N-methyl-D-aspartate receptor (NMDAR)

When the slow Ca2+ chelator EGTA was in the intracellular solution, Rapastinel/RAP elicited significant enhancement of NMDAR-gated current at a 1 μmol/l concentration, and significantly reduced current at 10 μmol/l. In contrast, when recording with the 40-500-fold kinetically faster, more selective Ca2+ chelator BAPTA, NMDAR current increased in magnitude by 84% as BAPTA washed into the cell, and the enhancement of NMDAR current by 1 μmol/l RAP was completely blocked. Interestingly, the reduction in NMDAR current from 10 μmol/l RAP was not affected by the presence of BAPTA in the recording pipette, indicating that this effect is mediated by a different mechanism. Conclusion: Extracellular binding of RAP to the NMDAR produces a novel, long-range reduction in affinity of the Ca2+ inactivation site on the NMDAR C-terminus accessible to the intracellular space. This action underlies enhancement in NMDAR-gated conductance elicited by RAP. [2]

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One micromole per liter [Rapastinel] enhances N-methyl-D-aspartate receptor conductance when EGTA, but not BAPTA, is the intracellular Ca2+ chelator. [2]
Whole-cell patch-clamp recordings of pharmacologically isolated NMDAR-gated currents were initially measured in CA1 pyramidal neurons where the intracellular recording solution contained 0.5 mmol/l EGTA, a slow Ca2+ chelator included to prevent excess increases in intracellular [Ca2+]. As shown in Fig. 2a–c, bath application of 1 µmol/l RAP for 30 min significantly increased the magnitude of NMDAR-gated currents evoked by Schaffer collateral stimulation {+76 ± 31.9% at −30 mV; two-way ANOVA for repeated measures [F(1,9) = 7.723; P = 0.0214]}. This result confirms our previous findings from both hippocampal CA1 [3] and medial prefrontal cortical [4] neurons in hippocampal slices. As we have shown previously at Schaffer collateral-CA1 synapses in hippocampus [3], this effect reversed upon drug washout.

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Ten micromole per liter [Rapastinel] suppresses N-methyl-D-aspartate receptor conductance when either EGTA or BAPTA is in the intracellular recording solution. [2]
Whole-cell patch-clamp recordings of pharmacologically isolated NMDAR-gated currents were initially measured in CA1 pyramidal neurons where the intracellular recording solution contained 0.5 mmol/l EGTA, a slow Ca2+ chelator included to prevent excess increases in intracellular [Ca2+]. As shown in Fig. 3a–c, bath application of 10 µmol/l RAP for 30 min significantly reduced the magnitude of NMDAR-gated currents evoked by Schaffer collateral stimulation {−23.1 ± 5.8% at −30mV; two-way ANOVA for repeated measures [F(1,12) = 15.19; P = 0.0021]}. This result also confirms our previous findings from both hippocampal CA1 [3] and medial prefrontal cortical [4] neurons in hippocampal slices, where similar concentrations produced reversible suppression of NMDAR-gated conductance when EGTA was used intracellularly..

Currently undergoing a Phase II clinical development program, Rapastinel Trifluacetate is an NMDA receptor modulator with partial agonist characteristics at the glycine site that is being investigated as an adjuvant treatment for severe depressive disorder. Prior to acute ketamine (30 mg/kg), mice given Rapastinel trifluoroacetate (1.0 mg/kg) exhibited a substantial preference (P<0.01) for unfamiliar things over familiar ones. Rapastinel trifluacetate increased the pleasurable 50-kHz USV [F(1,20)=12.4, P<0.05] and decreased the unpleasant 20-kHz USV [F (1),20)=6.8, P<0.05] in the USV test, exhibiting an antidepressant-like effect. In the open field, rapastinel trifluacetate also had anxiolytic effects as indicated by increased center time [F(1,20)=19.2, P<0.05], without changing line crossing [F(1,20) )=0.0, P>0.05] as a measure of locomotor activity.

