IC50: 25 nM (rat cortical neurons)[1]

CP-465022 (0.0001 μM-10 μM) reduces kainate-induced response in very slow way and dependents on compound concentration, demonstrating a predicted IC50 of 25 nM and practically complete inhibition at 3.2 µM[1]. Peak NMDA-induced currents are not significantly affected by CP-465022 1 µM for 10 min, but current measured at 8 s during NMDA application is reduced by 26%. In primary cultures of cortical and cerebellar granule neurons, CP-465,022 at 10 µM inhibits peak NMDA-induced currents by 36% and currents measured at 8 s by 70% d[1]. In cultured rat cerebellar granule neurons, CP-465022 1 µM for 10 min decreases peak NMDA currents with a mean suppression of 19% and NMDA currents assessed at 8 s by 45%, which is comparable to what is seen in cortical neurons[1]. In voltage-clamped rat hippocampus slices, CP-465022 (100 nM – 10 µM) exhibits inhibitory effects on Kainate-induced whole-cell currents. At 100 nM, CP465,022 suppresses kainate currents that form over 200 s, while at 500 nM and 1 µM, CP-465,022 almost entirely suppresses these currents (99.3%)[1].

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Mechanism of inhibition[1]
To determine whether CP-465,022-induced inhibition of AMPA receptors was competitive with respect to agonist, we examined the effects of CP-465,022 on currents in cortical neurons induced by a range of kainate concentrations. Responses induced by 10 μM to 10 mM kainate were significantly inhibited by 32 nM CP-465,022 (Fig. 4A). However, in the presence of 32 nM CP-465,022, the EC50 for kainate was similar to that in the absence of the compound (EC50 in absence=145 μM; EC50 in the presence of 32 nM CP-465,022=125 μM). In fact, the inhibitory effect of the compound was not overcome by a concentration of kainate (10 mM) that was approximately 70 times higher than the kainate EC50 in these cells. Thus, inhibition was not competitive with agonist.
We next tested whether CP-465,022 required open channels to inhibit kainate-induced responses. This was done by exposing cultured cortical neurons to CP-465,022 in the absence of agonist stimulation as shown in Fig. 4B. After establishing a baseline amplitude of kainate-induced currents, cells were exposed to either control solution, 10 nM CP-465,022, or 100 nM CP-465,022 for 10 min. During this 10-min period, there were no applications of kainate to the cells. The amplitudes of the first kainate-induced response after the 10-min antagonist exposure were dose-dependently decreased compared to control (Fig. 4B). The first kainate-induced responses after exposure to 10 nM and 100 nM CP-465,022 were inhibited 54±6% (n=3) and 88±4% (n=3), respectively. These levels of inhibition were similar to those produced by these concentrations of the compound during repeated kainate applications. Thus, inhibition by CP-465,022 develops in the absence of channel activation.
Finally, the voltage-dependence of the inhibitory efficacy of CP-465,022 was assessed in cortical neurons voltage clamped at −60 mV or +30 mV. The time course and extent of inhibition of kainate-induced responses by 100 nM CP-465,022 was similar under these two conditions with inhibition of 73±7% at −60 mV (n=3) and 70±5% at +30 mV (n=4) (Fig. 4C). This indicates that inhibition is not voltage dependent.

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Specificity of CP-465,022 for AMPA receptors[1]
The above data indicate that CP-465,022 is a potent, noncompetitive antagonist of AMPA receptors. We next investigated the specificity of the compound for AMPA receptors over that for other ligand-gated ionotropic receptors. The effect of CP-465,022 on NMDA receptor-mediated responses was examined in primary cultures of cortical and cerebellar granule neurons. NMDA receptor activation in cortical neurons was induced in the absence of Mg2+ by 100 μM NMDA plus 10 μM glycine (Fig. 5A). Incubation with CP-465,022 at 1 μM for 10 min had little effect on peak NMDA-induced currents (0±0% inhibition, n=3), but reduced current measured at 8 s during NMDA application by 26±5%. CP-465,022 at 10 μM inhibited peak NMDA-induced currents in cortical neurons by 36±4% and currents measured at 8 s by 70±10% (n=6). These levels of inhibition were apparently at steady state within 3–4 NMDA applications (3–4 min). We did not investigate further the difference in block by CP-465,022 of peak and sustained NMDA-induced currents. We also examined the effect of CP-465,022 on NMDA-induced currents in cultured rat cerebellar granule neurons (Fig. 5B). CP-465,022 at 1 μM for 10 min inhibited peak NMDA currents in these cells with mean inhibition of 19±11% and NMDA currents measured at 8 s by 45±9% (n=4), similar to what was observed in the cortical neurons.

