NMDA receptor

Dopamine metabolism, as reflected by the concentration of dihydroxyphenylacetic acid (DOPAC), in the medial prefrontal cortex was significantly increased following 30 min immobilisation stress or systemic administration of the benzodiazepine/GABA(A) receptor inverse agonist methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The response to stress was attenuated by pretreatment of rats with the benzodiazepine/GABA(A) receptor agonists diazepam and zolpidem. Furthermore, pretreatment with R-(+)-3-amino-1-hydroxypyrrolid-2-one (R-(+)-HA-966), a low efficacy partial agonist, and 7-chloro-4-hydroxy-3(3-phenoxy) phenylquinolin-2-(H)-one (L-701,324) a novel, high affinity, full antagonist at the glycine/NMDA receptor attenuated the response to both stress and DMCM. These results demonstrate that antagonists at the glycine/NMDA receptor complex are comparable with benzodiazepine/GABA(A) receptor agonists in their ability to prevent activation of the mesocortical dopamine system by stress and GABA(A) receptor inverse agonists. Results are discussed in relation to the interaction between glycine/NMDA receptor antagonists, the mesocorticolimbic dopamine system and stress related disorders[3].

In the forced swim test (FST) and tail suspension test (TST), L-701,324 (5–10 mg/kg; i.p.; once) shows antidepressant-like potential without influencing mice's locomotor activity [1]. In the chronic unpredictable mild stress (CUMS) depression model, L-701324 (5-10 mg/kg; i.p.; daily for 2 weeks) shows strong antidepressant-like effects, inhibits CUMS-induced europagenesis, and decreases BDNF signaling cascades in the hippocampus [1]. L-701324 (2.5–5 mg/kg; oral; once) decreases unconditioned and unconditioned anxiety-like behaviors while inhibiting NMDA receptor activity by blocking the NMDA/glycine-sensitive region on the NMDA receptor. circumstances including conditional conflict behavior[2].

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Antidepressants currently used in clinical practice have limitations such as low efficacy, slow onset and various adverse reactions. It has become necessary to develop novel antidepressants beyond monoaminergic drugs. L-701,324 is a potent NMDA receptor antagonist, and the purpose of this study was to investigate the possible antidepressant effects of L-701,324 in mice. Here, various methods including the forced swim test (FST), tail suspension test (TST), chronic unpredictable mild stress (CUMS) model of depression, western blotting and immunofluorescence, were used together. A single injection of L-701,324 exhibited antidepressant-like potential in the FST and TST without affecting the locomotor activity of mice. Repeated injection of L-701,324 not only prevented CUMS-induced depressive-like behaviors in mice, but also ameliorated the downregulating effects of CUMS on the hippocampal BDNF signaling cascade and neurogenesis. Furthermore, K252a, a potent inhibitor of the BDNF system, fully blocked the antidepressant-like activity of L-701,324 in mice. K252a administration also abolished the activating actions of L-701,324 on the hippocampal BDNF signaling cascade and neurogenesis in CUMS-treated mice. Collectively, these data indicated that L-701,324 possesses antidepressant-like activity in mice, which was mediated, at least in part, by promoting the hippocampal BDNF system. [1]

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The effects of the NMDA/glycine site antagonist, 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(1H)-quinolone (L-701,324), and the benzodiazepine receptor agonist, diazepam, were examined in the elevated plus-maze and in the Vogel's conflict test. Oral administration of L-701,324 caused a dose-dependent increase (2.5 and 5.0 mg/kg, -30 min) in the percent time spent in the open arms with no change in the total number of arm entries or in the percent entries into the open arms of the plus-maze. The same doses of L-701,324 increased punished responding in the Vogel's conflict test in a dose-dependent fashion, with no influence on unpunished drinking behavior. The anxiolytic-like effects of L-701,324 were obtained at doses which by themselves had no influence on the locomotor activity of the animals. Diazepam (2 mg/kg, i.p., -30 min) was slightly more effective than L-701,324 in the plus-maze situation, whereas the increase in punished drinking in the Vogel's test was of the same magnitude for both compounds. Our present results suggest that inhibition of NMDA receptor activity via a blockade of the NMDA/glycine-sensitive site at the NMDA receptor is accompanied by a reduction of anxiety-like behavior in both non-conditioned and conditioned conflict behavior situations. [2]

