GLYX-13, a NMDA receptor glycine-site functional partial agonist, induces antidepressant-like effects without ketamine-like side effects

Abstract

Recent human clinical studies with the NMDA receptor (NMDAR) antagonist ketamine have revealed profound and long-lasting antidepressant effects with rapid onset in several clinical trials, but antidepressant effects were preceded by dissociative side effects. Here we show that GLYX-13, a novel NMDAR glycine-site functional partial agonist, produces an antidepressant-like effect in the Porsolt, novelty induced hypophagia, and learned helplessness tests in rats without exhibiting substance abuse-related, gating, and sedative side effects of ketamine in the drug discrimination, conditioned place preference, pre-pulse inhibition and open-field tests. Like ketamine, the GLYX-13-induced antidepressant-like effects required AMPA/kainate receptor activation, as evidenced by the ability of NBQX to abolish the antidepressant-like effect. Both GLYX-13 and ketamine persistently (24 h) enhanced the induction of long-term potentiation of synaptic transmission and the magnitude of NMDAR-NR2B conductance at rat Schaffer collateral-CA1 synapses in vitro. Cell surface biotinylation studies showed that both GLYX-13 and ketamine led to increases in both NR2B and GluR1 protein levels, as measured by Western analysis, whereas no changes were seen in mRNA expression (microarray and qRT-PCR). GLYX-13, unlike ketamine, produced its antidepressant-like effect when injected directly into the medial prefrontal cortex (MPFC). These results suggest that GLYX-13 produces an antidepressant-like effect without the side effects seen with ketamine at least in part by directly modulating NR2B-containing NMDARs in the MPFC. Furthermore, the enhancement of 'metaplasticity' by both GLYX-13 and ketamine may help explain the long-lasting antidepressant effects of these NMDAR modulators. GLYX-13 is currently in a Phase II clinical development program for treatment-resistant depression.

Figure 1

GLYX-13 produces an antidepressant-like effect in multiple rat models of depression. (a) Mean±SEM floating time in the rat Porsolt test in 2–3 month-old Sprague Dawley rats treated with GLYX-13 (TPPT-NH₂; 1–56 mg/kg, IV), scrambled GLYX-13 (PTTP-NH₂; 3 mg/kg, IV), ketamine (10 mg/kg, IP), fluoxetine (20 mg/kg, SC), or sterile saline vehicle (1 ml/kg, IV) 30–60 min before testing, or (b) GLYX-13 (3 mg/kg, IV), ketamine (10 mg/kg, IV) or fluoxetine (20 mg/kg, SC) or saline vehicle-treated rats tested 24 h post dosing. (c) Mean±SEM latency to eat in the novelty induced hypophagia (NIH) test in rats dosed with GLYX-13 (3 mg/kg, IV), ketamine (10 mg/kg, IV) or saline and tested 1 h post dosing. (d) Mean±SEM escape failures in the footshock-induced learned helplessness test in 2–3-month-old male SD rats dosed with GLYX-13 (3 mg/kg, IV), fluoxetine (20 mg/kg, SC), or sterile saline vehicle (1 ml/kg IV; tail vein) 24 h before testing. Naïve control animals did not receive pre-shock or injection before LH testing. N=7–21 per group. *P<0.05 Fisher's PLSD post hoc test vs vehicle.

Figure 2

The AMPA/kainate antagonist NBQX blocks the antidepressant-like effect of GLYX-13. Mean (±SEM) floating time in the Porsolt test in animals pre-treated with NBQX (10 mg/kg, IP) before GLYX-13 (3 mg/kg, IV) dosing and tested 1 h post-dosing. The antidepressant-like effects of ketamine has also been shown to be blocked by NBQX (Maeng et al, 2008) n=11 per group. *P<0.05, Fisher's PLSD vs all other groups.

Figure 3

GLYX-13 but not ketamine produces an antidepressant-like effect in the rat Porsolt test when injected into the medial prefrontal cortex. Mean (±SEM) time (sec) spent immobile in the Porsolt test in 2–3-month-old male rats implanted with (a) medial prefrontal or motor cortex (dorsal control) cannulae and injected with GLYX-13 ( 0.1, 1, 10 μg/side) or sterile saline vehicle (0.5 μl/1 min) and tested 1 h post-dosing or rats given MPFC injections of ketamine (0.1, 1, 10 μg), GLYX-13 (1 μg), or saline and tested (b) 20 min and (c) 24 h post dosing. Animals received a 15-min training swim session 1 day before dosing. (d) Mean (± SEM) line crosses in the open field 20 min; following MPFC infusion of GLYX-13 (1 μg), ketamine (0.1 μg) or sterile saline vehicle. Given that 0.1 μg dose of ketamine increased locomotor activity, the Porsolt data for that dose were not included in the analysis given that increasing locomotor activity produces a false-positive antidepressant-like response. (e) a representative H&E-stained section depicting MPFC cannulae placement, arrow indicates injection site. n=5–10 per group. *P<0.05, Fisher's PLSD vs vehicle.