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Rapastinel (GLYX-13) is an N-methyl-d-aspartate receptor (NMDAR) modulator that has characteristics of a glycine site partial agonist. Rapastinel is a robust cognitive enhancer and facilitates hippocampal long-term potentiation (LTP) of synaptic transmission in slices. In human clinical trials, rapastinel has been shown to produce marked antidepressant properties that last for at least one week following a single dose. The long-lasting antidepressant effect of a single dose of rapastinel (3mg/kg IV) was assessed in rats using the Porsolt, open field and ultrasonic vocalization assays. Cognitive enhancement was examined using the Morris water maze, positive emotional learning, and contextual fear extinction tests. LTP was assessed in hippocampal slices. Dendritic spine morphology was measured in the dentate gyrus and the medial prefrontal cortex. Significant antidepressant-like or cognitive enhancing effects were observed that lasted for at least one week in each model. Rapastinel facilitated LTP 1day-2weeks but not 4weeks post-dosing. Biweekly dosing with rapastinel sustained this effect for at least 8weeks. A single dose of rapastinel increased the proportion of whole-cell NMDAR current contributed by NR2B-containing NMDARs in the hippocampus 1week post-dosing, that returned to baseline by 4weeks post-dosing. The NMDAR antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) blocked the antidepressant-like effect of rapastinel 1week post dosing. A single injection of rapastinel also increased mature spine density in both brain regions 24h post-dosing. These data demonstrate that rapastinel produces its long-lasting antidepressant effects via triggering NMDAR-dependent processes that lead to increased sensitivity to LTP that persist for up to two weeks. These data also suggest that these processes led to the alterations in dendritic spine morphologies associated with the maintenance of long-term changes in synaptic plasticity associated with learning and memory. [1]

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GLYX-13 (rapastinel), a tetrapeptide (Thr-Pro-Pro-Thr-amide), has been reported to have fast acting antidepressant properties in man based upon its N-methyl-D-aspartate receptor (NMDAR) glycine site functional partial agonism. Ketamine, a non-competitive NMDAR antagonist, also reported to have fast acting antidepressant properties, produces cognitive impairment in rodents and man, whereas rapastinel has been reported to have cognitive enhancing properties in rodents, without impairing cognition in man, albeit clinical testing has been limited. The goal of this study was to compare the cognitive impairing effects of rapastinel and ketamine in novel object recognition (NOR), a measure of declarative memory, in male C57BL/6J mice treated with phencyclidine (PCP), another NMDAR noncompetitive antagonist known to severely impair cognition, in both rodents and man. C57BL/6J mice given a single dose or subchronic ketamine (30 mg/kg.i.p.) showed acute or persistent deficits in NOR, respectively. Acute i.v. rapastinel (1.0 mg/kg), did not induce NOR deficit. Pre-treatment with rapastinel significantly prevented acute ketamine-induced NOR deficit. Rapastinel (1.0 mg/kg, but not 0.3 mg/kg, iv) significantly reversed both subchronic ketamine- and subchronic PCP-induced NOR deficits. Rapastinel also potentiated the atypical antipsychotic drug with antidepressant properties, lurasidone, to restore NOR in subchronic ketamine-treated mice. These findings indicate that rapastinel, unlike ketamine, does not induce a declarative memory deficit in mice, and can prevent or reverse the ketamine-induced NOR deficit. Further study is required to determine if these differences translate during clinical use of ketamine and rapastinel as fast acting antidepressant drugs and if rapastinel could have non-ionotropic effects as an add-on therapy with antipsychotic/antidepressant medications. [3]

