HeLa Cells (21). NR2B-SMe failed to compete with Cy3 dye for lysosome trapping in HeLa cells, in contrast with the positive control, loperamide (Supplemental Fig. 7). Experiments with 11C-NR2B-SMe in Rats and Human Tissues Stability of 11C-NR2B-SMe in Rat Whole Blood, Plasma, and Brain Ex Vivo and In Vitro. The radiochemical purity of 11C-NR2B-SMe was 98.4% throughout the experiment (duration ~ 3 h), after HPLC separation (Supplemental Fig. 8) and formulation (Supplemental Fig. 9). At least three radiometabolites eluted before 11C-NR2B-SMe in the reversed phase HPLC analyses of rat plasma ex vivo (Supplemental Fig. 10A). These were very minor in brain and myocardium ex vivo (Supplemental Fig. 10B and 10C). Unchanged radioligand at 30 min after injection accounted for 71.6% of radioactivity in rat plasma, 99.2% in brain, and 90% in myocardial tissue (Table 1). Brain and myocardium showed high ratios of radioligand concentration (SUV) to that in plasma (Table 2). Ex vivo radioactivity in plasma distributed 51% to blood cells (22). The radiometabolites in vitro are the same as those observed ex vivo (Supplemental Fig. 11). 11 11 Plasma Free Fraction of C-NR2B-SMe. The human plasma free fraction (fp) of C-NR2B-SMe was 0.82% ± 0.01% (n = 3). Evaluation of 11C-NR2B-SMe and Its Enantiomers in Rats with PET After intravenous injection of a bolus of racemic 11C-NR2B-SMe into rat, there was a rapid and high uptake of radioactivity in brain followed by a quick decline to a moderately high stable level (Fig. 2A and Supplemental Fig. 12A). Relatively high uptakes were seen in thalamus, striatum, and cortex (Fig. 2A). When rats were intravenously administered NR2B-SMe or the NR2B ligand ifenprodil, at 3.0 mg/kg at 10 min before 11C-NR2B-SMe, the peak whole brain and brain regional radioactivity uptake declined to a common low level at 90 min, corresponding to about 10% of peak values in the baseline experiment (Fig. 2A). Accordingly, summed PET images before and after ifenprodil treatment were strikingly

7 different (Fig. 2B). Administration of the NR2B ligand Ro-25-6981 at 10 min after radioligand resulted in dose-dependent faster whole brain radioactivity decline (Supplemental Figs. 12B). The separate homochiral radioligands gave slightly different time-activity curves (TACs) in whole brain (Fig. 3). The R-enantiomer (Fig. 3A) showed somewhat faster radioactivity decline from peak value than the S-enantiomer (Fig. 3B). Intravenous injection of the NR2B ligand Ro-25-691 at 10 min before either homochiral radioligand lowered whole brain retention of radioactivity. The effect was greater at the higher dose of 0.05 mg/kg than the lower dose of 0.01 mg/kg. As seen for the racemic radioligand, Ro- 25-6981 administered at 10 min after the R-enantiomer resulted in dose-dependent faster whole brain radioactivity decline (Supplemental Fig. 12C). The abilities of NR2B ligands to preblock whole brain radioactivity uptake before intravenous injection of 11C-(S)-NR2B-SMe into rats was studied in more detail. The TACs for 11C-NR2B-SMe and each enantiomer showed dose-dependent preblocking by NR2B-SMe itself (Fig. 3C) and by the NR2B ligands ifenprodil (Supplemental Fig. 13A) and CO101244 (Supplemental Fig. 13C). Sigmoidal dose- response curves (relative AUC 20–90 min vs dose) for NR2B-SMe (Fig. 3C), ifenprodil (Supplemental Fig. 13B) and CO101244 (Supplemental Fig. 13D), as well as for the NR2B ligand Ro-25-6981 (Fig. 3C) were readily fitted to provide ED50 values (nmol/kg) (Table 4). These ED50 values were in rank order of their NR2B binding potencies (1/Ki) in vitro (Table 4). σ1 Receptor antagonists, such as BD1047, NE100 and F4, showed minimal preblocking of radioactivity retention in whole rat brain before the administration of 11C-(S)-NR2B-SMe (Supplemental Fig. 14). No meaningful sigmoidal dose-response curves could be fitted for these data, showing that the

ED50 values for these ligands were much greater than 1000 nmol/kg, in contrast to their affinities for 1 receptors in vitro which are in the low nanomolar range (Table 5), or for the affinity of BD19147 at 2 receptors which is quite low at 47 nM (Table 5). The very high-affinity 1-receptor antagonist FTC146 only caused preblocking at a high dose of 1.25 mg/kg i.v. (Fig. 4A). The 1 ligand F3 also showed a dose-dependent preblocking effect (Supplemental Fig. 14). Dose-response curves could be fitted for

FTC146 (Fig. 4B) and F3 (Supplemental Fig. 14) and allowed ED50 values to be estimated. These ED50 values were found to be extremely high relative to their Ki values at σ1 receptors in vitro (Table 5). σ1 Receptor agonists, such as (+)-pentazocine, (±)-PPCC, PRE-084, and (+)-SKF10047 (Supplemental Fig. 15), showed very weak preblocking effects on radioactivity retention in whole rat brain before administration of 11C-(S)-NR2B-SMe. These data could not be fitted to sigmoidal dose-

response curves and therefore ED50 estimates were extremely high (> 1000 nmol/kg) (Table 6).

8 Exceptionally, the σ1 agonists TC1 (Fig. 5A) and SA4503 (Fig. 6A) showed dose-dependent preblocking

effects that could be fitted to sigmoidal dose-response curves (Figs. 5C and 6C respectively). The ED50 values for TC1 and SA4503 were estimated to be quite low at 45 and 29 nmol/kg, respectively (Table 6). However, the preblocking effects of these two 1 agonists were unaltered when the potent receptor antagonist, FTC146, was used to pharmacologically block σ1 receptors (Figs. 5 and 6). The corresponding dose-response curves (Figs. 5B, 6B, 5C and 6C) were congruent with those obtained in the absence of 1 receptor blocking with FTC146.

DISCUSSION We were able to resolve the enantiomers of NRB2B-SMe by chiral HPLC. Knowledge of the absolute configuration of NR2B-SMe could be valuable in future NR2B ligand design. We were able to assign the absolute configurations of NR2B-SMe enantiomers successfully with IR and VCD. 11C-NR2B-SMe and its enantiomers were readily prepared for intravenous injection into rats by treating NR2B-ester with 11C-methyl iodide under basic conditions. The radiolabeling process is likely reverse Michael addition of α,ß-unsaturated acrylic acid or ester (Supplemental Fig. 1) (23). 11C-NR2B- SMe decomposed when concentrated immediately after HPLC isolation, but the decomposition was minimal after ascorbic acid was added during the removal of mobile phase. A constant radiochemical purity of greater than 98% was then achieved. Initial PET experiments in rats used racemic 11C-NR2B-SMe. These experiments showed that 11C-NR2B-SMe readily entered brain, peaking at ~ 3 SUV in whole brain at about 2.5 min (Fig. 2A). Peak radioactivity was followed by a slow decline in radioactivity. Radioactivity retention in brain regions such as thalamus, cortex and cerebellum could be preblocked with NR2B-SMe itself and by the NR2B ligand ifenprodil (Fig. 2A). Thalamus and cortex are generally considered to be NR2B-rich regions. However, appreciable specific binding in cerebellum would not have been expected based on previous in vitro autoradiography of post mortem rat brain with NR2-specific antibody (24). Our finding of specific binding in cerebellum that can be blocked by ifenprodil is consistent with those of Krämer et al. (12), who observed high specific binding of their putative NR2B radioligand, 11C-Me-NB1, to rat cerebellum with both in vitro autoradiography and ex vivo biodistribution measurements. Yet deeper investigations are needed to verify that specific binding in cerebellum is to NR2B. The radioactivity in brain could be partially displaced by the NR2B radioligand Ro-25-6981, suggesting reversibility of radioligand binding to NR2B (Supplemental Fig. 12).

