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Review
, 15 (1), 47-56

The Development of Rapastinel (Formerly GLYX-13); A Rapid Acting and Long Lasting Antidepressant

Affiliations
Review

The Development of Rapastinel (Formerly GLYX-13); A Rapid Acting and Long Lasting Antidepressant

Joseph R Moskal et al. Curr Neuropharmacol.

Abstract

Background: Rapastinel (GLYX-13) is a NMDA receptor modulator with glycine-site partial agonist properties. It is a robust cognitive enhancer and shows rapid and long-lasting antidepressant properties in both animal models and in humans.

Methods: Rapastinel was derived from a monoclonal antibody, B6B21, is a tetrapeptide (threonine-proline-proline-threonine-amide) obtained from amino acid sequence information obtained from sequencing one of the hypervariable regions of the light chain of B6B21. The in-vivo and in-vitro pharmacology of rapastinel was examined.

Results: Rapastinel was found to be a robust cognitive enhancer in a variety of learning and memory paradigms and shows marked antidepressant-like properties in multiple models including the forced swim (Porsolt), learned helplessness and chronic unpredictable stress. Rapastinel's rapid-acting antidepressant properties appear to be mediated by its ability to activate NMDA receptors leading to enhancement in synaptic plasticity processes associated with learning and memory. This is further substantiated by the increase in mature dendritic spines found 24 hrs after rapastinel treatment in both the rat dentate gyrus and layer five of the medial prefrontal cortex. Moreover, ex vivo LTP studies showed that the effects of rapastinel persisted at least two weeks post-dosing.

Conclusion: These data suggest that rapastinel has significant effects on metaplasticity processes that may help explain the long lasting antidepressant effects of rapastinel seen in the human clinical trial results.