Whole-cell patch-clamp recordings [2]
Whole-cell patch-clamp recordings of excitatory postsynaptic currents (EPSCs) were obtained from CA1 pyramidal neurons using standard techniques. Infrared illumination coupled to digital image correction optics with a 63× water immersion objective was used to visualize cell bodies of CA1 pyramidal neurons in stratum pyramidale of hippocampal slices, allowing assessment of the health of the cells and recording from identified pyramidal neurons under visual guidance. Patch pipettes were pulled from standard-wall borosilicate glass with a P-97 Flaming/Brown Micropipette Puller, with tip resistances of 4–5 MΩ when filled with internal solution containing (in mmol/l): 125 Cs-methylsulfate, 8 NaCl, 5 Na-phosphocreatine, 5 MgCl2, 2 Na-ATP, 0.3 Na-GTP, either 0.5 EGTA or 5 BAPTA, 40 HEPES, and 5 QX314 (pH = 7.3, adjusted with CsOH, 285 mOsm). Access resistances were measured regularly during recording (typically less than 20 MΩ), especially before and after bath application of Rapastinel/RAP, and any cells that varied by more than 10% were excluded from further analysis. Synaptic EPSCs in CA1 pyramidal neurons were evoked by stimulation with a bipolar tungsten stimulating electrode placed in stratum radiatum (~100–300 µmol/l to the side of the recorded cell and 50–100 µmol/l from stratum pyramidale). Single stimulus (80 µs, 20–50 mA) evoked NMDAR-dependent EPSCs were amplified with a Multipatch 700B, filtered at 3 kHz and digitized at 10 kHz with an analog-to-digital board, and analyzed off-line with Clampfit. NMDAR currents were pharmacologically isolated by adding the following to the extracellular aCSF: 6-nitro-2,3-dioxo-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (10 µmol/l) and bicuculline (20 µmol/l), to block 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid and γ-aminobutyric acid receptors, respectively. For generation of current-voltage (I/V) relations, NMDAR currents were recorded at holding membrane potentials from −90 to 10 mV, at intervals of 10 mV.

Rapastinel was administered in 1 ml/kg 0.9% sterile saline vehicle. The dose of 3 mg/kg IV for rapastinel was chosen because it was the optimal antidepressant dose in Porsolt testing based on a previous dose-response (1–56 mg/kg IV) study (Burgdorf et al., 2013). [1]

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\n\nAnimals were tested 1 week post-dosing with Rapastinel (3 mg/kg IV) or 0.9% sterile saline (1 ml/kg) vehicle (Figure 1A), or received a dose of CPP (10 mg/kg IP) 1 hr before the 1 week test point (Figure 4B). Alternatively, animals received pre-treatment with CPP (10 mg/kg IP) 1 hr before rapastinel administration and were tested 1 hr after rapastinel administration (Results Section). The broad spectrum NMDAR glutamate site antagonist CPP was chosen for these studies because it does not produce an antidepressant response in the Porsolt test (Zhang et al., 2013) unlike the NMDAR channel blockers like ketamine, MK-801 or the NR2B-specific antagonist Ro25-6981 (Maeng et al., 2008, Burgdorf et al., 2013). [1]

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\n\nOpen Field Test: Open field testing was performed as previously described (Burgdorf et al., 2009). Time spent in the open compartment has been shown to be increased by some classes of anxiolytic/antidepressant compounds (Prut and Belzung, 2003). Testing consisted of placing an animal in a 40 cm × 40 cm × 20 cm high opaque plexiglas open field cage divided into 9 equally sized 13.3 cm × 13.3 cm sections under red lighting for 10 min. Between animals, boli and urine were removed from the apparatus. Animals were videotaped, and line crosses and time spent in the center chamber were scored offline by a blind experimenter with high inter-rater reliability (Pearson’s r > .9).\n\nAnimals were tested 1 week post-dosing with Rapastinel (3 mg/kg IV) or 0.9% sterile saline vehicle (1 ml/kg) .\n\n

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\nHigh frequency recordings of ultrasonic vocalizations were captured using a condenser microphone amplified by a bat detector (D980, Pettersson Elektronik, Sweden) and recorded with a Fostex FR2 field recorder (192 kHz sampling rate, 24 bit) onto compact flash cards as .wav files, as described previously (Burgdorf et al., 2008). Ultrasonic vocalizations were scored manually in a blind manner. Hedonic 50-kHz USVs, defined as having a peak frequency of greater than 40-kHz and a bandwidth greater than 18-kHz, were scored along with 20-kHz USVs (peak frequency 20–25 kHz, duration greater than 100 ms) as described in (Burgdorf et al., 2008). High inter- and intra-rater reliability for these measures (Pearson's r>.90) has been established for this method (Burgdorf et al., 2008). Rates of hedonic 50-kHz USVs during the interstimulus interval were reported.\n\nAnimals were tested 1 week post-dosing with Rapastinel (3 mg/kg IV) or 0.9% sterile saline vehicle (1 ml/kg). [1]\n\n

