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.

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]

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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Animals 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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Open 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). Animals were tested 1 week post-dosing with Rapastinel (3 mg/kg IV) or 0.9% sterile saline vehicle (1 ml/kg) .

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High 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. Animals were tested 1 week post-dosing with Rapastinel (3 mg/kg IV) or 0.9% sterile saline vehicle (1 ml/kg). [1]

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Contextual Fear Conditioning Test Contextual 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. Animals 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]

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Animals 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]

[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.

Rapastinel has been used in trials studying the treatment of Depressive Disorder, Major and Obsessive-Compulsive Disorder (OCD).

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The studies presented here were undertaken to further elucidate the mechanisms responsible for the long lasting antidepressant effects seen in humans after a single dose of Rapastinel. In each of the seven different paradigms tested, rapastinel was found to have a significant effect that lasted for at least one week. These included tests associated with depression/anxiety including the forced swim, open field, and ultrasonic vocalization tests in which rapastinel had a robust antidepressant-like and/or anxiolytic-like effect as well as in tests associated with learning, including alternating + maze, positive emotional learning, Morris water maze and contextual fear extinction. Interestingly, the dose of rapastinel found to give optimal antidepressant- /anxiolytic-like effects (3 mg/kg IV) was also found to produce optimal cognitive enhancement. This suggests a mechanistic link between the long-lasting antidepressant-like effects of rapastinel and its cognitive enhancing effects and lends support to the idea that the antidepressant- /anxiolytic-like effects of rapastinel operate through a mechanism shared with the mechanisms associated with learning and memory.

Since Rapastinel is an NMDA receptor modulator, it is also reasonable to assume that its long-lasting antidepressant- /anxiolytic-like effects involve an NMDAR mediated process akin to LTP; particularly since we have previously reported that rapastinel does in fact enhance the magnitude of LTP in rat hippocampal slice preparations (Zhang et al., 2008), and that the NMDAR-specific, glutamate site antagonist CPP also prevented the antidepressant-like effects of rapastinel. It should also be noted that the increase in the fraction of mature dendritic spines and stubby spine densities in the dentate gyrus and in layer V of the medial prefrontal cortex as well as the increase in NMDA receptor expression seen at 24 hrs have been shown to be causally linked to LTP formation (Bosch and Hayashi, 2012, Burgdorf et al., 2013, Bosch et al., 2014). Mushroom spines and stubby spines are both functionally classed as large, mature spines that express NMDAR-dependent Ca2+ conductances and activity-dependent structural stability, with stubby spines producing the greatest net influx of NMDAR dependent Ca2+ (Noguchi et al., 2005, Hasegawa et al., 2015).

Thus, the most appealing way to couch these data as a whole is in the framework of metaplasticity (Abraham and Bear, 1996). This the only model that posits that modulation of the ability to induce synaptic plasticity in an already established neural circuit can be affected by prior stimulation and is typically measured by changes in the threshold for induction of LTP or LTD (Abraham, 2008). Metaplasticity is now a well-established phenomenon having been shown in a variety of brain regions and induced by a variety of stimuli (Wexler and Stanton, 1993, Stanton, 1995, Abraham, 2008, Hulme et al., 2013). Of particular interest here, Richter-Levin and Maroun (Richter-Levin and Maroun, 2010) have characterized an NMDA receptor-dependent, emotionally modulated form of metaplasticity in the medial prefrontal cortex. This resonates very well with the studies reported here in that Rapastinel treatment showed a long lasting behavioral effect in many models associated with synaptic plasticity as well as in physiology studies showing marked increases in the magnitude of LTP induced by a sub-maximal stimulus that lasted at least two weeks with a single dose of rapastinel and at least 8 weeks with repeat dosing. Rapastinel has also been shown to produce a long-lasting facilitation of LTP in the MPFC (Burgdorf et al., 2015). In addition to producing a long-lasting selective increase in the contribution of NR2B-containing NMDAR to total NMDA current, rapastinel also produced a long-lasting increase in both NR2B NMDAR and GLUR1 AMPAR cell surface expression, with both receptors necessary for long-lasting behavioral effects of rapastinel (Burgdorf et al., 2013). In contrast, a study by Hall et al. (2007) that examined synapse development in embryonic cortical neurons in culture, found that enhanced NR2B NMDAR activation can suppress LTP. If their observations extend to intact adult synapses, this would suggest that GLYX-13 increases NR2B and AMPAR expression via different mechanisms, which could explain why repeat dosing is necessary to maximally enhance magnitude of LTP.