CP-465,022 potently and efficaciously inhibited AMPA receptor-mediated hippocampal synaptic transmission and the induction of seizures. However, at comparable doses, CP-465,022 failed to prevent CA1 neuron loss after brief global ischemia or to reduce infarct volume after temporary middle cerebral artery occlusion. Conclusions: Given the high selectivity of CP-465,022 for AMPA over kainate and N-methyl-D-aspartate subtypes of glutamate receptors, the lack of neuroprotective efficacy of the compound calls into question the neuroprotective efficacy of AMPA receptor inhibition after ischemia.[2]

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AMPA Receptor-Mediated Synaptic Transmission In Vivo [2]
Stimuli applied to the Schaeffer collateral/commissural pathway evoked a reproducible population spike in the contralateral CA1 region of approximately 3 mV (Figure 1A). This response is mediated by AMPA and NMDA receptors on the CA1 neurons, as has been established in hippocampal slice preparations. CP-465,022 efficaciously and reversibly decreased the amplitude of the evoked population spike (Figure 1A). When administered as a 1-minute intravenous infusion, this effect was dose dependent (Figure 1B). Maximal inhibition was at 1 mg/kg, which reversed within approximately 30 minutes. After subcutaneous administration, inhibition was maintained for much longer periods (Figure 1C). Maximum inhibition was observed at 15 mg/kg SC of racemic CP-465,022 (equivalent to 7.5 mg/kg of the resolved S enantiomer); at higher doses, lethality was sometimes observed. Subcutaneous administration of the quinoxalinedione AMPA receptor antagonist YM-90K also reversibly decreased the amplitude of the evoked population spike. At a maximally effective dose of 56 mg/kg SC (Figure 1C), the magnitude of inhibition was similar to that caused by CP-465,022. However, inhibition by YM-90K developed more quickly and was of shorter duration than for CP-465,022.

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Pentylenetetrazole-Induced Seizures [2]
Administration of pentylenetetrazole (100 mg/kg IP) to rats results in a characteristic syndrome of clonic seizures followed by tonic seizures and lethality within 30 minutes. CP-465,022 administered subcutaneously 60 minutes before pentylenetetrazole dose-dependently increased the latency to and decreased the incidence of pentylenetetrazole-induced clonic seizures, tonic seizures, and lethality (Figure 3, Table). Complete protection was observed across all 3 measures at 10 mg/kg. Efficacy at this dose was observed with pretreatment times up to 4 hours (data not shown). YM-90K also increased the latency to and increased the incidence of pentylenetetrazole-induced seizures and lethality, albeit with less potency than CP-465,022 (Table). A dose of YM-90K that completely inhibited tonic seizures and lethality when given 30 minutes before pentylenetetrazole (56 mg/kg SC) was ineffective if given 2 hours before pentylenetetrazole (data not shown).

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Locomotor Activity [2]
CP-465,022 dose dependently decreased horizontal and vertical locomotor activity, with ED50 values of 11.9 and 6.6 mg/kg, respectively (Table). YM-90K produced similar effects but with less potency (Table).

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Global Ischemia [2]
Ten minutes of global cerebral ischemia results in a substantial loss of hippocampal CA1 pyramidal neurons after 7 days.19 The ability of CP-465,022 to prevent this neuronal loss was assessed at 2 dose levels. In 1 group, CP-465,022 was administered at 5 mg/kg SC at the time of reperfusion and at 2 mg/kg SC 4 hours later. A second group was similarly dosed but with 10 and 4 mg/kg of CP-465,022. The comparator group received vehicle injections. After 7 days, the brief global ischemia caused a loss of 81% of CA1 neurons in the vehicle-treated group (Figure 4). CP-465,022 at either of the doses tested failed to reduce the observed neuronal loss.