Figures

Fig. (1)
Fig. (1)
Three ways of looking at rapastinel. Based on combining NMR studies of rapastinel in solution with a nuclear Overhauser analysis, it appears that rapastinel exists as a rigid β-1 type structure that also has a triple ring structure held by hydrogen bonds. Thus a simple seeming tetrapeptide actually has a quite rich 3-dimensional structure under physiological conditions.
Fig. (2)
Fig. (2)
Rapastinel activates NMDA receptor activity and readily crosses the blood–brain barrier. (A) Comparison of Rapastinel NMDA receptor modulatory activity compared with D-cycloserine. NMDA receptor modulatory activity was measured by monitoring [3H]MK-801 binding in well washed rat forebrain membrane preps in the presence of glutamate but not glycine. Both Rapastinel and D-cycloserine showed an optimal increase in [3H]MK-801 binding in the range of 1-10 µM. Each data point represents the average of two experiments each done in triplicate (n=6) with the values for each data point varying by no more than 3-5%. Baseline MK-801 binding was 0.6 ± 0.06 pmol/mg protein. (B) Blood–brain-permeability studies were performed using [3H]-Rapastinel, along with the appropriate controls, was injected into rats IV and binding was measured in brain homogenates using liquid scintillation spectrometry. Using these methods a brain uptake index (Oldendorf, 1970) of 80 ± 15 was obtained suggesting that Rapastinel readily crossed the blood–brain barrier. Data were adapted with permission from [14].
Fig. (3)
Fig. (3)
Rapastinel is a cognitive enhancer. (A-C) The effects of an optimal cognitive enhancing dose of rapastinel (1 mg/kg, IV, 15 min post-dosing) in young adult (3 months) or learning-impaired aged (27 month old) rats in hippocampal dependent trace eyeblink conditioning (A), alternating T-maze (B), and Morris water maze (C) tests. (D) GLYX-13 (1 mg/kg, IV, 15 min post-dosing) in young adult (3 months) rats facilitated positive emotional learning as measured by rates of hedonic 50-kHz USVs in response to a conditioned stimulus that predicts heterospecific rough-and-tumble play. Data are expressed as Mean ± SEM. * P<0.05 Fisher's PLSD post hoc test vs. vehicle. Data adapted with permission from [14, 25, 26].
Fig. (4)
Fig. (4)
Rapastinel has antidepressant-like properties without psychotomimetic side effects in rats. (A-D) The antidepressant-like effects of an optimal antidepressant-like dose of rapastinel (3 mg/kg, IV) in the rat Porsolt test measured at 1 hr (A) or 24 hrs (B) post-dosing, the novelty induced hypophagia test 1 hr post-dosing (C), or the learned helplessness test 24 hrs post dosing (D). Rapastinel does not show ketamine-like discriminative stimulus effects (E) or sedative effects (F) in rats trained to discriminate 10 mg/kg ketamine from saline. (G-H) Rapastinel (10 mg/kg, IV) does not show ketamine-like rewarding effects in the conditioned place preference (G), or sensory-motor gating deficits in the pre-pulse inhibition (H) tests in rats. Data are expressed as Mean ± SEM. * P<0.05 Fisher's PLSD post hoc test vs. vehicle. Data adapted with permission from [27].
Fig. (5)
Fig. (5)
Rapastinel produces a rapid-acting and long-lasting antidepressant-like effect in a Chronic Unpredictable Stress (CUS) paradigm. (A) Schematic demonstrating the timeline for CUS exposure, drug administration, and behavioral testing. Numbers in parentheses represent days after drug administration. Rats were exposed to CUS and administered a single dose of Rapastinel (3 mg/kg, IV) or 0.9% sterile saline vehicle (1 ml/kg, IV) on day 21. Mean ± SEM floating time in the (B) Porsolt forced swim test (FS) in CUS treated rats treated with a single dose of Rapastinel (3 mg/kg, IV; n = 10), saline vehicle (n = 10), or No CUS exposed control rats (n = 9) tested 1 hr, 1 day, 1 week, and 2 weeks post-dosing. Mean ± SEM (C) latency to feed in the novelty induced hypophagia (NIH) test (2 days post-dosing), (D) sucrose preference (SP) in the sucrose preference test, 3 days post-dosing, and (E) change in body weight (2 weeks post-dosing compared to CUS day 0). * P < .05 Fisher’s PLSD post hoc test vs. No CUS group or CUS vehicle group. Data adapted with permission from [29].
Fig. (6)
Fig. (6)
Rapastinel facilitates MPFC-dependent positive emotional learning and fear extinction in CUS-treated rats. (A) Mean ± SEM hedonic USVs in response to a conditioned stimuli that predicts heterospecific play 3 hrs post-dosing with Rapastinel (3 mg/kg, IV) or 0.9% sterile saline vehicle (1 ml/kg, IV) in 2-3 month old adult male SD rats exposed to a depressogenic regimen of chronic unpredictable stress (CUS; 2 stressors / day for 21 consecutive days) or non-stressed control rats (No CUS). A similar increase in PEL was seen in animals tested 2 weeks post-dosing. (B-C) Mean ± SEM % time freezing in CUS treated animals dosed with Rapastinel (3 mg/kg IV) or sterile saline vehicle (1 ml/kg IV) 1 hr before extinction day 1. Three shocks (0.5 mA, 1 s) were administered 90, 210, and 330 sec after animals were placed in the conditioning chamber on D0. During extinction, rats were submitted to a 5 min non-reinforced test trial every 24 hrs for 6 consecutive days (D1-6), and on day 14 post-conditioning (consolidation trial). Freezing was quantified via FreezeView software and was measured at baseline (30-60 sec) on D0, and during the last 3 min of each extinction trial. N = 9 - 14 rats per group. * P < .05 Fisher’s PLSD post hoc test vs. No CUS group (A-B), * P < .05 within subjects t-test, 2 tailed (C). Data adapted with permission from [29].