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Effects of the glycine/NMDA receptor antagonists R-(+)-HA-966 and L-701,324 on the stress induced increase of mesocortical DOPAC concentration [3]
Pre-treatment of rats with either R-(+)-HA-966 (20 mg/kg, i.p.) or L-701,324 (5 but not 1 mg/kg, i.p.) significantly attenuated the increase of DOPAC concentration in medial prefrontal cortex following 30 min immobilisation stress without affecting DOPAC concentration per se (Fig. 3a–c). In contrast, pre-treatment with L-701,357 (10 mg/kg, i.p.), the inactive enantiomer of L-701,324, did not significantly affect either basal or stress induced increase of DOPAC concentration in the medial prefrontal cortex (Fig. 3d). The concentration of dopamine in the medial prefrontal cortex was unaffected by stress or drug treatment (data not shown).

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Effects of the glycine/NMDA receptor antagonist L-701,324 on the increase of mesocortical DOPAC concentration induced by the benzodiazepine/GABAA receptor inverse agonist DMCM [3]
Pre-treatment of rats with L-701,324 (5 mg/kg, i.p.) significantly attenuated the increase of DOPAC concentration in medial prefrontal cortex following administration of the GABAA receptor inverse agonist DMCM (5 mg/kg, i.p.). In contrast, pre-treatment with L-701,357 (10 mg/kg, i.p.), the inactive regioisomer of L-701,324, did not affect the increase of medial prefrontal cortex DOPAC concentration by DMCM (Fig. 4). The concentration of dopamine in medial prefrontal cortex was unaffected by DMCM or L-701,324 (data not shown).

Animal/Disease Models: Male C57BL/6 J mice (7 weeks old) in chronic unpredictable mild stress (CUMS) [1]
Doses: 5 and 10 mg/kg
Route of Administration: intraperitoneal (ip) injection; one time/day, continuous 2-week
Experimental Results: diminished immobility in C57BL/6 J mice. The expression of BDNF, pTrkB and pCREB was increased in the hippocampus.

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Animal/Disease Models: Male C57BL/6 J mice, forced swimming test (FST) and tail suspension test (TST) (7 weeks old) [1]
Doses: 5 and 10 mg/kg
Route of Administration: intraperitoneal (ip) injection;
Experimental Results: diminished immobility of C57BL/6 J mice in FST and TST.

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Animal/Disease Models: Male SD (SD (Sprague-Dawley)) rat (280-300 g) [2]
Doses: 2.5 and 5 mg/kg
Route of Administration: Oral;
Route of Administration: Oral.
Experimental Results: Dose-dependent increase in percentage of time spent with arms open. Increased punishment responses in a dose-dependent manner in the Vogel conflict test.

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L-701,324 was dissolved in normal saline containing 1% DMSO (vehicle) and intraperitoneally (i.p) injected (10 mL/kg). The dosages of L-701,324 (5 and 10 mg/kg), fluoxetine (20 mg/kg) and K252a (25 μg/kg) were determined according to previous reports and our pilot study [1].

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Forced swim test (FST) [1]
Naïve C57BL/6 J mice were given a single i.p. injection of L-701,324, fluoxetine or vehicle 30 min before this test. Mice were individually placed in a transparent glass tank (containing 15 cm high pure water, 25 ± 1 °C) for 6 min. A stopwatch was used to record the duration of immobility for each mouse during the last 4 min. The water was replaced after each trial. Immobility was defined as the mouse was floating in the water without struggling or having only slight movements to keep its nose above the water. This test was recorded with the observer unaware of the experimental grouping.

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Tail suspension test (TST) [1]
Naïve C57BL/6 J mice were given a single i.p. injection of L-701,324, fluoxetine or vehicle 30 min before this test. The tail tip of each mouse was individually glued to a rail 60 cm above the floor, and hung for 6 min. The immobility (completely motionless) duration of each mouse during the 6-min period was recorded. This test was recorded with the observer unaware of the experimental grouping.