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Focal Ischemia [2]
The neuroprotective effect of CP-465,022 was also examined in a rat temporary MCAO model.16 Animals were subjected to unilateral MCAO and after 90 minutes were administered 5 mg/kg SC of CP-465,022 or vehicle. After 30 minutes, artery clips were removed, and, after an additional 3.5 hours, animals received a second administration of CP-465,022 at 2 mg/kg SC or vehicle. Body temperatures were maintained at 37°C for 6 hours after the initiation of occlusion, and CP-465,022 had minimal effects on other physiological parameters during this period. Twenty-four hours after the initiation of occlusion, the volume of cortical infarction was 141±46 mm3 (mean±SD) in vehicle-treated animals (Figure 4). CP-465,022 caused a small decrease in infarct volume (to 124±49 mm3), but this was not significantly different from the vehicle control group. A second group of 4 animals received a higher dose of CP-465,022 (10 mg/kg SC) under identical conditions; however, these animals all died within 1 hour of compound administration.

Whole-cell recordings in cortical, hNT, and cerebellar granule neurons[1]
The rate of inhibition of kainate-induced currents by CP-465,022 was concentration dependent and inhibition was very slowly reversible upon washout. Thus, potency estimations were arrived at using the following protocol. Cells were chosen for analyses if, prior to antagonist exposure, the current amplitude in response to kainate did not vary by more than ±5% over three consecutive kainate pulses. After antagonist exposure, the current amplitude was taken to have stabilized when two consecutive agonist pulses resulted in current amplitudes that did not vary by more than ±5%. For high concentrations of CP-465,022 (e.g. 1 μM), current amplitudes reached this criteria with the first two agonist pulses, whereas for lower concentrations of compound, current stabilization occurred after 3–4 pulses. The percent inhibition was then taken as the difference in current amplitude for the pulse prior to antagonist application and the second pulse after current stabilization post-antagonist application. Percent inhibition data was fit to the equation: where F is the amplitude of the response expressed as a fraction of control, x is the concentration of compound, IC50 is the half-maximal inhibition, and h is the Hill coefficient.

CP-465,022 and YM-90K were dissolved in 10% Captisol (sulfobutylether β-cyclodextrin. [2]

Plasma Levels of CP-465,022[2]
Cannulas were inserted into the jugular vein of fasted rats (n=5) under ketamine/xylazine (70:30) anesthesia. The following day, animals were administered 10 mg/kg SC of racemic CP-465,022. Plasma samples were obtained before dosing and at 0.25 to 6 hours afterward. Plasma concentrations of CP-465,022 were determined by high-performance liquid chromatography with UV detection.

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Global Ischemia[2]
Rats were subjected to brief global cerebral ischemia as previously described.18,19 Under 1% to 2% halothane anesthesia, both common carotid arteries were isolated, and a ligature was gently placed around each vessel. The vertebral arteries were electrocauterized, and a ligature was passed ventral to the cervical and paravertebral muscles but dorsal to the trachea, esophagus, external jugular veins, and common carotid arteries. On the following day, aneurysm clips were put on carotid arteries, resulting in occlusion of the 4 major vessels supplying the cerebrum (4-vessel occlusion). Loss of consciousness was seen within 10 to 15 seconds. The ligature surrounding the paravertebral musculature was then tightened to prevent the opening of collateral blood flow channels. Body temperature was maintained at 37.5°C throughout the ischemic period. After 10 minutes of ischemia, animals were checked for unresponsiveness and dilated pupils. The aneurysm clips were removed, the vessels were inspected for patency, and the wound was closed with a surgical clip. Immediately after reperfusion and 4 hours afterward, animals were administered CP-465,022 or vehicle by subcutaneous injection. Animals were allowed to survive for 7 days, after which the animals were perfusion-fixed with 4% buffered formaldehyde. The number of normal and abnormal (dead) CA1 neurons was counted, and results were expressed as the percentage of dead neurons, as previously described.