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\nContextual Fear Conditioning Test [1]
\nContextual fear conditioning and extinction testing was conducted as previously described (Akirav et al., 2009), and the first extinction tests occurred 1 hr post-dosing. On the contextual fear training day (D0), animals were placed in a Coulbourn instruments shock chamber (40 × 40 × 40 cm) for 400 seconds and received three 0.5 mA 1 sec footshocks delivered to the floor bars at 90, 210, and 330 second timepoints. During extinction, rats were subjected to daily 5 min non-reinforced (no shock) extinction trials for 6 days after training. Freezing was quantified via FreezeFrame software; at baseline (30–60 sec) on D0, and during the last 3 min of each extinction trial.\n\nAnimals received a single dose of Rapastinel (3 mg/kg IV) or 0.9% sterile saline vehicle (1 ml/kg) 24 hrs before the first extinction session. [1]\n\n

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\nAnimals received a single dose of Rapastinel (3 mg/kg IV) or 0.9% sterile saline vehicle (1 ml/kg) 24 hrs, 1 week, 2 weeks, or 4 weeks before slice recordings. Alternatively, animals received repeated doses of rapastinel once every 2 weeks and tested 24 hrs after the final dose, or tested 4 weeks after the last dose. [1]\n

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\n\nDrugs [3]
\nRapastinel, PCP, and ketamine were dissolved in 0.9% sterile saline (Sal). PCP and ketamine were administered intraperitoneally (ip), at a volume of 10 mL/kg body weight. Rapastinel was given intravenously (iv). The dose of rapastinel (1.0 mg/kg) was chosen because it produced optimal enhancement in learning in both young adult and aged rats [11] and in a trace eye blink conditioning task in rabbits. The doses for ketamine (30 mg/kg) and PCP (10 mg/kg) were chosen based on prior studies, which showed that these doses induce significant cognitive impairment in mice and rats. The dose for lurasidone (0.1 mg/kg) was chosen based on prior NOR studies in C57BL/6J mice which determined the effective dose of lurasidone to restore NOR in subchronic PCP-treated mice.\n\n

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\nDrug treatment [3]
\nFor acute drug treatments, Rapastinel (1.0 mg/kg, iv), lurasidone (0.1 mg/kg, i.p.) or ketamine (30 mg/kg, ip) were administered 30 min prior to the acquisition trial of the NOR task (described below) to the subchronic ketamine or subchronic PCP-treated animals. For subchronic drug treatments, 7–10 mice/cohort were randomly assigned to Sal, PCP, or ketamine. The Sal-treated mice received 0.9% NaCl; the drug treatment groups received either PCP (10 mg/kg; ip), or ketamine (30 mg/kg; ip) twice daily for 7 days. This was followed by a 7 day washout period during which time, mice were left undisturbed in the home cage until initiation of habituation (see below).

[1]. The long-lasting antidepressant effects of rapastinel (GLYX-13) are associated with a metaplasticity process in the medial prefrontal cortex and hippocampus. Neuroscience. 2015 Nov 12;308:202-11.

[2]. Extracellular application of the N-methyl-D-aspartate receptor allosteric modulator rapastinel acts remotely to regulate Ca2+ inactivation at an intracellular locus. Neuroreport. 2022 May 4;33(7):312-319.

[3]. GLYX-13 (rapastinel) ameliorates subchronic phencyclidine- and ketamine-induced declarative memory deficits in mice. Behav Brain Res. 2016 Feb 15;299:105-10.