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Open field test (OFT) [1]
This test was carried out in a darkroom. Naïve C57BL/6 J mice were given a single i.p. injection of L-701,324, fluoxetine or vehicle 30 min before this test. Mice were individually placed in the floor of an open field apparatus (100 × 100 × 45 cm; 25 squares, 20 × 20 cm for each square) and allowed to explore freely for 5 min. The apparatus was illuminated with a red bulb (50 W) on the ceiling. The number of squares each mouse crossed during the 5-min period was recorded. This test was recorded with the observer unaware of the experimental grouping. After each trail, the floor was cleaned.

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Chronic unpredictable mild stress (CUMS) [1]
Briefly, 8 stressors were adopted in this study: damp bedding (24 h), cage tilting (12 h), restraint (1 h), shaking (30 min), 4 °C exposure (1 h), day/night inversion, food deprivation (23 h) or water deprivation (23 h). All these stressors were randomly given for 6 weeks, and administration of L-701,324/fluoxetine/vehicle was performed daily during the last 2 weeks. The control mice were left undisturbed except general handing (e.g. regular cage cleaning) and drug treatment. After CUMS, FST, TST and sucrose preference test were performed together to assay the depressive-like behaviors of animals.

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Elevated plus-maze experiments [2]
The plus-shaped maze was made of wood and positioned on a height of 50 cm above the ßoor in a quiet laboratory surrounding. Two opposite arms were open (50 ´ 10 cm) and the other two were enclosed with walls (50 ´ 10 ´ 40 cm). Experiments were carried out in a darkened and quiet room with a constant light of 15 W, located 80 cm above the maze and directed towards the apparatus. The light levels on the open and enclosed arms were equal. Three days before the experiment, each rat was handled every day for 5 min. Animals were brought in their home cage into a separate silent room for 60 min before the experiment. Before the start of the pluz-maze behavior recordings, each animal was placed into a novel environment, represented by a conventional Skinner box, for 5 min. The plus-maze experiment was initiated by placing the rat into the center of the plus-maze facing an open arm, after which the number of entries and time spent in each of the two arms were recorded for a period of 5 min by an independent observer with no knowledge of the drug treatment protocol. An “arm entry” was recorded when the rat entered the arm with all four paws into the arm. The maze was carefully cleaned with tap water after each test session and with a weak alcohol washing solution after Þnishing all the experimental sessions of the day. The open-arm activity was quantiÞed as a) time spent in the open arms, as well as b) number of entries into the open arms. The glycine receptor antagonist, L-701,324, was given per os (PO) 30 min prior to the test.

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Vogel’s conßict test [2]
The drinking training sessions and the conßict-suppressed drinking experiments (for protocol, see Möller et al. 1997) were conducted in two standard boxes for operant behavior but specially designed for shock-induced suppression of drinking in rats. The whole experimental period consisted of 3 days. In the morning of the Þrst day, the drinking water was removed from the home cage of the animals. During the second and third day, the subjects were placed in the test apparatus and allowed a 12-min period of free drinking with no electric shocks delivered. After this 2-day training period, most animals showed a stable baseline of number of licks recorded during the training session. A few animals refused to drink and were removed from the experiments. On the day of the experiments, the animals were randomly divided into a control group (receiving solvent), and an experimental group receiving the drug of interest. Thirty minutes after drug administration, the animals were placed into the apparatus and the experimental session was initiated as soon as the animals had completed 20 licks on the water bottle. After this, continued drinking triggered the delivery of electric shocks in cycles of 5 s, with 4-s intervals with a shock current set at 0.2 mA. The number of punished and unpunished “drinking episodes” was recorded during a 12-min period, and the number of punished drinking episodes was taken as a measure of suppressed drinking behavior. All recordings were carried out between 12 a.m. and 6 p.m. in order to avoid large diurnal variations in the results. The benzodiazepine receptors agonist, diazepam, was given in a dose of 2.0mg/kg intraperitoneally (IP), which previously has been found to produce a reliable anti-conßict actions in rats. The glycine receptor antagonist, L-701,324 was given PO in doses of 2.5 and 5.0mg/kg, 30min prior to the start of the behavioral recordings.