9 At 30 min after intravenous administration, unchanged radioligand represented virtually all rat brain radioactivity (> 99%), a finding that was highly favorable to pursuing further radioligand characterization. Parent 11C-NR2B-SMe represented close to 70% of radioactivity in plasma at 30 min after injection of 11C-NR2B-SMe, showing peripheral metabolism in vivo was relatively slow (Table 1). 11C-NR2B-SMe showed high accumulation in NR2B-rich heart (25) as well as in brain relative to blood (Table 2). 11C-NR2B-SMe also showed high metabolic stability in rat whole blood, plasma, and brain in vitro (Table 1). Because 11C-NR2B-SMe was strongly retained in rat brain at baseline, we investigated whether trapping in lysosomes might be responsible. Lysosomes are membrane-bound organelles found in nearly all animal cells (26). With a pH ranging from 4.5 to 5.0, the interior of the lysosomes is acidic whereas cytosol is slightly basic (pH 7.2). Protonated weak bases may accumulate within lysosomes, in some cases reaching 100 to 1000-fold higher concentrations than the extracellular concentration, a phenomenon known as "lysosomotropism (27), or "proton pump effect" (28). The accumulation of compounds in

lysosomes may be estimated from compound pKa (29). NR2B-SMe is monobasic. The optimal pKa range for lysosomal trapping of a monobasic compound is between 6 and 10. Therefore, we measured the pKa of 11C-NR2B-SMe. We also investigated the uptake of NR2B-SMe into HeLa cell lysosomes in vitro. 11 The apparent pKa of C-NR2B-SMe was found to be 5.04 ± 0.01 (n = 3) (Supplemental Fig. 6). Therefore, 11C-NR2B-SMe will be protonated to only a very low extent (0.63%) at the physiological pH of cytosol (pH = 7.2) but will be protonated to a greater extent (76–50%) in lysosomes (pH = 4.5 to 5.0). 11 The pKa of C-NR2B-SMe however suggested a low risk for lysosomal trapping. Conclusive evidence against lysosomal trapping of 11C-NR2B-SMe came from experiments in HeLa cells where NR2B-SMe competes with the cyanine dye Cy3 for cell uptake. The greenish yellow fluorescence of the dye did not change upon addition of NR2B-SMe, just as with addition of DMSO only, whereas the dye was excluded from the cells with loperamide (50 µM), a known ligand for lysosome trapping (30). The lipophilicity of a PET radioligand, as indexed by logD at pH 7.4, is a key property that influences many aspects of PET radioligand behavior in vivo (31), including brain entry, metabolism, and 11 protein binding. We found the logD7.4 of C-NR2B-SMe to be 3.41 which is close to that predicted by computation (2.98), and in the range for many successful PET radioligands for CNS targets. The stability of 11C-NR2B-SMe in human brain homogenate and human plasma and the plasma

free fraction (fP) in vitro were of interest with respect to possible eventual radioligand application in human

10 subjects. 11C-NR2B-SMe was virtually unchanged when exposed to these tissues for 30 min at RT in vitro, suggesting that radiometabolites would not be generated in human brain in vivo. The plasma free fraction of a PET radioligand can be an important parameter for the use of a PET 11 radioligand for quantifying a protein target in human subjects. The fp value for C-NR2B-SMe in human plasma was relatively low, but measurable with good precision (0.82% ± 0.01%, n = 3). Generally, PET radioligands should not be used as racemates because their enantiomers may show differences in binding affinities for their targets, and possibly other differences in, for example, kinetics and metabolism. Given the encouraging PET results and encouraging in vitro measures with racemic 11C- NR2B-SMe, we proceeded to evaluate the enantiomers of 11C-NR2B-SMe. Each homochiral radioligand showed similar brain uptake to the racemic radioligand in rat. The S-enantiomer showed slower decline in whole brain radioactivity concentration from peak value than the R-enantiomer. The NR2B ligand Ro- 25-6981 was effective in blocking the brain retention of radioactivity from both enantiomers, suggesting they both show specific binding to NR2B (Fig. 3). As also seen for the racemic radioligand, radioactivity in brain from the R-enantiomer could be displaced with Ro-25-6981 in a dose-dependent manner, thereby confirming the reversibility of specific binding (Supplemental Fig. 12). Because 11C-(S)-NR2B-SMe showed the stronger retention in rat brain, subsequent experiments were performed with this radioligand. They were primarily directed at exploring the specificity of the PET signal. In total, we used three NR2B ligands (Ro-25-6981, CO-101244, and ifenprodil) and NR2B-SMe itself as pre-blocking agents at various doses for PET experiments on 11C-(S)-NR2B-SMe in rat. Each

ligand gave data that could be fitted to a dose-response curve for the estimation of ED50 values in vivo.

The ED50 values were in rank order of the Ki values in vitro (Table 6). These results are therefore consistent with 11C-NR2B-SMe selectively occupying the NR2B binding site in rat brain in vivo. σ1 receptors are transmembrane proteins expressed in many different tissues. They are particularly concentrated in certain regions of the CNS (32) and may function as a chaperone to NMDA receptors (33,34). Five antagonists of the σ1 receptor (FTC146, BD1047, NE100, F3, and F4) had very limited preblocking effects on the PET radioligand 11C-NR2B-SMe (Fig. 4, and Supplemental Fig. 14, Table 5), further suggesting that 11C-NR2B-SMe does not occupy σ1 receptors. Four of six tested σ1 receptor

agonists had only weak pre-blocking effects (i.e., high ED50 values) (Supplemental Fig. 14, Table 6). However, two putative selective σ1 agonists, TC1 and SA4503, had strong effects on the brain uptake of 11C-NR2B-SMe (Figs. 5, 6, Table 6). We considered that these two ligands might have off-target binding to NR2B receptors. Therefore, we designed pre-blocking PET experiments with TC1 or SA4503 and 11C-

11 (S)-NR2B-SMe in which σ1 receptors would be pharmacologically blocked with the high-affinity

antagonist FTC146 (KD = 2.5 pM). For these experiments, we selected an FTC146 dose of 0.05 mg/kg (i.v.) which we estimated would achieve an exceptionally high peak brain concentration of 134 nM, that should assuredly occupy all 1 receptors, even if the brain free fraction was exceptionally low. This estimate was based on published data (18F-FTC146 peak brain uptake: ~ 1 SUV in mice) (35,36) (see Supplemental Information for estimation). The effects of TC1 and SA4503 on brain uptake of 11C-(S)- NR2B-SMe were the same as in the absence of FTC146 (Figs. 5 and 6, respectively). Therefore, preblockingof1 receptors did not prevent interactions of TC1 and SA4503 with the NR2B receptor. This strongly suggests that these 1 agonists have some affinity for NR2B receptors. Also, these data further suggest that 11C-(S)-NR2B-SMe does not have appreciable off-target binding to 1 receptors in rat brain in vivo.

CONCLUSIONS Racemic 11C-NR2B-SMe binds selectively and with high affinity to the NR2B subunit. Absolute configurations were successfully assigned to the enantiomers of NR2B-SMe. 11C-NR2B-SMe and its enantiomers were readily prepared as prospective radioligands for PET imaging of NR2B subunits within NMDA receptors. In normal rats, these radioligands show high initial brain radioactivity uptake, followed by slow washout. The radioactivity in brain is predominantly unchanged radioligand and is not trapped in lysosomes. 11C-(S)-NR2B-SMe shows slower decline in brain concentration than its antipode. Brain uptake of this radioligand is reversible and can be pre-blocked up to 90% with a variety of NR2B ligands in a dose-dependent way. Brain uptake of 11C-NR2B-SMe cannot be pre-blocked by σ1 receptor antagonists, consistent with the radioligand occupying the NR2B binding site, not the σ1 receptor. However, brain uptake of 11C-(S)-NR2B-SMe can be pre-blocked by two of six σ1 receptor agonists, consistent with a direct interaction of the two agonists with NR2B. Altogether, this study suggests that 11C-(S)-NR2B-SMe has adequate NR2B-specific PET signal in rat brain to warrant yet deeper investigation of its specific binding across brain regions, including cerebellum, and further evaluation as a radioligand in higher species.

12 DISCLOSURE This study was funded by the Intramural Research Program of the National Institute of Mental Health, National Institutes of Health: projects ZIAMH002795 and ZIAMH002793. The authors have no conflict of interest to declare.

ACKNOWLEDGMENTS This research was supported by the Intramural Research Program of NIH, specifically NIMH. We thank Dr. Mary Herman (CDBD, NIMH) for post mortem human brain tissues. We also thank the NIH PET Department for carbon-11 production, PMOD Technologies for providing the image analysis software and PDSP for performing assays: the PDSP is directed by Bryan L. Roth, PhD, with project officer Jamie Driscoll (NIMH) at the University of North Carolina Chapel Hill (contract # NO1MH32004).

13 KEY POINTS

QUESTION: Can we image the brain NR2B receptor using PET?

PERTINENT FINDINGS: PET imaging using normal rats has showed that 11C-NR2B-SMe in both racemic and chiral forms has specific signal which can be pre-blocked and displaced using specific ligands specifically targeting NR2B receptors, but not using ligands specifically targeting sigma-1 receptors.

