Binding characterization of N‐(2‐chloro‐5‐thiomethylphenyl)‐N′‐(3‐[3H]3methoxy phenyl)‐N′‐methylguanidine ([3H]GMOM), a non‐competitive N‐methyl‐D‐aspartate (NMDA) receptor antagonist

Abstract Labeled with carbon‐11, N‐(2‐chloro‐5‐thiomethylphenyl)‐N′‐(3‐methoxyphenyl)‐N′‐methylguanidine ([11C]GMOM) is currently the only positron emission tomography (PET) tracer that has shown selectivity for the ion‐channel site of N‐methyl‐D‐aspartate (NMDA) receptors in human imaging studies. The present study reports on the selectivity profile and in vitro binding properties of GMOM. The compound was screened on a panel of 80 targets, and labeled with tritium ([3H]GMOM). The binding properties of [3H]GMOM were compared to those of the reference ion‐channel ligand [3H](+)‐dizocilpine maleate ([3H]MK‐801), in a set of concentration‐response, homologous and heterologous inhibition, and association kinetics assays, performed with repeatedly washed rat forebrain preparations. GMOM was at least 70‐fold more selective for NMDA receptors compared to all other targets examined. In homologous inhibition and concentration‐response assays, the binding of [3H]GMOM was regulated by NMDA receptor agonists, albeit in a less prominent manner compared to [3H]MK‐801. Scatchard transformation of homologous inhibition data produced concave upward curves for [3H]GMOM and [3H]MK‐801. The radioligands showed bi‐exponential association kinetics in the presence of 100 μmol L−1 l‐glutamate/30 μmol L−1 glycine. [3H]GMOM (3 nmol L−1 and 10 nmol L−1) was inhibited with dual affinity by (+)‐MK‐801, (R,S)‐ketamine and memantine, in both presence and absence of agonists. [3H]MK‐801 (2 nmol L−1) was inhibited in a monophasic manner by GMOM under baseline and combined agonist conditions, with an IC50 value of ~19 nmol L−1. The non‐linear Scatchard plots, biphasic inhibition by open channel blockers, and bi‐exponential kinetics of [3H]GMOM indicate a complex mechanism of interaction with the NMDA receptor ionophore. The implications for quantifying the PET signal of [11C]GMOM are discussed.

The ionotropic NMDA receptors possess at least six distinct sites for the recognition of endogenous ligands, including sites for the agonist L-glutamate, the co-agonists glycine and D-serine, and modulators of receptor function as diverse as polyamines, steroids, metal ions, and protons. 4 Of the multiple binding sites, considerable effort has been directed on imaging the site within the pore of the NMDA receptor ion-channel. Due to its unique physical location, the intrachannel site is primarily accessible when the NMDA receptors are in an open conformation, that is activated by the agonists L-glutamate and glycine/D-serine. 5,6 Thus, PET tracer uptake at the ion-channel site should be proportional to the amount of activated NMDA receptors, permitting the evaluation of their functional state in vivo. 7. N, N΄-di-substituted guanidines were first described as open channel blockers of the NMDA receptors by electrophysiology, in 1989. 8 To date, diarylguanidine analogs constitute the most promising class of compounds for the functional imaging of NMDA receptors. Unlike early derivatives of phencyclidine and (+)-dizocilpine maleate (MK-801), which were unsuitable for PET due to high lipophilicity and non-specific uptake, [9][10][11] 15 are NMDA-targeting tracers of moderate lipophilicity and high affinity (Figure 1), which have exhibited quantifiable brain uptake in human PET studies). [16][17][18][19] [ 11 C]GMOM, in particular, is currently the only diarylguanidine tracer that has shown selectivity for the NMDA receptors in vivo, as evidenced by a F I G U R E 1 Structures and affinity values of NMDA receptor PET tracers used in human imaging studies reduction in the tracer's net influx rate following the administration of an S-ketamine challenge to healthy human volunteers. 19 Despite showing considerable potential as a PET tracer, [ 11 C] GMOM remains poorly characterized in terms of its in vitro binding profile. GMOM is known to possess a high-affinity binding site that is associated with the reference ion-channel blocker of NMDA receptors, [ 3

| Selectivity profile of GMOM
The pharmacological selectivity of GMOM was evaluated at CEREP S.A. (Poitiers, France), using a broad screen of 79 targets, comprising neurotransmitter receptors and transporters, and ion-channels (Table S1). Binding of GMOM to the ifenprodil site of NMDA receptors was evaluated in-house, using a previously detailed assay. 20 A percent (%) inhibition of control specific binding was initially calculated for each target, using a single concentration of GMOM (10 μmol L −1 ). The affinity of GMOM for targets showing ≥50% inhibition of control binding in the primary screen was subsequently derived from full concentration-response inhibition curves, which were obtained by using at least eight concentrations of GMOM. Hill coefficients (nH) and half-maximal inhibitory concentrations (IC 50 ) were derived from the competition binding experiments by non-linear regression analysis, using software developed at CEREP (Hill software  dark cycle (lights on: 8 AM) and controlled temperature (21 ± 2°C) and humidity conditions (50 ± 10%). Food and water were available ad libitum (Teklad Global 16% Protein Rodent Diet, Harlan Laboratories).

| Animals and membrane preparation
Extensively washed, crude synaptic membranes were prepared as detailed previously. 22 Animals were killed by decapitation, and the cerebral cortices and hippocampi immediately removed and homogenized in a 15-fold excess (v/w) of ice-cold 0.32 mol L −1 sucrose, using a DUALL tissue grinder (20 strokes, 800 rpm; VWR, Amsterdam, the Netherlands). The homogenate was centrifuged at 1000 g for 10 min-  incubations in assay buffer, using 10 μmol L −1 unlabeled GMOM to define NSB (pH range: 6.5-8.5). Based on these preliminary studies, the methods described below represent optimized binding conditions.

| Regulation of radioligand binding by NMDA receptor agonists/antagonists
The effects of L-glutamate and glycine on the binding of 10 nmol L −1 Experiments were conducted in triplicate, 3-4 independent times.  All competitors were dissolved as 10 mmol L −1 stock solutions in DMSO, with the exception of (R,S)-ketamine, which was prepared as 1 mol L −1 stock solution in dH 2 O, containing 10% ethanol. Experiments were repeated 3-9 independent times. IC 50 values were derived from the concentration-inhibition curves by fitting the raw data to the one-or two-sites competition model using GraphPad Prism software.

| Heterologous inhibition studies
All values were calculated as logIC 50 and reported as the IC 50 .

| Synthesis of [ 3 H]GMOM
Five to ten MBq of [ 3 H]GMOM were obtained per synthesis, with radiochemical yields ranging from 11 to 22%. The specific activity of the product was 3.13 TBq/mmol (84.5 Ci/mmol), and its radiochemical and chemical purity >99% (Figure 2).

| Homologous inhibition assays
Saturation binding parameters were obtained by self-inhibition of      Table 3.

| Association kinetics
Association curves were constructed in the presence of 100 μmol

| Non-linear Scatchard plots
Linear transformation of homologous inhibition data produced con-  Observations to support this statement include GMOM's selectivity profile at 3 nmol L −1 and 10 nmol L −1 , the limited ability of MK-801, ketamine and memantine to discriminate between NMDA receptor subtypes, 44   imaging studies, are unlikely to target a single intra-channel site that is primarily associated with the activated state of NMDA receptors.