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Focal Ischemia[2]
Rats were subjected to temporary MCAO as previously described.16 The right common carotid artery was first isolated and permanently occluded. The right MCA was then exposed by a subtemporal approach and occluded with clips. Ninety minutes after occlusion and an additional 4 hours afterward, animals were administered CP-465,022 or vehicle by subcutaneous injection. Occlusion was maintained for an additional 30 minutes after the first compound administration (for a total of 2 hours of occlusion), and then the clips were removed. Temperature was maintained at 37°C throughout surgery and treatment. After survival for 22 hours, animals were killed by decapitation, and the brains were rapidly removed and frozen. Coronal brain sections (20 μm thick) were cut at 500-μm intervals, fixed in 90% ethanol, and stained with hematoxylin and eosin. Infarcted areas for each section were traced, and a total infarct volume was calculated by summation of infarct area in sequential sections and multiplication by the interval thickness between sections.

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Pentylenetetrazole-Induced Seizures[2]
Compounds (e.g. CP-465,022)were administered 30 or 60 minutes before pentylenetetrazole (100 mg/kg IP). Rats were then observed for 30 minutes, and latencies to clonic seizures, tonic seizures, and lethality were recorded. Data were analyzed with Kruskal-Wallis ANOVA followed by Mann-Whitney U tests.

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Spontaneous Locomotor Activity[2]
Rats were placed into chambers (30 cm3) equipped with photocells housed in sound-attenuating cabinets. Compounds (e.g. CP-465,022) were administered to unhabituated animals immediately before placement into chambers, and horizontal (crossovers) and vertical (rears) locomotion was recorded for 12 hours. Data were analyzed with ANOVA followed by Dunnett’s t tests for multiple comparisons with a control.

Atropisomers may be racemized thermally, a process which can render them unsuitable for drug development (at least as a single chiral entity). However, CP-465,022 is thermally stable in human plasma at 37 °C for up to 7 days [1].

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Plasma Levels of CP-465,022 [2]
Plasma levels of racemic CP-465,022 after subcutaneous administration of 10 mg/kg are indicated in Figure 2. Levels reached a peak within approximately 30 minutes of dosing and then declined slowly with a half-life of approximately 4 hours. Qualitative observation indicated that the animals became ataxic within 30 minutes of dosing and remained so for approximately 4 hours.

[1]. Functional characterization of CP-465,022, a selective, noncompetitive AMPA receptor antagonist. Neuropharmacology. 2002 Feb;42(2):143-53.

[2]. CP-465,022, a selective noncompetitive AMPA receptor antagonist, blocks AMPA receptors but is not neuroprotective in vivo. Stroke. 2003 Jan;34(1):171-6.

The hypothesis that aberrant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor activity contributes to epileptogenesis and neurodegeneration has prompted the search for AMPA receptor antagonists as potential therapeutics to treat these conditions. We describe the functional characterization of a novel quinazolin-4-one AMPA receptor antagonist, 3-(2-chloro-phenyl)-2-[2-(6-diethylaminomethyl-pyridin-2-yl)-vinyl]-6-fluoro-3H-quinazolin-4-one (CP-465,022). This compound inhibits AMPA receptor-mediated currents in rat cortical neurons with an IC(50) of 25 nM. Inhibition is noncompetitive with agonist concentration and is not use- or voltage-dependent. CP-465,022 is selective for AMPA over kainate and N-methyl-D-aspartate receptors. However, the compound is found to be equipotent for AMPA receptors composed of different AMPA receptor subunit combinations. This is indicated by the finding that CP-465,022 is equivalently potent for inhibition of AMPA receptor-mediated responses in different types of neurons that express different AMPA receptor subunits. Thus, CP-465,022 provides a new tool to investigate the role of AMPA receptors in physiological and pathophysiological processes. [1]