IMPLICATIONS FOR PATIENT CARE: 11C-NR2B-SMe has potential as a PET radioligand to measure the distribution of NR2B receptors in human brain.

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18 11C-(S)-NR2B-SMe 11 4 250 C-(S)-NR2B-SMe Baseline 3 200 0.01 mg/kg Ro-25-6981 0.05 mg/kg 150 2 0.25 mg/kg 100 1.25 mg/kg 1 50

Conc. radioactivity (SUV) radioactivity Conc. 0 Relative AUC (20-90 min) 0

0 20406080100 Time (min)

Figure 1. Panel A: Ro-25-6981 dose-dependently pre-blocks rat whole brain radioactivity uptake from the intravenous injection of 11C-(S)-NR2B-SMe. Panel B: The area-under-the-curve (AUC) between 20 and 90 min from panel A for 11C-(S)-NR2B-SMe at different dose of pre-blocking agent Ro-25-6981, highly selective antagonist for NR2B receptor. Data are for n = 1.

A 11C-NR2B-SMe

4 Baseline: Thalamus Cerebellum 2 Cortex Ifenprodil predose: Thalamus Cortex Conc. radioactivity (SUV) radioactivity Conc. 0 Cerebellum

0 20406080100 Time (min)

Figure 2. PET imaging of rat brain with racemic 11C-NR2B-SMe. Panel A: PET TACs for selected brain regions in rat after intravenous injection of 11C-NR2B-SMe showing brain radioactivity accumulation is blocked by pre-administration of ifenprodil at 3.0 mg per kg (i.v.). Panel B: Summed transaxial PET images (60−90 min) from 11C-NR2B-SMe at baseline and after preblock with NR2B ligand ifenprodil. Data are for n = 1.

Figure 3. Blocking of whole brain radioactivity uptake in rat by dosing with the NR2B ligand Ro-25- 6981 before intravenous injection of 11C-(R)-NR2B-SMe (Panel A), 11C-(S)-NR2B-SMe (Panel B), and fitted dose-response curves derived from panel B for NR2B-SMe and from panel A in Fig. 1 for NR2B ligand Ro-25-6981 (Panel C). Data are for n = 1.

Figure 4. Panel A: Low to moderate pre-administered intravenous doses of the highly potent 1 receptor antagonist FTC146 do not reduce whole brain radioactivity in rats after intravenous injection of 11C-(S)- NR2B-SMe. Panel B: Fitted dose-response curve for FTC146 derived from panel A data.. Baseline data are mean for n = 2. Error bar represents range. Other data are for n = 1.

Figure 5. Panel A: Intravenous pre-administration of TC1 dose-dependently reduces rat whole brain radioactivity uptake from i.v. injection of 11C-(S)-NR2B-SMe. Panel B: Intravenous pre-administration of TC1 in rats that have been simultaneously pre-treated with FTC146 (0.05 mg/kg, i.v.) at 10 min before 11C-(S)-NR2B-SMe dose-dependently reduces whole brain radioactivity uptake. Panel C: Fitted dose- response curves from panel A and B data. Curves in panels A and B are highly congruent, as are curves in panel C, showing no effect of 1 receptor blockade by FTC146. Baseline data are mean for n = 2. Error bar represents range. Other data are for n = 1.

Figure 6. Panel A: Pre-administered intravenous doses of SA4503 reduced whole brain radioactivity uptake in rats from the intravenous injection of 11C-(S)-NR2B-SMe. Panel B: Simultaneous intravenous pre-administration of SA4503 and FTC146 (0.05 mg/kg, i.v.) at 10 min before 11C-(S)-NR2B-SMe dose- dependently reduced whole brain radioactivity uptake . Panel C: Fitted dose-response curves derived from panel A and B data. Curves in panels A and B are highly congruent, as are curves in panel C, showing no effect of 1 receptor blockade by FTC146. Data are for n = 1.

23 Table 1. Ex vivo and In vitro stabilities of 11C‐NR2B‐SMe in rat tissues.a Measurements were made at 30 min after intravenous injection of radioligand ex vivo and at 30 min after incubation of radioligand at 37 °C in vitro. Stabilities are represented by the ratios of the percentage of the parent radioligand in the tissue to the fractional radioligand purity at the start of the study.

Radioactive Ex vivo In vitro sample stability stability (%) (%) Whole blood 89.8 Plasma 71.6 90.4 Brain 99.2 97.0 Myocardium 90.0 a The formulated radioligand was initially 98.4% pure and found to be 99.2% intact 3 h later at the end of other measurements.

Table 2. Concentrations of 11C‐NR2B‐SMe in rat tissues and tissue to plasma radioactivity ratios measured ex vivo at 30 min after intravenous injection. Radioactive Radioactivity Tissue to plasma sample concentration radioactivity ratio (SUV) Whole blood 0.047 1.6 Plasma 0.03 1.0 Brain 2.59 86.3 Myocardium 0.30 10.1

24 Table 3. Pharmacological parameters for 11C‐NB1 and 11C‐NR2B‐SMe.

Radioligand NR2B  ligand  Ki 2 ligand 2 Ki Ki binding binding inhibition inhibition at 10 M (nM) at 10M (nM) (nM) (%) (%) 11C‐NB1 (12) 9.8 89 182 88 554 11C‐NR2B‐SMe 2.2 84 90

Table 4. In vitro and in vivo pharmacological parameters for NR2B ligands.

Ligand Tissue NR2B Reference Ki for Ki for other ED50 a Ki assay radioligand for NR2B targets in vivo NR2B Ki assay (nM) (nM) (nmol/kg) NR2B‐SMe Oocytes [3H]Ifenprodil 2.0 9.5 Ro‐25–6981 [3H]MK801 9.0 (37) 29 CO101244 Oocytes Electric current 43 (38) 74 Ifenprodil Rat [3H]Ifenprodil 46.3 (39) 1 = 11 (39) 211 2 = 1.1 a For preblocking of binding of 11C‐NR2B‐SMe in rat whole brain measured in this study.

25 Table 5. In vitro and in vivo pharmacological parameters for σ1 receptor ligands (not identified as agonists).

c 1 Tissue for Ki Ki for  Ki for σ2 ED50 in vivo Ligand assays (nM)a (nM)b (nmol/kg) FTC146 Rat liver 0.0025 (35,36) 364 2571 homogenate BD1047 Guinea pig 0.9 (40) 47 >1000 brain NE100 Guinea pig 1.03 (41) 211 >1000 brain F3 Rat brain 0.79 (42) 277 138 homogenate F4 Rat brain 2.30 (42) 327 >1000 homogenate a Reference radioligand 3H‐pentazocine, except 18F‐FTC146 for FTC146. b Reference radioligand 3H‐DTG. c For preblocking of 11C‐(S)‐NR2B‐SMe rat whole brain uptake measured in this study.

Table 6. In vitro and in vivo pharmacological parameters for putative σ1 receptor agonists.

c Ligand Tissue for Ki assays Ki for 1 Ki for 2 ED50 in vivo (nM)a (nM)b (nmol/kg) TC1 Guinea pig brain 10 (43) 370 45 SA4503 Guinea pig membranes 17.4 (44,45) 1784 29 Rat brain membranes 4.6 (36,39) 63 (+)‐Pentazocine Guinea pig brain 13.7 (44,46) 2875 >1000 (±)‐PPCC Guinea pig brain 1.5 (47) 50.8 >1000 PRE‐084 Guinea pig brain 44 (48,49) >1000 (+)‐SKF10047 Guinea pig brain 48 (50) 625 >1000 a Reference radioligand 3H‐pentazocine, except 3H‐(+)‐SKF‐10047 for PRE‐084. b Reference radioligand 3H‐DTG. c For preblocking of 11C‐(S)‐NR2B‐SMe rat whole brain uptake measured in this study.

26 Evaluation of 11C-NR2B-SMe and its Enantiomers as Radioligands for Imaging the NR2B Subunit within the NMDA Receptor Complex in Rats

Supplemental Information

Lisheng Cai, 1 Jeih-San Liow,1 Cheryl L. Morse,1 Sanjay Telu,1 Riley Davies,1 Michael P. Frankland,1 Sami S. Zoghbi,1 Ken Cheng,2 Matthew D. Hall,2 Robert B. Innis,1 Victor W. Pike1

1Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA. 2NCATS Chemical Genomics Center (NCGC), National Institutes of Health, Rockville, MD 20850, USA.