L-701,324 was given PO as a suspension prepared using a 0.5% solution of methyl cellulose.

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L-701,324 (1 and 5 mg/kg, i.p.), L-701,357 (10 mg/kg, i.p.) or vehicle (0.5% carboxy methylcellulose in 0.9% saline, 1 ml/kg, i.p.). Thirty minutes later rats were either injected with DMCM (5 mg/kg, i.p.) or vehicle (1 ml/kg, i.p.) and killed 30 min later or in the stress studies either left in the home cage or immobilised for 30 min and immediately killed. Brains were removed, the medial prefrontal cortex dissected, frozen on solid CO2, and stored at −70°C. All brain samples were analysed for dopamine and the acidic metabolite dihydroxyphenylacetic acid (DOPAC) by high pressure liquid chromatography (HPLC) with electrochemical detection (Hutson et al., 1991). Briefly, tissue samples were homogenised in 10 vols. of 0.4 M perchloric acid containing 0.1% cysteine, 0.01% sodium metabisulphite and 0.01% sodium ethylene diaminetetraacetic acid (NaEDTA) and centrifuged at 3000×g/10 min. The HPLC system comprised an HPLC Technology Techsphere 3μ ODS column (4.6 mm×7.5 cm). The mobile phase consisted of 0.07 M KH2PO4, 0.0035% NaEDTA, 0.023% octyl sodium sulphate and 12.5% methanol, pH 2.75 at a flow rate of 1 ml/min. Dopamine and metabolites were detected using an Antec electrochemical detector (Presearch) with the working electrode set at +0.65 V relative to a silver/silver chloride reference electrode. [3]

[1]. Antidepressant-like activity of L-701324 in mice: A behavioral and neurobiological characterization. Behav Brain Res. 2021 Feb 5;399:113038.

[2]. A characterization of anxiolytic-like actions induced by the novel NMDA/glycine site antagonist, L-701,324. Psychopharmacology (Berl). 1998 Jan;135(2):175-81.

[3]. L-701,324, a glycine/NMDA receptor antagonist, blocks the increase of cortical dopamine metabolism by stress and DMCM. Eur J Pharmacol. 1997 May 20;326(2-3):127-32.

7-chloro-4-hydroxy-3-(3-phenoxyphenyl)-1H-quinolin-2-one is a member of quinolines.

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Dopamine metabolism, as reflected by the concentration of dihydroxyphenylacetic acid (DOPAC), in the medial prefrontal cortex was significantly increased following 30 min immobilisation stress or systemic administration of the benzodiazepine/GABA(A) receptor inverse agonist methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The response to stress was attenuated by pretreatment of rats with the benzodiazepine/GABA(A) receptor agonists diazepam and zolpidem. Furthermore, pretreatment with R-(+)-3-amino-1-hydroxypyrrolid-2-one (R-(+)-HA-966), a low efficacy partial agonist, and 7-chloro-4-hydroxy-3(3-phenoxy) phenylquinolin-2-(H)-one (L-701,324) a novel, high affinity, full antagonist at the glycine/NMDA receptor attenuated the response to both stress and DMCM. These results demonstrate that antagonists at the glycine/NMDA receptor complex are comparable with benzodiazepine/GABA(A) receptor agonists in their ability to prevent activation of the mesocortical dopamine system by stress and GABA(A) receptor inverse agonists. Results are discussed in relation to the interaction between glycine/NMDA receptor antagonists, the mesocorticolimbic dopamine system and stress related disorders.[3]