Rapastinel has been used in clinical trials to study the treatment of depression, major depressive disorder, and obsessive-compulsive disorder (OCD). This study aims to further elucidate the mechanism of the durable antidepressant effect observed in humans after a single dose of Rapastinel. Significant efficacy with at least one week was found in seven different testing paradigms. These paradigms included depression/anxiety-related tests such as forced swimming, open field tests, and ultrasonic vocalization tests, in which Rapastinel demonstrated significant antidepressant-like and/or anti-anxiety-like effects; and learning-related tests such as the alternating maze, positive emotion learning, the Morris water maze, and situational fear extinction tests. Interestingly, the study found that the optimal dose of Rapastinel (3 mg/kg intravenously) producing the best antidepressant/anxiety-like effect also produced the best cognitive enhancement. This suggests a mechanistic link between lapastin's durable antidepressant-like effects and its cognitive-enhancing effects, supporting the idea that lapastin's antidepressant/anxiety-like effects operate through mechanisms shared with those related to learning and memory. Since lapastin is an NMDA receptor modulator, it is reasonable to hypothesize that its durable antidepressant/anxiety-like effects involve an NMDAR-mediated process similar to LTP; notably, we have previously reported that lapastin does indeed enhance the amplitude of long-term potentiation (LTP) in rat hippocampal slices (Zhang et al., 2008), and that the NMDAR-specific glutamate site antagonist CPP also blocks the antidepressant-like effects of lapastin. Furthermore, it should be noted that the increased proportion of mature dendritic spines and the density of short dendritic spines in the dentate gyrus and medial prefrontal cortex layer V, as well as the increased NMDA receptor expression observed at 24 hours, have all been confirmed to be causally related to LTP formation (Bosch and Hayashi, 2012; Burgdorf et al., 2013; Bosch et al., 2014). Mushroom-shaped and short, thick spines are functionally classified as large mature spines, expressing NMDAR-dependent Ca2+ conductance and activity-dependent structural stability, with the short, thick spines generating the largest net inflow of NMDAR-dependent Ca2+ (Noguchi et al., 2005; Hasegawa et al., 2015). Therefore, summarizing these data within the framework of metaplasticity (Abraham and Bear, 1996) is the most compelling interpretation. This is the only model that hypothesizes the ability of established neural circuits to induce synaptic plasticity is influenced by prior stimuli and is typically measured by changes in the LTP or LTD induction threshold (Abraham, 2008). Metaplasticity is now a recognized phenomenon, confirmed in multiple brain regions and induced by a variety of stimuli (Wexler and Stanton, 1993; Stanton, 1995; Abraham, 2008; Hulme et al., 2013). Of particular note is the discovery by Richter-Levin and Maroun (Richter-Levin and Maroun, 2010) of an NMDA receptor-dependent, emotion-regulated form of metaplasticity in the medial prefrontal cortex. This aligns well with the findings reported in this paper, where Rapastinel treatment has demonstrated durable behavioral effects in many models related to synaptic plasticity. Physiological studies have also shown that a single dose of Rapastinel significantly enhances the magnitude of submaximal stimulation-induced long-term potentiation (LTP), with the effect lasting at least two weeks and at least eight weeks with repeated administration. Rapastinel has been shown to promote long-term potentiation (LTP) in the medial prefrontal cortex (MPFC) (Burgdorf et al., 2015). In addition to selectively increasing the contribution of the NR2B subunit NMDAR to total NMDA currents over a long period, Rapastinel also increases the cell surface expression of NR2B NMDAR and GLUR1 AMPAR, both of which are essential for the long-term behavioral effects of Rapastinel (Burgdorf et al., 2013). Conversely, a study by Hall et al. (2007) found that enhanced NR2B NMDAR activation inhibited LTP in cultured embryonic cortical neurons. If their observations also apply to intact adult synapses, this suggests that GLYX-13 increases NR2B and AMPAR expression through a different mechanism, which could explain why repeated dosing is required to maximize the magnitude of LTP enhancement. It is now well understood that metaplasticity regulation at both the receptor level (e.g., by altering channel dynamics, receptor number and type, and transport) and the biochemical level (late and therefore persistent transcription- and translation-dependent metaplasticity) can significantly influence synaptic activity associated with learning and memory. Therefore, a plausible hypothesis for the long-term antidepressant properties of Rapastinel in humans is that it induces a promoting effect on NMDA receptor-triggered, AMPA receptor-dependent metaplasticity processes associated with LTP-like mechanisms (Moskal et al., 2005; Zhang et al., 2008; Burgdorf et al., 2013; Moskal et al., 2014). This differs from the antidepressant effects induced by NMDA receptor antagonists and suggests a novel aspect of the mechanistic basis of glutamatergic modulators' antidepressant action. [1]