Contents

Title Brief Description Page Material and Methods Materials S4 Instruments and General Methods S4 Chiral Separation S5 In Vitro Binding Assay S6 Radiosynthesis S7

LogD7.4 and pKa Measurement S8 Radiometabolites of 11C-NR2B-SMe in Rat Tissues In Vitro S9 Distribution of 11C-NR2B-SMe in Rat Blood In Vitro S10 Experiments with 11C-NR2B-SMe and Human Tissues In S10 Vitro PET imaging in rats S11 Results Properties of NR2B-SMe S12 Radiochemistry S13 Experiments with 11C-NR2B-SMe in Rats and Human Tissues S14 Supplemental Table 1 Experimental Parameters used for radiometabolite analysis S15 Supplemental Table 2 Numerical comparison between calculated IR and VCD S16 spectra Supplemental Figure 1 Radiosynthesis of 11C-NR2B-SMe S17 Supplemental Figure 2 Chiral HPLC of the enantiomers of NR2B-SMe S18 Supplemental Figure 3 Chiral HPLC of the enantiomers of NR2B-ester S19 Supplemental Figure 4 Measured IR and VCD spectra of (–)-NR2B-SMe S20 Supplemental Figure 5 Comparison of VCD (upper frame) and IR (lower frame) S20 spectra measured for (–)-NR2B-SMe Supplemental Figure 6 Radiochromatogram from the HPLC separation of 11C-NR2B- S21 SMe Supplemental Figure 7 Radiochromatogram from the HPLC analysis of formulated S21 11C-(S)-NR2B-SMe Supplemental Figure 8 pH-Dependence of the distribution of 11C-NR2B-SMe S22 between cyclohexane and sodium phosphate buffers Supplemental Figure 9 Experiments in HeLa cells show that NR2B-SMe is not S22 trapped in lysosomes Supplemental Figure 10 Radiochromatograms from HPLC analysis of rat tissues at 30 S23 min after intravenous injection of 11C-(S)-NR2B-SMe

measured ex vivo Supplemental Figure 11 Radiochromatograms from HPLC analysis of rat tissues at 30 S24 min after incubation with 11C-(S)-NR2B-SMe in vitro Supplemental Figure 12 PET imaging of rat brain with 11C-NR2B-SMe S25 Supplemental Figure 13 In vivo dose-dependent preblocking of 11C-(S)-NR2B-SMe by S26 NR2B ligands (ifenprodil and CO101224) in rats Supplemental Figure 14 In vivo dose-dependent preblocking of 11C-(S)-NR2B-SMe by S27 σ1 antagonists (BD1047, F3, F4, and NE100) in rats Supplemental Figure 15 In vivo dose-dependent preblocking of 11C-(S)-NR2B-SMe by S28 σ1 agonists [(+)-pentazocine, (±)-PPCC, PRE-084, and (+)- SKF10047] References S29

MATERIALS AND METHODS

Materials

Common reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI), Fluka Chemical Co. (Milwaukee, WI), Acros (Hampton, NH), or Strem Chemicals (Newburyport, MA), and were used without further purification. Water was purified with a Milli-Q synthesis system (Millipore; Bedford, MA). Common solvents were obtained from Fisher Scientific (Pittsburgh, PA). The precursors and reference non-radioactive versions of the radioligands were synthesized in-house, and the methods will be reported separately. Blocking agents were purchased commercially, as follows: NR2B antagonists, Ro-25–6981, CO101244 hydrochloride, and ifenprodil were from Tocris (Minneapolis, MN); σ1 receptor antagonists BD1047 and NE100 were from Tocris, F3 and F4 from ChemShuttle (Hayward, CA); FTC146 was a gift from Dr. Frederick T. Chin (Stanford University, CA); σ1 receptor agonists, TC1, SA4503, (+)-pentazocine, (±)-PPCC, PRE-084, and (+)-SKF10047, were from Tocris. Sprague Dawley rats were obtained from Taconic Farm (Rensselaer, NY).

Instruments and General Methods

Radioactivity measurements. γ-Radioactivity from carbon-11 (> 40 kBq) was measured with a calibrated dose calibrator (Atomlab 300; Biodex Medical Systems; Shirley, NY). Low levels of radioactivity (< 40 kBq) were measured with a calibrated automatic well-type γ-counter (model 1480 Wizard; Perkin-Elmer; Waltham, MA) having an electronic window set between 360 and 1800 keV (counting efficiency, 51.8%). All carbon-11 radioactivity data were corrected for decay with a half-life of 20.395 min (1).

Carbon-11 radioactivity concentration in tissue was expressed as standardized uptake value (SUV), defined as:

(body weight /tissue weight) × (tissue activity/injected activity)

HPLC for radiometabolite analysis (general HPLC method A). HPLC (high performance liquid chromatography) was performed on an X-Terra C18 column (10 µm, 7.8 × 300 mm; Waters Corp.; Milford, MA) housed within a compression module (Radial-Pak RCM-100; Waters Corp.) having a

sentry pre-column that was eluted with MeOH:H2O:Et3N (82.5:17.5:0.1 by vol.) at 4.0 mL/min. Eluted compounds were detected with an in-line photodiode-array absorbance detector (λ = 245 nm; Beckman

Coulter; Brea, CA) in series with a flow-through Na(Tl) scintillation detector-rate-meter (Bioscan; Washington, D.C.). Samples were injected onto the HPLC through nylon filters (13 mm × 0.45 µm; Iso- Disk; Supelco, Bellefonte, PA). Recovery of all radioactivity from the HPLC column was checked by injection of absolute methanol (2 mL) at the end of the chromatography with continued monitoring for radioactivity. Radiochromatograms were collected and stored with Bio-Chrome Lite software (Bioscan) and analyzed after decay correction.

Post mortem human brain tissue. Post mortem brain tissues were obtained from the Human Brain Collection Core (HBCC), NIH. Experiments with these materials were performed under the regulations of the Ethics Committee of the National Institutes of Health.

Chiral Separations

Samples of racemate solution were injected onto an (S,S)-Whelk-O1 column (21.1 × 250 mm; 5 µm; Regis Technologies Inc.; Morton Grove, IL) that was eluted at 80 mL/min with ethanol containing diethylamine (0.2% v/v) in liquid carbon dioxide (40:60 v/v) with eluate monitored for absorbance at 220 nm. Each enantiomer was isolated from the appropriate HPLC fraction by evaporation of mobile phase under reduced pressure at 40 oC. The purity of each enantiomer was determined with HPLC on an (S,S)-Whelk-O1 column (4.6 × 250 mm; 5 µm; Regis Technologies Inc.) eluted with hexane-0.2% diethylamine in ethanol (50:50 v/v) at 1.5 mL/min with eluate monitored for absorbance at 220 nm.

Chiral resolution of NR2B-SMe. NR2B-SMe (0.288 g) was dissolved in a mixture of ethanol and methanol (75:25 v/v; 45 mL). Samples of this solution (1.75 mL) were injected onto the semi-

preparative size chiral HPLC column. (+)-NR2B-SMe (65 mg; tR = 4.5 min) and (−)-NR2B-SMe (95

mg; tR = 7.3 min) were accumulated from these injections (Supplemental Fig. 2). Each enantiomer was free of its antipode (i.e., had an ee of 100%).

Chiral resolution of NR2B-ester. NR2B-ester (0.130 g) was dissolved in ethanol (15 mL). Samples of this solution (0.90 mL) were loaded onto the semi-preparative size (S,S)-Whelk-O1 column

to give (+)-NR2B-ester (50 mg; tR = 6.2 min) and (−)-NR2B-ester (94 mg; tR = 10.9 min) (Supplemental Fig. 3). Each enantiomer was free of its antipode (i.e., had an ee of 100%).

Optical rotation measurements. Each enantiomer of NR2B-SMe was dissolved in ethanol to give a stock solution of 1.0 mg/mL, and the optical rotation of the solution was measured at 20 oC, 20 giving [α] D = + 10° (c 1.00, EtOH) for (+)-NR2B-SMe, and − 8.0° for (−)-NR2B-SMe.

Absolute configuration determination. Each enantiomer of NR2B-SMe was dissolved in chloroform to give a stock solution of 11.2 mg in 0.175 mL that was then placed in a BaF2 cell (path length: 100 µm). The IR and vibrational circular dichroism (VCD) spectra of each enantiomer in the range 1000 to 1800 cm-1 were acquired experimentally over 18 h with ChiralIR with DualPEM (BioTool Inc.; Jupiter, FL) at a resolution of 4 cm-1 and a photoelastic modulator setting of 1400 cm-1. The spectra were then calculated using Gaussian 09 at the B3LYP/6-31G(d) level with a total of 147 low- energy conformers identified and used for Boltzmann summation (Supplemental Figs. 4 and 5).