It is now clear that the regulation of metaplasticity at the receptor level (e.g., by changes in channel kinetics, receptor expression in both number and type, and trafficking) and biochemically (late stage and thus persistent transcription and translation-dependent metaplasticity) can have significant effects on synaptic activity associated with learning and memory. Thus, a plausible hypothesis for how Rapastinel displays long-term antidepressant properties in humans is that it induces an NMDA receptor-triggered, AMPA receptor-dependent, facilitation of metaplasticity processes associated with an LTP-like mechanism (Moskal et al., 2005, Zhang et al., 2008, Burgdorf et al., 2013, Moskal et al., 2014). This is different from the antidepressant effects induced by NMDA receptor antagonists and suggests a novel aspect of the mechanistic underpinnings of the antidepressant effects of glutamatergic modulators.[1]

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Background: A novel N-methyl-D-aspartate receptor (NMDAR) allosteric modulator, Rapastinel (RAP, formerly GLYX-13), elicits long-lasting antidepressant-like effects by enhancing long-term potentiation (LTP) of synaptic transmission. RAP elicits these effects by binding to a unique site in the extracellular region of the NMDAR complex, transiently enhancing NMDAR-gated current in pyramidal neurons of both hippocampus and medial prefrontal cortex.

Methods: We compared efficacy of RAP in modulating Schaffer collateral-evoked NMDAR-currents as a function of kinetics of the Ca2+ chelator in the intracellular solution, using whole-cell patch-clamp recordings. The intracellular solution contained either the slow Ca2+ chelator EGTA [3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecane-1,14-dioic acid, 0.5 mmol/l] or the 40-500-fold kinetically faster, more selective Ca2+ chelator BAPTA {2,2',2″,2‴-[ethane-1,2-diylbis(oxy-2,1-phenylenenitrilo)] tetraacetic acid, 5 mmol/l}. NMDAR-gated currents were pharmacologically isolated by bath application of the 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptor antagonist 6-nitro-2,3-dioxo-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (10 μmol/l) plus the GABA receptor blocker bicuculline (20 μmol/l).

Results: 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 Rapastinel/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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In this study, we compared the ability of the NMDAR modulatory compound Rapastinel/RAP (formerly GLYX-13) to modulate NMDAR-gated whole-cell currents in hippocampal CA1 pyramidal neurons filled with the slow Ca2+ chelator EGTA versus the 40–500-fold faster chelater BAPTA. We found that BAPTA occluded the enhancement of NMDAR currents by a low concentration of RAP but did not prevent the suppression of NMDAR current produced by a 10-fold higher concentration of the drug. Taken together, these data support the existence of high- and low-affinity extracellular actions of RAP on the NMDAR, which mediate the enhancement and suppression of channel conductance, respectively. Moreover, the effect of intracellular infusion of BAPTA on the activity of an extracellularly applied drug leads to the conclusion that RAP produces its enhancement of NMDAR-gated current via a long-range downregulation of the affinity of an intracellular binding site for Ca2+ near the channel pore.[2]

413.4686

413.227

C, 52.29; H, 7.56; N, 16.94; O, 23.22

117928-94-6

Rapastinel Trifluoroacetate;1435786-04-1

14539800

White to off-white solid powder

-2.8

5

7

7

29

659

6

C[C@H]([C@@H](C(=O)N1CCC[C@H]1C(=O)N2CCC[C@H]2C(=O)N[C@@H]([C@@H](C)O)C(=O)N)N)O

GIBQQARAXHVEGD-BSOLPCOYSA-N

InChI=1S/C18H31N5O6/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/h9-14,24-25H,3-8,19H2,1-2H3,(H2,20,26)(H,21,27)/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

Rapastinel; GLYX 13; GLYX-13; GLYX13; Rapastinelum; GLYX-13 peptide; UNII-6A1X56B95E; L-Threoninamide, L-threonyl-L-prolyl-L-prolyl-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ≥ 32 mg/mL (~77.39 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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