Figure 4

GLYX-13 does not show ketamine-like discriminative stimulus effects. Mean (±SEM) (a) percentage ketamine-lever responding and (b) rates of responding for different doses of ketamine (IP and SC) and GLYX-13 (SC) in rats trained to discriminate 10 mg/kg ketamine (Ket), IP, from saline (Sal). Values above Sal and Ket are the results of control tests conducted before testing each dose–response curve. Values above Sal/Sal and Sal/Ket are the results of similar control tests performed, following administration of 2 ml saline SC, 30 min before the session start to mimic conditions of GLYX-13 testing. n=7–8 per group. *P<0.05 Fisher's PLSD post hoc test vs vehicle.

Figure 5

GLYX-13 does not show ketamine-like rewarding, sensory-motor gating, or sedative side effects. (a) Ketamine (10 mg/kg, IV), but not GLYX-13 (10 mg/kg, IV), induced conditioned place preference as measured by % time in drug paired chamber. (b) Ketamine (10 mg/kg, IP), but not GLYX-13 (10 mg/kg, IV), decreased sensory-motor gating as measured by prepulse inhibition. (c) A sedating dose of ketamine (10 mg/kg, SC), but not GLYX-13 (10 mg/kg, IV), reduced locomotor activity in the open field as measured by line crosses. N=8–11 per group. *P<0.05 Fisher's PLSD post hoc test vs vehicle. Data are expressed as Mean (±SEM).

Figure 6

GLYX-13 and ketamine increased the expression of cell surface NR2B and GluR1 proteins in the rat MPFC and hippocampus 24 h post dosing. Mean±SEM protein levels in the medial prefrontal cortex (MPFC) or hippocampus as measured by Western analyses in 2–3-month-old SD rats treated with GLYX-13 (3 mg/kg, IV) or sterile saline vehicle (1 ml/kg IV; tail vein) 24 h before sacrifice. MPFC or hippocampus slices were incubated with Sulfo-NHS-SS-Biotin to label surface protein, and biotinylated protein was precipitated with avidin-agarose beads. Protein samples were analyzed by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes probed with NR2B-, GluR1-, and β-actin-specific antibodies. The sample sizes for each group are, NR2B MPFC vehicle n=11, GLYX-13 n=11, ketamine n=8; NR2B hippocampus vehicle n=7, GLYX-13 n=6, ketamine n=6; GluR1 MPFC and hippocampus n=6 per group. *P<0.05, Fisher's PLSD post hoc test vs vehicle.

Figure 7

GLYX-13 and ketamine increase hippocampus NR2B current 24 h post dosing. (left panel) Representative Schaffer collateral-evoked EPSCs in a CA1 pyramidal neuron, treated 24 h earlier with vehicle (control), GLYX-13 (3 mg/kg, IV) or ketamine (10 mg/kg, IV). Traces are shown before (black), after bath application of the NR2B-selective NMDAR antagonist ifenprodil (IFE;10 μM; red), and after co-application of ifenprodil plus the NR2A-NMDAR-selective antagonist NVP-AM077 (NVP; 100 nM; blue). The scale for the x- and y-axes are an inset in the top panel. (right panel) Mean±SEM NMDA receptor-dependent single shock-evoked EPSCs in the presence of the NR2B-selective NMDAR antagonist ifenprodil (10 μM), in CA1 pharmacologically isolated NMDA current in rats that were dosed with GLYX-13 (3 mg/kg, IV) ketamine (10 mg/kg, IV), or sterile saline vehicle (IV), 24 h before in vitro NMDA current measurement. n=5–6 per group. *P<0.05, Fisher's PLSD vs vehicle.

Figure 8

GLYX-13 and ketamine induce metaplasticity 24 h post dosing. GLYX-13 (3 mg/kg, IV) or ketamine (10 mg/kg, IV) 24 h post dosing enhances the magnitude of ex-vivo long-term potentiation (LTP) of synaptic transmission at Schaffer collateral-CA1 synapses. Plot of mean±SEM Schaffer collateral-evoked normalized field EPSP slopes in hippocampal slices from rats treated 24 h earlier with vehicle (control; black circles), GLYX-13 (GLYX-13; blue circles) or ketamine (ketamine; red squares). Three submaximal bouts of high-frequency Schaffer collateral stimulation (2 × 100 Hz/800 ms) were applied at the arrows to elicit incremental LTP. Slices from both GLYX-13 and ketamine pre-treated rats exhibited significantly larger LTP compared with controls. n=8–10 per group, *P<0.05, **P<0.01, Fisher's PLSD post hoc test vs vehicle.