In Vitro Binding Assay

NR2B-SMe was assayed by the National Institutes of Health (NIH) Psychoactive Drug Screening

Program (PDSP) for Ki at the NR2B subtype as an index of binding affinity (2). Thus, a suspension of transient transfected mouse fibroblast cell membrane homogenates was prepared as reported (2) at a concentration of ~500,000 cells per mL. This suspension was sonicated again, and aliquots (100 µL) were added to each of four tubes (total 48 in a rack). A solution of [3H]ifenprodil (2.22 TBq/mmol, 0.01 kBq/μL; Perkin–Elmer) in PBS (100 µL) was added to each tube. Non-radioactive ifenprodil or other displacer was dissolved in DMSO to give a 1-mM stock solution, which was further diluted with DMSO to give solutions ranging in known concentration from 10-5 to 10-10 M. Then 10 µL of each solution was added to a separate tube. The content of each tube was then diluted to 1.0 mL with PBS, vortexed, and incubated at 37 °C for 2 h. After separation of tube contents with a cell harvester, the filter paper (GF/B; Whatman, Derwood, MD), pretreated with 0.5% polyethyleneimine solution, was washed with PBS (3 mL × 3). Each filter was then placed in a 7-mL plastic vial. Scintillation fluid (4 mL) was added to each vial. The scintillation vials were incubated overnight and then counted for radioactivity. The data were analyzed with Prism 7 version 7.03 (GraphPad Software; San Diego, CA) with ‘One site competition’ curve-fitting. Ki values were calculated according to the Cheng-Prusoff equation (3): Ki =

IC50/(1 + [L]/KD) where [L] is the concentration (0.4 nM) and KD the equilibrium dissociation constant (7.6 nM) of the reference radioligand. The latter was determined with ‘Scatchard analysis’ of homologous displacement from multiple runs with self-displacement from membrane homogenates.

Pharmacological screen. NR2B-SMe was submitted to the PDSP for assessment of binding affinity against a wide range of other receptors and binding sites. A full listing of these receptors and binding sites is given later in Results. Detailed assay protocols are available at the PDSP web site (http://pdsp.cwru.edu).

Experiments in HeLa cells. Briefly, cryopreserved HeLa cells, originally obtained from American Type Culture Collection (ATCC), were thawed and diluted to give 106 cells per mL.

Fluorescent microscopy experiments. Cells (0.5 mL) were added to 8-chamber slides and allowed to adhere overnight. The cyanine dye Cy-3 (200 nM) was added to each well, except for background wells, and left for 2 h. Controls included incubations with nigericin (10 µM) and monensin (20 µM), which are ionophores that uncouple the proton gradient present in lysosomes, and incubations with ammonium chloride (10 mM), which raises lysosomal pH through buffering. The cells were subsequently washed thrice with Dulbecco's PBS (D-PBS) and immediately mounted with a coverslip for imaging. Samples were visualized with epifluorescence microscopy, conducted as previously reported (4). An Eclipse 80i epifluorescence microscope (Nikon Instruments Inc.; Melville, NY) equipped with a 40-magnitude oil-immersion objective at the excitation and emission wavelengths for

Cy-3 (λex 550 nm, λem 570 nm, respectively) was used. Images were captured using an ORCA ER camera (Hamamatsu; Hamamatsu City, Japan) and analyzed with Metamorph version 7.0 (Molecular Devices; Sunnyvale, CA). Background images of non-fluorescently labeled cells were acquired to correct for auto-fluorescence. Images were scaled identically to allow comparison.

Quantitative experiments. HeLa cells (50,000 cells per well) were plated in collagen-coated black-clear-bottom 96-well microtiter plates (Corning Inc.; Corning, NY), as reported previously (4). Before the assay, the multifunction enhancing plating medium was removed and the wells were rinsed twice with prewarmed (37 °C) PBS (10 mM) (100 µL). Stock solutions of NR2B-SMe in methanol were prepared and diluted with supplemented modified Chee’s medium such that the final concentrations of each incubate were 1, 5, 10, 50, 100, and 500 µM. Cy-3 was added to each sub-stock solution such that the final incubate concentration of Cy-3 was 50 nM. Cells were incubated at a final

volume of 100 µL in triplicate for 30 min at 37 °C under 95% humidity and 5% CO2. The incubation matrix was then aspirated, and the plate was rinsed twice with PBS (10 mM; 200 µL) at RT using a Genesis EVO automated liquid handling system (Tecan; Morrisville, NC) before solubilizing the cells with acetonitrile (100 µL). Samples were then analyzed for Cy-3 fluorescence with a Synergy plate reader (BioTek; Winooski, VT).

Radiosynthesis

Production of 11C-iodomethane. No-carrier-added (NCA) 11C-carbon dioxide (~ 50 GBq) was produced by irradiating nitrogen (initially ~ 225 psi) containing oxygen (1%) for 20 min with a proton beam (16 MeV, 40 µA) from a PETtrace cyclotron (GE Medical Systems, Milwaukee, WI). The 11C- carbon dioxide (> 40 GBq) was converted into 11C-iodomethane by reduction to 11C-methane and then high temperature iodination (5) within a PETtrace Microlab module (GEMS PET Systems AB; Piscataway, NJ).

Preparation of 11C-(R)-NR2B-SMe and 11C-(S)-NR2B-SMe. The procedure used to make 11C- NR2B-SMe was used with (R)-NR2B-ester and (S)-NR2B-ester to give 11C-(R)-NR2B-SMe and 11C-(S)- NR2B-SMe, respectively. Chiral HPLC analysis of residual carrier after radioactivity decay showed that no racemization occurred during the radiosyntheses.

LogD7.4 and pKa Measurement

1-Octanol that was to be used in logD7.4 measurement was pre-equilibrated overnight with sodium phosphate buffer (0.15 M; pH 7.4). 11C-NR2B-SMe was dissolved in saline (0.9% w/v) containing ethanol (10% v/v) at a concentration of 159 MBq/mL. The radiochemical purity of the radioligand was measured with general HPLC method A. Radioligand solution (~ 307 MBq; 900 µL) was added to sodium phosphate buffer (0.15 M; pH 7.4; 16.1 mL) and mixed well. Aliquots (1 mL) were distributed to each of eight borosilicate disposable culture tubes (13 × 100 mm). Six tubes were extracted with buffer pre-treated 1-octanol (1 mL) by vortexing for 1 min, and then centrifuged for 1 min. The organic and aqueous phases were separated. Samples of organic phase (50 µL) and aqueous phase (50 µL) were taken and separately measured for radioactivity. The remaining two tubes, containing radioactivity in buffer, were analyzed with HPLC to check for any decrease in radiochemical purity over the time-span of the experiment. The radioactivity in the aqueous phase had a counting error of 0.4 ± 0.04% (n = 6) at one standard deviation. Each aqueous phase was analyzed with general HPLC method A to determine

radioligand purity (NR2B-SMe, tR ~ 6.4 min) for use as a correction factor for the γ-counter counts. No correction for the γ-counting of the organic phase was needed because no radiochemical impurity was

ever detected. LogD7.4 was calculated as log[(cpm/mL for 1-octanol phase)/(cpm/mL for aqueous phase)].

LogD7.4 was also computed with Pallas for Windows software (CompuDrug Inc.; Bal Harbor, Fl)

11 For pKa measurement, the distribution of C-NR2B-SMe between sodium phosphate buffers (0.15 M) of known pH and cyclohexane was measured following the methodology for logD determination.

Radiometabolites of 11C-NR2B-SMe in Rat Tissues In Vitro

A rat without injection of radioligand served as a control for experiments. Anticoagulated blood, plasma, and frontal brain were harvested from a single rat.

For Brain. The brain (1.7 g) was homogenized in twice its volume of cold saline. Formulated 11C-NR2B-SMe (20 µL; 3.7 MBq) was added to the homogenate, incubated at 37 °C for 1 h, and then measured for radioactivity. Then an aliquot (450 µL) was placed in acetonitrile (720 µL) along with carrier NR2B-SMe (5 µg) and mixed well. Water (100 µL) was then added, mixed well, measured for radioactivity, and centrifuged at 10,000 g for 1 min. The supernatant was analyzed with general HPLC method A. The precipitate was measured for radioactivity to determine the recovery of radioactivity into the supernatant that had been injected onto HPLC.