Fig. (7)
Fig. (7)
A single dose of rapastinel produces antidepressant-like (A-C) or cognitive enhancing (D-G) effects in multiple models 1 week post-dosing. Male 2-3 month old Sprague Dawley rats were pretreated with a single dose of rapastinel (3 mg/kg, IV) or sterile saline vehicle (0.9%, 1 ml/kg) and were tested 1 week post-dosing in the following behavioral model of depression: (A) Porsolt forced swim test with floating time (sec) quantified during the 5 min test; (B) open field test with total number of line crosses and time (sec) spent in the center compartment of the open field being measured during the 10 min test; (C) Ultrasonic vocalization (USVs) test with total hedonic and aversive USVs measured during the 3 min test session. Alternatively, animals received behavioral tests of learning and memory, 1 week post dosing (D-E) or were dosed 24 hrs before the first of 5 (F) or 6 (G) daily test sessions: (D) % of alternating trials in the spontaneous alternating closed arm plus maze test; (E) hedonic ultrasonic vocalizations in response to a conditioned stimuli that predicts heterospecific play in the USVs test; (F) Path length to find the hidden platform in the movable platform version of the Morris water maze test; (G) % freezing during contextual fear extinction. N = 8-21 rats per group. Mean ± SEM, * P < .05 ANOVA or Fisher’s PLSD post hoc test Rapastinel vs. vehicle. Data adapted with permission from [30].
Fig. (8)
Fig. (8)
The therapeutic-like effects of Rapastinel are due to activation of NMDA receptor dependent synaptic plasticity. (A) The induction of the antidepressant-like effect of rapastinel is blocked by the NMDAR antagonist CPP. Mean (±SEM) floating time in the Porsolt forced swim test in 2-3 month old male SD rats pretreated with the NMDAR receptor antagonist CPP (10 mg/kg ip; or saline vehicle ip) 1 hr before rapastinel (3 mg/kg, IV) dosing and tested 1 hr post dosing with rapastinel. (B) A single in vivo dose of rapastinel (3 mg/kg iv; filled blue circles) in 2-3 month old male SD rats significantly enhanced the magnitude of long-term potentiation (LTP) of synaptic transmission compared to vehicle treated controls (open black circles), tested in vitro 24 hrs post-dosing at Schaffer collateral-CA1 synapses after 1, 2 and 3 sub-maximal high-frequency stimulus trains (2x100Hz/800ms). (C) Rapastinel (3 mg/kg, IV; 24 hrs post-dosing) increased the density (spines / 10 µM of dendrite) of the most electrophysiologically active spine type (stubby spines) in the dentate gyrus (primary apical, 100-150 µM from the dendrite) or MPFC layer 5 tufts. (D) Representative laser-scanning confocal micrographs of layer 5 MPFC dendrites from rapastinel and vehicle treated animals. Data was adapted with permission from [30].
Fig. (9)
Fig. (9)
Rapastinel is an NMDAR functional glycine site partial agonist. (A) The effects of rapastinel on NMDA currents in oocytes. Cells were injected with e1/z1 cRNA, voltage-clamped at-80 mV in the presence of 100 μM NMDA, varying concentrations of rapastinel, and no exogenous glycine. Data are expressed as a percentage of the current elicited by 10 μM glycine in the same cell, usually in the same trial. Results from nine cells injected with e1/z1 cRNA; error bars indicate SEM. (B) The effects of rapastinel (0.1–100 μM) on LTP induced by a high frequency stimulus train (3 × 100 Hz/500 ms) at Schaffer collateral–CA1 synapses. Each point represents mean ± SEM of normalized field e.p.s.p. slope. (C) Pharmacologically isolated NMDAR currents were recorded from whole cell patch clamp recordings of CA1 pyramidal neurons in slices. Rapastinel by itself (left-most symbols) elicited increased NMDA receptor channel current to 20% the maximum current elicited by the full agonist D-serine. Rapastinel added in increasing concentrations shifted the dose-response of D-serine to the right. Kp = 1.3 µM was calculated using the Stephenson method [31]. Data was adapted with permission from [14, 32].
Fig. (10)
Fig. (10)
Proposed model of rapastinel’s mechanism of action. Rapastinel has a very short (approximately 7 min) half-life in plasma. It binds directly to NMDA receptor subtypes triggering a conformational change that reads out as if rapastinel is a glycine site partial agonist leading directly to increases in intracellular calcium and hypothesized changes in intracellular kinases/phosphatases that alter both the phosphorylation state of the NMDA receptor as well as the phosphoproteome. These processes in turn alter the cell-surface expression of NMDA receptor subtypes, in particular there is an increase in NR2B expression which in turn interacts with AMPA receptors that leads to LTP-like synaptic plasticity processes associated with learning and memory formation. By 24 hrs, significant alterations in mature dendritic spine formation can also be observed.

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