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In summary, the present study indicates that CP-465,022 is a novel, noncompetitive AMPA receptor antagonist. This compound differs from the prototypical AMPA receptor antagonist NBQX and other quinoxalinediones in specificity for AMPA compared to kainate receptors. In this regard, CP-465,022 is similar to the prototypical noncompetitive AMPA receptor antagonist GYKI 52466 and the related 2,3-benzodiazepines. In fact, we have previously reported that the binding site for CP-465,022 and related quinazolinones overlaps with that of the 2,3-benzodiazepines (Menniti et al., 2000). However, CP-465,022 is significantly more potent than this latter class (e.g. the IC50 of GYKI 53655 for inhibition of AMPA receptor-mediated responses in various preparations is 1.5–6 μM; Bleakman et al., 1996). Furthermore, CP-465,022 has excellent pharmaceutical properties and solubility (when formulated as a methanesulfonate salt, the solubility of CP-465,022 exceeds 150 mg/ml in water at pH 4.7), and readily gains access to the brain after peripheral administration (Seymour et al., 1998). Thus, CP-465,022 provides a new tool to study the role of AMPA receptors in physiological and pathophysiological conditions.[1]

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Background and purpose: Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor inhibition has been hypothesized to provide neuroprotective efficacy after cerebral ischemia on the basis of the activity in experimental ischemia models of a variety of compounds with varying selectivity for AMPA over other glutamate receptor subtypes. CP-465,022 is a new, potent, and selective noncompetitive AMPA receptor antagonist. The present study investigated the ability of this compound to reduce neuronal loss after experimental cerebral ischemia to probe the neuroprotective potential of AMPA receptor inhibition.
Methods: To demonstrate that CP-465,022 gains access to the brain, the effects of systemic administration of CP-465,022 were investigated on AMPA receptor-mediated electrophysiological responses in hippocampus and on chemically induced seizures in rats. The compound was then investigated for neuroprotective efficacy in rat global and focal ischemia models at doses demonstrated to be maximally effective in the electrophysiology and seizure models.[2]

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In summary, the results presented here suggest that AMPA receptor inhibition alone may not be sufficient to account for the robust efficacy profile of the quinoxalinediones against ischemia-induced neuronal damage. On the other hand, the similar profiles of central nervous system depressant activity observed between CP-465,022 and YM-90K indicate that AMPA receptor inhibition does account for the central nervous system depressant activity of the quinoxalinediones. If AMPA receptor inhibition is not obligate for neuroprotective efficacy, then it may be possible to identify a glutamate receptor target that provides neuroprotection at a reduced side effect burden. CP-465,022 provides an important new tool in pursuing this line of research.[2]

C26H25CL2FN4O

498.138

1785666-59-2

CP-465022;199655-36-2;CP-465022 maleate;199656-46-7

53251536

Typically exists as solid at room temperature

1

5

7

34

727

0

CCN(CC)CC1=NC(=CC=C1)/C=C/C2=NC3=C(C=C(C=C3)F)C(=O)N2C4=CC=CC=C4Cl.Cl

YKYDGCRJPYLXHY-GVYCEHEKSA-N

InChI=1S/C26H24ClFN4O.ClH/c1-3-31(4-2)17-20-9-7-8-19(29-20)13-15-25-30-23-14-12-18(28)16-21(23)26(33)32(25)24-11-6-5-10-22(24)27;/h5-16H,3-4,17H2,1-2H3;1H/b15-13+;

3-(2-chlorophenyl)-2-[(E)-2-[6-(diethylaminomethyl)pyridin-2-yl]ethenyl]-6-fluoroquinazolin-4-one;hydrochloride

CP 465022 HYDROCHLORIDE; 1785666-59-2; CP465022 HCl; CP-465022 hydrochloride; 3-(2-Chlorophenyl)-2-[2-[6-[(diethylamino)methyl]-2-pyridinyl]ethenyl]-6-fluoro-4(3H)-quinazolinone hydrochloride; CP-465,022 (hydrochloride); CP 465,022; 3-(2-Chlorophenyl)-2-(2-(6-((diethylamino)methyl)pyridin-2-yl)vinyl)-6-fluoroquinazolin-4(3H)-one hydrochloride;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)