For Whole Blood. Formulated 11C-NR2B-SMe (10 µL; 1.85 MBq) was added to whole blood (200 µL). The sample was mixed well and incubated at 37 °C for 1 h. Water (300 µL) was added to lyse open all cells. Then an aliquot (450 µL) was removed to a new vial and acetonitrile (720 µL) was added. The mixture was mixed well, measured for radioactivity, and centrifuged at 10,000 g for 1 min. The supernatant was analyzed with HPLC as described for brain. For Plasma. Plasma was separated from blood cells. Formulated 11C-NR2B-SMe (10 µL; 1.85 MBq) was added to plasma (500 µL), mixed well, and incubated at 37 °C for 1 h. The mixture was measured for radioactivity. Then an aliquot (450 µL) was placed in acetonitrile (720 µL) along with carrier NR2B-SMe (5 µg) and mixed well. Water (100 µL) was then added, mixed well, measured for radioactivity, and centrifuged at 10,000 g for 1 min. The supernatant was analyzed with HPLC as described for brain. The precipitate was measured for radioactivity to determine radioactivity recovery into the supernatant that had been injected onto HPLC.

The concentration (SUV) of any radioactive species (parent radioligand or radiometabolite) in tissue was calculated as the total radioactivity concentration in tissue (SUV) multiplied by the fraction of radioactivity represented by the radioactive species in the whole analytical chromatogram. Finally, the metabolic stabilities of 11C-NR2B-SMe in rat tissues in vitro were calculated by dividing the percentage of parent radioligand in the tissue sample measured with HPLC by the initial fractional radiochemical purity of the radioligand.

Distribution of 11C-NR2B-SMe in Rat Blood in vitro

Formulated 11C-NR2B-SMe (1.85 MBq; ~ 10 µL) was added to anticoagulated rat whole blood (2.5 mL), mixed well, and incubated at 37 °C for 30 min. A sample of whole blood (50 µL) was measured for radioactivity. The remaining whole blood was centrifuged at 1,800 g for 2.5 min and an aliquot (50 µL) was removed for γ-counting. Then, an aliquot (450 µL) of the plasma was removed to a new vial. Acetonitrile (720 µL) was added, mixed well, and measured for radioactivity. The mixture was centrifuged at 10,000 g for 1 min. The supernatant was analyzed with general HPLC method A. The precipitate was measured for radioactivity to determine the recovery of radioactivity into the supernatant that was injected onto HPLC. The relative cellular blood (red blood cells, white blood cells, and platelets) partitioning of 11C- NR2B-SMe was calculated with the following formula:

% Cells = [(Cwhole blood – Cplasma) × (1–Hcrt)]/Cwhole blood

where Cwhole blood is the concentration of radioactivity in whole blood, Cplasma is the concentration of radioactivity in plasma, and Hcrt is the hematocrit (Table 2). Hcrt was determined with a StatSpin® CritSpin™ Microhematocrit system (Beckman Coulter) using heparinized capillary tubes (12 × 40 mm) with blood samples drawn after the injection of the vehicle, immediately before injection of 11C-NR2B- SMe.

Experiments with 11C-NR2B-SMe and Human Tissues In Vitro

Metabolic Stabilities of 11C-NR2B-SMe in Human Brain Homogenate and Human Plasma in vitro. The tissues had been stored frozen at −70 °C but were thawed for analysis. 11C-NR2B-SMe (~37 kB; 10.0 µL) was added to thawed tissue (650 µL), mixed well, and incubated for 30 min at room

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temperature. Subsequent processing and analysis were as described for rat ex vivo experiments in the main text.

Human Plasma Free Fraction. Plasma free fraction (fp) was measured as described previously (6). Briefly, formulated 11C-NR2B-SMe (74 kBq; 11 µL) was mixed with human plasma (650 µL), incubated at RT for 10 min, and then filtered gravimetrically through ultrafiltration membranes (Centrifree; Millipore). If necessary, samples were left to decay to within the optimal radioactivity range for accurate γ-counter measurement.

PET Imaging in Rats

Evaluation of Radioligands in Normal Rats with PET. For radioligand injection, a polyethylene catheter (PE 10) was inserted into the penile vein of a male rat and was secured with tissue adhesive and tape. For dynamic scanning, the rats were secured with tape in the scanner and kept under 1.5% isoflurane anesthesia via a nose cone. Body temperature was monitored with a rectal probe. All PET scans were performed on a microPET Focus 220 scanner (Siemens Medical Solutions; Knoxville, TN) (7,8). Data were acquired in listmode and reconstructed with Fourier rebinning + 2D filtered backprojection. A transmission scan was acquired before radioligand injection for attenuation correction. Dynamic scanning began at the time of a one-minute intravenous bolus injection of the respective NCA PET radioligand (55 ± 19 MBq; volume 0.2 to 1.0 mL) and last for 100 min. The injected mass dose was 3 ± 1 nmol/kg. Challenging agents were administered either i.v. 10 min before radioligand for preblocking studies or 10 min after radioligand for displacement studies. Tomographic images were analyzed with pixel-wise modeling computer software (PMOD 2.6; PMOD Group, Zurich, Switzerland). Regions-of-interest in brain were delineated, and time-activity curves in SUV calculated.

Estimation of FTC concentration in rat brain for a 0.05 mg/kg i.v. dose and occupapcy of σ1- receptors. The estimation of concentration in brain is based on 1 SUV representing a uniform concentration of drug throughout the body. 0.05 mg/kg for a compound (NR2B-SMe; M.Wt. 373) in a typical 440 g rat giving 1 SUV equates to 134 nM. The uptake of 18F-FTC146 in mouse brain is about 1 (9). The logD of FTC146 has been measured to be very low (1.46) (10). Compounds with low lipophilicity in general do not have low plasma or brain free fractions. Even if the brain free fraction were exceptionally low, say

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1%, the rat brain concentration would be 1.34 nM. If the true KD in vivo is 100-fold less than the KD in

liver homogenate, the KD would be 2.5 × 100 pM = 0.25 nM. Therefore, even in this worst-case scenario (unrealistically low free fraction in brain and 100-fold weaker affinity in brain in vivo than in liver in vitro), FTC146 should still occupy the preponderance of σ1 receptors. RESULTS

Properties of NR2B-SMe

Absolute Configuration. Initially, the absolute configuration of each enantiomer of NR2B-SMe was unknown. NR2B-SMe was resolved with chiral HPLC and the optical rotations of the separate enantiomers were measured. (−)-NR2B-SMe and (+)-NR2B-SMe were both thick syrups, and our attempts to crystallize their TFA salts failed. This precluded X-ray crystallography for determination of absolute configuration and so we resorted to the use of VCD (11). Solvent-corrected IR and VCD spectra for (−)-NR2B-SMe and (+)-NR2B-SMe were obtained (Supplemental Figs. 4 and 5). Conformations of the R-enantiomer were searched at the molecular mechanics level on the entire molecule and optimized using a B3LYP “functional” on a 6-31G(d) basis set with Gaussian 09. During calculation, the SMe group was removed (truncated). Truncation is a common technique for reducing the number of low energy conformations to be calculated. Groups are only truncated when they are far away from any chiral center, and not likely to influence the VCD spectrum. For NR2B-SMe, there are over 500 low energy conformers. Without the SMe group, the number of conformers that were within 1.5 kcal/mol of the lowest-energy conformer dropped to 147. The excellent agreement between experimental and calculated VCD spectra (Supplemental Table 2) strongly indicated that the SMe group does not contribute significantly to the VCD spectra, generating a confidence level of 99% (Table S2). This is a measure of the degree of agreement between the measured and calculated spectra, not the likelihood that the assignment is correct. VCD and IR spectra were calculated on the optimized geometries of these conformers. Their Boltzmann summation was compared with the observed spectra of (−)-NR2B-SMe. Given the agreement between calculation and experiment, the absolute configuration of (−)-NR2B-SMe was assigned to be R. The same methodology showed (+)-NR2B-SMe has S-configuration.