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The finding that L-701,324 treatment promoted hippocampal neurogenesis is exciting and indicated that L-701,324 may be a pro-neurogenic compound. Given the well-established correlation between BDNF and neurogenesis, L-701,324 is very likely to regulate neurogenesis by enhancing the expression of hippocampal BDNF. It is also possible that L-701,324 can modulate some well-known pro-neurogenic factors such as sex determining region Y box 2 and paired box protein 6, which needs further investigation. The neurobiology of depression is rather complex. In addition to BDNF dysfunction, monoamine hypofunction and HPA hyperfunction, recent studies have increasingly reported other targets implicated in depression such as peroxisome proliferation-activated receptor alpha, vascular endothelial growth factor, salt-inducible kinase 2 and ΔFosB. Although the experimental results involving K252a suggested that BDNF was involved in the antidepressant-like actions of L-701,324, we could not exclude protein targets other than BDNF. In summary, L-701,324 has antidepressant-like efficacy in mice, which was mediated, at least in part, by enhancing the hippocampal BDNF signaling cascade. [1]

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The present findings that L-701,324, a full antagonist at the glycine/NMDA receptor, attenuated both stress and benzodiazepine/GABAA receptor inverse agonist induced changes of cortical dopamine metabolism are consistent with evidence implicating glutamate and, in particular, the NMDA receptor complex in the response to stress and anxiety. Thus, behavioural studies have demonstrated that antagonists at the glutamate, glycine and ion channel sites of the NMDA receptor complex demonstrate anxiolytic activity in both conditioned and non-conditioned tests in rodents (Trullas et al., 1989; Corbett and Dunn, 1991; Dunn et al., 1992; Kehne et al., 1991; Faiman et al., 1994). Results in the present study also support the previous finding that antagonists at the strychnine insensitive glycine site of the NMDA receptor interact with mesocorticolimbic dopamine neurones but only when those neurones are activated. Thus, both R-(+)-HA-966 and L-701,324 have been shown not only to lack the ability of the non-competitive ion channel blockers phencyclidine and MK801 to increase mesocorticolimbic dopamine metabolism and induce hyperlocomotion but in addition to markedly attenuate these effects of PCP and MK801 (Bristow et al., 1993, Bristow et al., 1996a; Hutson et al., 1991, Hutson et al., 1995). Based on these and other studies it has been suggested that glycine/NMDA receptor antagonists display an atypical neuroleptic-like profile in rodents. Given the importance of the frontal cortex in the pathophysiology of schizophrenia and that stress may be a mitigating factor in the relapse of schizophrenic patients (Dohrenwend and Egri, 1981) and the exacerbation of schizophrenic symptoms (Bebbington et al., 1993), the present findings emphasize the previous suggestion that glutamate systems may be important in schizophrenia and that this class of compound may be a novel approach for the treatment of schizophrenia. Whether such compounds would be clinically effective in the treatment of schizophrenia or anxiety related disorders remains to be determined. [3]

C21H14CLNO3

363.7938

363.066

C, 69.33; H, 3.88; Cl, 9.75; N, 3.85; O, 13.19

142326-59-8

54682505

Typically exists as White to off-white solids at room temperature

1.4±0.1 g/cm3

584.7±50.0 °C at 760 mmHg

307.4±30.1 °C

0.0±1.7 mmHg at 25°C

1.680

5.5

2

3

3

26

558

0

ClC1C([H])=C([H])C2C(=C(C(N([H])C=2C=1[H])=O)C1C([H])=C([H])C([H])=C(C=1[H])OC1C([H])=C([H])C([H])=C([H])C=1[H])O[H]

FLVRDMUHUXVRET-UHFFFAOYSA-N

InChI=1S/C21H14ClNO3/c22-14-9-10-17-18(12-14)23-21(25)19(20(17)24)13-5-4-8-16(11-13)26-15-6-2-1-3-7-15/h1-12H,(H2,23,24,25)

7-chloro-4-hydroxy-3-(3-phenoxyphenyl)quinolin-2(1H)-one

L701,324; L701324; L 701324; L 701,324; 142326-59-8; L-701,324; L-701,324; 7-chloro-4-hydroxy-3-(3-phenoxyphenyl)quinolin-2(1H)-one; CHEMBL31741; 2(1H)-Quinolinone, 7-chloro-4-hydroxy-3-(3-phenoxyphenyl)-; 7-CHLORO-4-HYDROXY-3-(3-PHENOXYPHENYL)-1H-QUINOLIN-2-ONE; I9WY146163; L-701324; L-701,324.

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 : ≥ 34 mg/mL (~93.46 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.)