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Background: A novel N-methyl-D-aspartate receptor (NMDAR) allosteric modulator, Rapastinel (RAP, formerly known as GLYX-13), induces a durable antidepressant-like effect by enhancing long-term potentiation (LTP) of synaptic transmission. These effects are generated by RAP binding to a unique site in the extracellular region of the NMDAR complex, transiently enhancing NMDAR-gated currents in pyramidal neurons of the hippocampus and medial prefrontal cortex.
Methods: We used whole-cell patch-clamp recordings to compare the potency of RAP in modulating Schaffer collateral-induced NMDAR currents with the kinetics of Ca2+ chelators in intracellular solution. Intracellular solutions contain either the slow-acting Ca2+ chelator EGTA [3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecane-1,14-diacid, 0.5 mmol/L] or the more kinetically fast and selective Ca2+ chelator BAPTA {2,2',2″,2‴-[ethane-1,2-diylbis(oxo-2,1-phenylenenitrile)]tetraacetic acid, 5 mmol/L}. NMDAR can be pharmacologically isolated by adding the 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid receptor antagonist 6-nitro-2,3-dioxo-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (10 μmol/L) and the GABA receptor blocker bicoline (20 μmol/L) to the perfusion. Gated Current.
Results: In the presence of the slow Ca2+ chelator EGTA in the intracellular fluid, Rapastinel/RAP significantly enhanced the NMDAR gated current at a concentration of 1 μmol/L, while significantly reducing the current at a concentration of 10 μmol/L. Conversely, when recording was performed using BAPTA, a Ca2+ chelator with kinetics 40-500 times faster and higher selectivity, the NMDAR current amplitude increased by 84% as BAPTA was washed into the cells, while the enhancing effect of 1 μmol/L RAP on the NMDAR current was completely blocked. Interestingly, the reduction in NMDAR current induced by 10 μmol/L RAP was unaffected by the presence of BAPTA in the recording electrode, indicating that this effect is mediated by a different mechanism.
Conclusion: Extracellular binding of Rapastinel/RAP to NMDAR leads to NMDAR gated current. Novel long-range reduction of affinity for C-terminal Ca2+ inactivation sites that are in contact with the intracellular space. This effect underlies RAP-induced enhancement of NMDAR-gated conductance. [2] In this study, we compared the ability of the NMDAR-regulating compound Rapastinel/RAP (formerly GLYX-13) to modulate NMDAR-gated whole-cell currents in hippocampal CA1 pyramidal neurons filled with slow Ca2+ chelators EGTA and 40-500 times faster chelators BAPTA. We found that low concentrations of RAP blocked the enhancement of NMDAR currents by BAP, but did not prevent the inhibition of NMDAR currents by 10 times higher concentrations of RAP. In summary, these data support the existence of high-affinity and low-affinity extracellular effects of RAP on NMDAR, mediating enhancement and inhibition of channel conductance, respectively. Furthermore, the effect of intracellular BAPTA infusion on the activity of extracellularly applied drugs suggests that RAP downregulates intracellular Ca2+ near the channel pore over a long range. The affinity of the binding site is used to enhance the NMDAR gating current. [2]

C20H32F3N5O8

527.491995811462

527.22

1435786-04-1

Rapastinel;117928-94-6

71311638

White to off-white solid powder

6

12

7

36

742

6

C(F)(F)(F)C(=O)O.C(N1CCC[C@H]1C(=O)N[C@H](C(=O)N)[C@H](O)C)([C@@H]1CCCN1C(=O)[C@@H](N)[C@H](O)C)=O

BCWVNVCGYWOAAK-GDLIIDCZSA-N

InChI=1S/C18H31N5O6.C2HF3O2/c1-9(24)13(19)18(29)23-8-4-6-12(23)17(28)22-7-3-5-11(22)16(27)21-14(10(2)25)15(20)26;3-2(4,5)1(6)7/h9-14,24-25H,3-8,19H2,1-2H3,(H2,20,26)(H,21,27);(H,6,7)/t9-,10-,11+,12+,13+,14+;/m1./s1

(2S)-1-[(2S)-1-[(2S,3R)-2-amino-3-hydroxybutanoyl]pyrrolidine-2-carbonyl]-N-[(2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl]pyrrolidine-2-carboxamide;2,2,2-trifluoroacetic acid

Rapastinel Trifluoroacetate; 1435786-04-1; GLYX-13 trifluoroacetate; Rapastinel (Trifluoroacetate); (2S)-1-[(2S)-1-[(2S,3R)-2-amino-3-hydroxybutanoyl]pyrrolidine-2-carbonyl]-N-[(2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl]pyrrolidine-2-carboxamide;2,2,2-trifluoroacetic acid;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~236.97 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.94 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.94 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.94 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)