Assessment of Binding Affinity against a Wide Range of Other Receptors and Binding Sites. NR2B-SMe was submitted to the National Institute of Mental Health Psychoactive Drug Screening program (NMIH-PDSP) for assessment of binding affinity against a wide range of other receptors and

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binding sites (5HT1A, 1B, 1D, 1E, 2A, 2A agonist, 2A antagonist, 2B, 2B agonist, 2B antagonist, 2C, 2C agonist, 2C antagonist, 3, 5A, 6, 7; α2;

α2β2, α2β2, α3β2, α3β4, α4β2, α4β4, α7; α1A, 1A agonist, 1B, 1B agonist, 1B antagonist, 2A-2C; AMPA*; β1-3; BZPa1- 2+ 3, a5, a6, rat brain site; Ca -Channel; CB1-2, D1, 2, 2 agonist, 2 antagonist, 3-5; DAT; DOR; EP1-4; GABAA,B; GPR40,41,43;

H1-4; HERG; IMIDAZOLINE 1; KA-R*; KOR, KORagonist, antagonist; M1, 1 agonist, 1 antagonist, 2, 3, 3 agonist, 3 + antagonist, 4, 5, 5 agonist, 5 antagonist; MDR 1; mGluR1A,2,4,5,5-cloned,5-rat brain,6,8; MOR; Na -Channel; NET; NK1, 1

antagonist, 2, 2 antagonist, 3, 3 antagonist; NMDA; NMDA/MK801; NT-1; OXYTOCINagonist, antagonist; PAR1agonist, antagonist; PCP; PKCA, B, D, E, G; PTHR2, Purinergicagonist, antagonist; SET; σ1,2; V1, 1 agonist, 1 antagonist, 2, 2 agonist, 2

antagonist, 3, 3 agonist, 3 antagonist, VMAT1, 2). Detailed assay protocols are available at the NIMH-PDSP web site (http://pdsp.cwru.edu). At 10 µM concentration, inhibition was greater than 10% for only a few binding sites and receptors. These were the calcium channel (10.6%), hERG channels (66.3%), guinea pig σ1 (83.7%), and PC12 cell σ2 (90.0%). 11 11 LogD7.4 and pKa of C-NR2B-SMe. The measured logD7.4 of C-NR2B-SMe at RT in 1-octanol

was 3.41 ± 0.08 (n = 6) and in fair agreement with the value (clogD7.4) of 2.98 computed with Pallas

software. The apparent pKa was 5.03 ± 0.01 (n = 3) (Supplemental Fig. 8).

Radiochemistry

Treatment of NR2B-ester with 11C-iodomethane under basic conditions (Supplemental Fig. 1) gave > 95% radiochemically pure 11C-NR2B-SMe in 10−13% yield after formulation for intravenous administration, and with high molar radioactivity (52−74 GBq/µmol). The most effective base for the radiosynthesis was tetra-n-butyl ammonium hydroxide. Other bases, including KOH and the phosphazene base P1-t-Bu-tris(tetramethylene), gave complicated mixtures of radiolabeled products.

11C-(R)-NR2B-SMe and 11C-(S)-NR2B-SMe were synthesized from their respective homochiral precursors. The identity of each enantiomer was confirmed by analysis of the formulated product after decay with radio-chiral HPLC, with and without co-injection of the reference enantiomer.

Stability of 11C-NR2B-SMe in the Formulated Dose. The radiochemical stability of 11C-NR2B- SMe was found to depend on the composition of the formulation. The radiochemical purity of 11C- NR2B-SMe ranged between 95 and 99% immediately after radiosynthesis. However, the fraction collected from HPLC contains 0.1% TFA. When the pH of this fraction was not increased immediately before drying by rotary evaporation at 80 oC, the purity measured for the formulated radioligand was only about 90%. Protection with ascorbic acid was therefore used immediately after HPLC radioligand

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purification to maintain weakly acidic and reducing conditions. The formulated radioligand was then radiochemically stable for at least 3 h at RT (99.9 ± 0.3%; n = 4), and stable in PBS (10 mM) for at least 2.5 h. There was virtually no adsorption of radioligand to the walls of glass test tubes when in PBS (10 mM) (98.8 ± 0.3%; n = 4, remained in solution). The error in the γ-counter measurements of samples with least counts was 0.3 ± 0.12% (n = 6).

Experiments with 11C-NR2B-SMe in Rats and Human Tissues Stability of 11C-NR2B-SMe in Rat Whole Blood, Plasma, and Brain ex vivo and in vitro. 11C- NR2B-SMe was slightly unstable in rat whole blood and plasma in vitro, with only small amounts

(~10%) of a single less lipophilic radiometabolite (tR ~ 3.6 min) detected after incubation for 30 min at 37°C (Table 1 and Supplemental Fig. 11). 11C-NR2B-SMe was especially stable in brain homogenate, with only 3% conversion into radiometabolite. Metabolic Stability of 11C-NR2B-SMe in Human Brain Homogenate and Human Plasma. 11C- NR2B-SMe was stable in human brain homogenate (99.5%) and human plasma (100%) at RT for 30 min.

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Supplemental Table 1. Experimental parameters used for the metabolite analysis of 11C-NR2B-SMe in whole blood, plasma, and brain of one rat in vitro at 37°C and one rat ex vivo, and recoveries of radioactivity into MeCN from tissues.

Sample In vitro Ex vivo

Injected radioactivity (MBq) 40.1

Rat weight (g) 441 438

Molar activity at injection time (MBq/µmol) 21.6

Mass injected (nmol/kg) 4.32

Experiment duration (min) 30 30

Recoveries (%): into MeCN from: Plasma 91.9 94.2

Whole blood 90.2

Brain 85.2 87.9

Myocardium 75.0

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Supplemental Table 2. Numerical comparison describing the similarity in the range of 1000–1800 cm- 1 between the calculated IR and VCD spectra for the R-enantiomer at the B3LYP/6-31G(d) level and the observed IR and VCD spectra for (–)-NR2B-SMe.

Numerical comparison Observed

Scaling factor 0.964

IR similarity (%) 84.7

VCD similarity: aƩ(%) 71.324

Enantiomeric similarity: bΔ(%) 64.458

Confidence level (%) 99

aƩsingle VCD similarity: gives the similarity between calculated and observed VCD spectra. bΔ: enantiomeric similarity index: gives the difference between the value of Ʃ for both enantiomers.

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n-Bu R S 4NOH in R S MeOH - DMF CO2Me CO2 NR2B-ester - HO OH R = N 11CH I 3 - MeO R S R S RT, 5 min 11CH 3 + 11C-NR2B-SMe - O O

Supplemental Figure 1. Radiosynthesis of 11C-NR2B-SMe. The enantiomers were prepared in the same manner from the respective homochiral precursors.

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(+)-NR2B-SMe

(–)-NR2B-SMe

Time (min)

HPLC conditions: (S,S)-Whelk-01 column (250 × 4.6 mm; 5 µm) eluted with hexane/ethanol (1:1 v/v) containing 0.1% diethylamine at 1.5 mL/min. Injection volume 50 µL; Retention times: (+)-NR2B-SMe, 4.51 min; (–)-NR2B-SMe, 7.32 min. Supplemental Figure 2. Chiral HPLC of the enantiomers of NR2B-SMe.

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(+)-NR2B-ester

(–)-NR2B ester

Time (min)

HPLC conditions: (S,S)-Whelk-O1 column (250 × 4.6 mm; 5 µm) eluted with hexane/ethanol (1:1 v/v) containing 0.1% diethylamine at 1.5 mL/min. Injection volume 50 µL; Retention times: (+)-NR2B-ester, 6.19 min; (–)-NR2B-ester, 10.93 min. Supplemental Figure 3. Chiral HPLC of the enantiomers of NR2B-ester.

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VCD (upper frame; blue line) and IR (lower frame; red line) spectra measured for (–)-NR2B-SMe in CDCl3 (11.2 mg in 0.175 mL); 0.1 mm path-length cell with BaF2 windows, with 18 h collections for samples and solvent. Instrument was optimized at 1400 cm-1. Solvent-subtracted IR and enantiomer-subtracted VCD spectra are shown. Uppermost trace is the VCD noise spectrum. Supplemental Figure 4. Measured IR and VCD spectra of (–)-NR2B-SMe.

Supplemental Figure 5. Comparison of VCD (upper frame) and IR (lower frame) spectra measured for (–)-NR2B-SMe (left axes; red lines) with the calculated Boltzmann-averaged spectra of the calculated conformations for (R)-NR2B-SMe (right axes, blue lines).

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Black line: absorbance at 255 nm. Red line: radioactivity detector response. Supplemental Figure 6. Radiochromatogram from the HPLC separation of 11C-NR2B-SMe after radiolabeling. Y-axes are on linear scales. See full text for HPLC conditions.

Blue line: absorbance at 255 nm. Purple line: radioactivity detector response. Supplemental Figure 7. Radiochromatogram from the HPLC analysis of formulated 11C-(S)-NR2B- SMe. Y-axes are on linear scales. See full text for HPLC conditions.

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100

75 Cyclohexane phase Aqueous phase 50

concentration 25 Relative radioactivity 0

3 5 7 9 11 pH

Supplemental Figure 8. pH-Dependence of the distribution of 11C-NR2B-SMe between cyclohexane and sodium phosphate buffers. Error bars are mean ± SD, and are within the symbol size if not shown. The red arrow indicates the apparent pKa of 11C-NR2B-SMe. The curves are fitted with Prism software.

Supplemental Figure 9. Experiments in HeLa cells show that NR2B-SMe is not trapped in lysosomes. Panel A: Image showing absence of NR2B-SMe cellular uptake. Panel B: Image showing cell uptake of loperamide in control experiment. Panel C: Quantitative experiment showing that NR2B-SMe does not compete with Cy3 for uptake into HeLa cells. Error bars are mean ± SD (n = 3).

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Supplemental Figure 10. HPLC radiochromatograms of rat plasma (panel A), brain (panel B), and mycocardium (panel C) sampled at 30 min after the intravenous injection of 11C-NR2B2B-SMe, measured ex vivo. The radioligand represented 71.6% of the radioactivity in plasma, 97% in brain, and 90% in mycocardium. Y-axis (radioactivity; cps) is on linear scale. See full text for HPLC conditions.

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Whole blood in vitro Plasma in vitro

Time (min) Time (min) Rat brain homogenate in vitro

Time (min) Supplemental Figure 11. Radiochromatograms from HPLC analysis of rat tissues at 30 min after incubation with 11C-(S)-NR2B-SMe in vitro. Y-axes (radioactivity; cps) are on linear scale. See full text for HPLC conditions.

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11 A C-NR2B-SMe B 11C-NR2B-SMe

3 3 Baseline 2 2 Ro-25-6981: Baseline 2.5 mg/kg NR2B-SMe 1.25 mg/kg 1 1 Ro-25-6981 0.25 mg/kg Ifenprodil Conc. radioactivity (SUV) radioactivity Conc. 0 (SUV) radioactivity Conc. 0

0 20 40 60 80 100 0 20 40 60 80 100 Time (min) Time (min)

C 11C-(R)-NR2B-SMe

3 Ro-25-6981

1 Baseline Ro-25-6981: 0.05 mg/kg

Conc. radioactivity (SUV) radioactivity Conc. 0 0.25 mg/kg 1.25 mg/kg 0 20 40 60 80 100 Time (min)

Supplementary Figure 12. PET imaging of rat brain with 11C-NR2B-SMe and 11C-(R)-NR2B-SMe. Panel A: PET TACs showing that radioactivity accumulation in rat whole brain after intravenous injection of 11C-NR2B-SMe is blocked by pre-administration of NR2B-SMe, or the NR2B ligand ifenprodil at 3.0 mg per kg (i.v.). Data are for n = 1. Panel B. Effect of dose of NR2B ligand Ro-25- 6981 on radioactivity displacement from rat whole brain after intravenous injection of 11C-NR2B-SMe. Ro-25-6981 was administered intravenously at 10 min after radioligand. Baseline data are mean for n = 2. Error bar represents range. Other data are for n = 1. Panel C. Increasing doses of the NR2B ligand Ro-25-6981 increase radioactivity displacement from rat whole brain after intravenous injection of 11C- (R)-NR2B-SMe. Ro-25-6981 was administered intravenously at 10 min after radioligand. Baseline data are mean for n = 2. Error bar represents range. Other data are for n = 1.

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Putative NR2B ligands show dose-dependent preblocking of radioactivity uptake in rat whole brain before intravenous injection of 11C-(S)-NR2B-SMe:

A Ifenprodil B Ifenprodil 4 1.0

3 0.8 Baseline 0.6 2 Ifenprodil: 0.01 mg/kg 0.4 1 0.05 mg/kg 0.25 mg/kg 0.2 1.25 mg/kg Conc. radioactivity (SUV) radioactivity Conc. 0 (20-90Relative min) AUC 0.0

0 20 40 60 80 100 -10 -9 -8 -7 -6 -5 Time (min) Log[Ifenprodil (nmol/kg)]

C CO101244 D CO101224 5 1.0

4 0.8 Baseline 3 0.6 CO101244: 2 0.01 mg/kg 0.4 0.05 mg/kg 1 0.2 0.25 mg/kg Relative AUC (20-90Relative min) AUC Conc. radioactivity (SUV) radioactivity Conc. 0 1.25 mg/kg 0.0

0 20 40 60 80 100 -10 -9 -8 -7 -6 -5 Time (min) log[C101224 (nmol/kg]

Supplementary Figure 13. In vivo dose-dependent preblocking of 11C-(S)-NR2B-SMe by NR2B ligands (ifenprodil and CO101224) in rats. Left panels are the PET time-activity curves for radioactivity uptake whole brain at the cited doses of the pre-administered NR2B ligands. Each right panel is the fitted dose-response curve from the data in the corresponding left panel.

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Putative σ1 receptor antagonists show weak or absent preblocking of radioactivity uptake in rat whole brain before intravenous injection of 11C-(S)-NR2B-SMe:

A B BD 1407 BD1407

3 1.0 0.8

2 0.6

Baseline 0.4 1 BD1407: 0.2 0.01 mg/kg

0.05 mg/kg (20-90Relative min) AUC Conc. radioactivity (SUV) radioactivity Conc. 0 0.0

0 20 40 60 80 100 -10 -9 -8 -7 -6 Time (min) Log [BD1047 (nmol/kg)]

C F4 D F4 4 1.4 1.2 3 1.0

2 0.8 0.6 1 Baseline 0.4

F4: AUC Normalized 0.01 mg/kg 0.2 Conc. radioactivity (SUV) radioactivity Conc. 0 0.05 mg/kg 0.0

0 20 40 60 80 100 -10 -9 -8 -7 -6 Time (min) Log[F4(nmol/kg)}

E NE100 F NE100 3 1.2 1.0 2 0.8 0.6 1 Baseline 0.4 NE100: 0.2 0.01 mg/kg Conc. radioactivity (SUV) radioactivity Conc. 0 0.05 mg/kg (20-90Relative min) AUC 0.0

0 20 40 60 80 100 -10 -9 -8 -7 -6 Time (min) Log[NE100 (nmol/kg)]

G F3 H F3 4 1.0

3 0.8 Baseline 0.6 2 F3: 0.01 mg/kg 0.4 1 0.05 mg/kg 0.25 mg/kg 0.2 1.25 mg/kg Conc. radioactivity (SUV) radioactivity Conc. 0 (20-90Relative min) AUC 0.0

0 20 40 60 80 100 -10 -9 -8 -7 -6 -5 Time (min) log[F3 (nmol/kg)] Supplementary Figure 14. In vivo dose-dependent preblocking of 11C-(S)-NR2B-SMe by σ1 antagonists (BD1047, F3, F4, and NE100) in rats. Left panels: PET time-activity curves for whole brain radioactivity uptake at the cited doses of pre-administered σ1 antagonists. Each right panel is the dose- response curves from the data in the corresponding left panel. Solid line in right panel is fitted and dashed lines unfitted. Data are for n = 1.

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Putative σ1 receptor agonists show very weak or absent preblocking of radioactivity uptake in rat whole brain before intravenous injection of 11C-(S)-NR2B-SMe:

(+)-Pentazocine A (+)-Pentazocine B 4 1.0

3 0.8

2 0.6 0.4 1 Baseline 0.01 mg/kg 0.2 0.05 mg/kg Conc. radioactivity (SUV) radioactivity Conc. 0 (20-90Relative min) AUC 0.0

0 20 40 60 80 100 -10 -9 -8 -7 -6 Time (min) Log[(+)-Pentazocine(nmol/mg)]

C (±)-PPCC (±)-PPCC 4 D 1.0

3 0.8

2 0.6

Baseline 0.4 1 0.01 mg/kg 0.05 mg/kg 0.2 Conc. radioactivity (SUV) radioactivity Conc. 0 (20-90Relative min) AUC 0.0

0 20 40 60 80 100 -10 -9 -8 -7 -6 Time (min) log[(±)-PPCC (nmol/kg)]

F E PRE-084 PRE-084 4 1.0

3 0.8

0.6 2 0.4 Baseline 1 0.01 mg/kg 0.2 0.05 mg/kg Relative AUC (20-90Relative min) AUC

Conc. radioactivity (SUV) radioactivity Conc. 0 0.0

0 20 40 60 80 100 -10 -9 -8 -7 -6 Log[PRE-084 (nmol/kg)] Time (min)

G (+)-SKF10047 H (+)-SKF10047 3 1.0

0.8 2 0.6

1 0.4 Baseline 0.01 mg/kg 0.2 0.05 mg/kg Conc. radioactivity (SUV) radioactivity Conc. 0 (20-90Relative min) AUC 0.0

0 20 40 60 80 100 -10 -9 -8 -7 -6 Time (min) Log[SKF10047(nmol/kg)] Supplementary Figure 15. In vivo dose-dependent preblocking of 11C-(S)-NR2B-SMe by σ1 agonists ((+)-pentazocine, (±)-PPCC, PRE-084, and (+)-SKF10047). Left panels: PET time-activity curves for whole brain radioactivity uptake in rat at the cited doses of pre-administered σ1 agonists. Each right panel is the unfitted dose-response curve from the data in the corresponding left panel.

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