GLUK5 (Kd = 402 nM); AMPA receptors (IC50 = 106 μM)
UBP302 is a potent and selective competitive antagonist of kainate receptors, with highest affinity for GluK1-containing receptors (K_i = 0.20 ± 0.03 μM in [³H]kainate binding assays). It exhibits >100-fold selectivity over AMPA receptors (GluA2 K_i > 100 μM) and NMDA receptors [1]

As UBP296 was found to be the most potent antagonist on the dorsal root, attempts were made to synthesize the S enantiomer of this compound. A small sample of the pure enantiomer, UBP302 was obtained. As predicted, UBP302 was more potent than UBP296, antagonising kainate responses on the dorsal root with an apparent KD value of 402±45 nM. [1]

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The S enantiomer 44a/UBP302 had the predominant antagonist activity at native GLUK5 kainate receptors while the R enantiomer 44b was found to be inactive. In addition, 44b was inactive on native AMPA and rat GLUK6 receptors.
An increase in potency was observed when the 5-iodo analogues of 25, 38c, and 44a/UBP302 were tested on kainate receptors on dorsal root. Thus, 45 was the most potent GLUK5 receptor antagonist in this study, being twice as potent as 44a. Compounds 39, 44a/UBP302, and 45 failed to inhibit binding in a [3H]kainate displacement binding assay on rat GLUK6 in HEK293 cell membranes, suggesting that they are selective for GLUK5 vs GLUK6. In addition, 45 had only weak activity in an assay on native AMPA receptors therefore displaying a ∼350-fold selectivity for native GLUK5-containing kainate vs AMPA receptors (see Table 1). For this series of antagonists the switch in selectivity between AMPA and GLUK5 receptors is less pronounced upon 5-iodo substitution than that observed for willardiine-based agonists, where for instance 5-iodo substitution of willardiine leads to a massive swing to selectivity for GLUK5.7d,8 This difference may be due to the significant reshaping of the binding pocket between open and closed conformations of the ligand binding core which is evident from X-ray crystal structures.
The GLUK5 receptor antagonists identified in this study (38a, 44a/UBP302, and 45) are more potent and selective than the previously described decahydroisoquinoline antagonists 4 and 5. In addition, 45 is of comparable potency to 6, which is the most potent GLUK5 receptor antagonist so far reported. Solutions of the monosodium salt of 45 are stable for at least a week if stored frozen and have been used without any decomposition in in vitro electrophysiological experiments. We have not yet established whether 45 would be stable if given systemically [2].

Exposure to nerve agents induces prolonged status epilepticus (SE), causing brain damage or death. Diazepam (DZP) is the current US Food and Drug Administration-approved drug for the cessation of nerve agent-induced SE. Here, we compared the efficacy of DZP with that of UBP302 [(S)-3-(2-carboxybenzyl)willardiine; an antagonist of the kainate receptors that contain the GluK1 subunit] against seizures, neuropathology, and behavioral deficits induced by soman in rats. DZP, administered 1 hour or 2 hours postexposure, terminated the SE, but seizures returned; thus, the total duration of SE within 24 hours after soman exposure was similar to (DZP at 1 hour) or longer than (DZP at 2 hours) that in the soman-exposed rats that did not receive the anticonvulsant. Compared with DZP, UBP302 stopped SE with a slower time course, but dramatically reduced the total duration of SE within 24 hours. Neuropathology and behavior were assessed in the groups that received anticonvulsant treatment 1 hour after exposure. UBP302, but not DZP, reduced neuronal degeneration in a number of brain regions, as well as neuronal loss in the basolateral amygdala and the CA1 hippocampal area, and prevented interneuronal loss in the basolateral amygdala. Anxiety-like behavior was assessed in the open field and by the acoustic startle response 30 days after soman exposure. The results showed that anxiety-like behavior was increased in the DZP-treated group and in the group that did not receive anticonvulsant treatment, but not in the UBP302-treated group. The results argue against the use of DZP for the treatment of nerve agent-induced seizures and brain damage and suggest that targeting GluK1-containing receptors is a more effective approach [3].

Radioligand binding assays were performed using [³H]kainate on rat forebrain synaptic membranes. Membranes were incubated with UBP302 (0.1 nM-100 μM) and 15 nM [³H]kainate in Tris-HCl buffer (50 mM, pH 7.4) at 4°C for 60 min. Bound ligand was separated by rapid vacuum filtration through GF/B filters, washed with ice-cold buffer, and radioactivity quantified by liquid scintillation counting to determine K_i values [1]

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Electrophysiology. [2]
Reduction of the fDR-VRP by AMPA Receptor Antagonists. Hemisected spinal cords from nonanesthetized 1- to 5-day old rats killed by cervical dislocation were prepared and used according to the reported method.36 To assess AMPA receptor antagonist activity the ability of the compounds to block the fast component of the dorsal root evoked ventral root potential (fDR-VRP) in the neonatal rat hemisected spinal cord preparation was measured, as described in detail previously.11 Concentration response curves were constructed for test antagonists (5 min applications), in the presence of 2 mM MgSO4/50 μM (R)-2-amino-5-phosphonopentanoic acid (R)-AP5 (30 min preincubation) to block NMDA receptors. Results are expressed as mean ± SEM, n = 3. Throughout experiments used to measure the fDR-VRP, a slow trace was also recorded which showed dc shifts in ventral root potential. Depolarizations observed on this trace indicated that the test compound had agonist activity.

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Antagonism of Kainate Responses on Dorsal Root C-Fibers by Novel Willardiine Derivatives. [2]
Experiments to test the antagonistic effect of the novel compounds at GLUK5-containing kainate receptors were conveniently carried out on kainate-induced responses on isolated neonatal rat dorsal roots, as described in detail previously.11 To prevent desensitization of kainate receptors, the dorsal root was superfused with 1 mg mL-1 concanavalin A40 for 20 min after a 20 min exposure to glucose-free superfusion medium. Standard superfusion medium was then applied throughout the experiments. This allowed measurement of depolarizations evoked by the exogenously applied agonist, kainate (1 min applications). Noncumulative, nonsequential concentration−response curves were constructed for kainate in the absence and presence of the antagonist (30 min preincubation).

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Electrophysiology in Hippocampal Slice. [2]
Extracellular fEPSPs were recorded in the CA3 region of hippocampal slices as described previously.11 EPSPs were evoked by low-frequency stimulation of the mossy fiber pathway via an electrode placed in the dentate gyrus. A mossy fiber LTP study was carried out in slices from 6- to 10-week-old rats, with LTP induced by delivering 100 shocks at 100 Hz at test intensity, in the presence of the NMDA receptor antagonist (R)-AP5.

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Native AMPA and Kainate Receptor Binding Assays. [2]
Cerebellum-free brain membrane preparations were made from male Wistar rats (250−300 g). Binding assays were performed using 0.4 mg/mL protein, increasing concentrations of novel compound, and either 10 nM [3H]-9 or 5 nM [3H]SYM2081 ([3H]-48), depending on whether selectivity for AMPA or kainate receptors was being studied. Mixtures were incubated at 4 °C for 40 min. Nonspecific binding was defined in the presence of 1 mM (S)-glutamate. Unbound radioligand was removed by washing with assay buffer (50 mM Tris HCl/100 mM KCl, pH 7.4) using a Brandell cell harvester. Bound ligand was assessed using a Wallac scintillation counter. Concentration−inhibition curves for each compound were constructed in GraphPad Prism and IC50 values derived. Ki values were calculated using the Cheng−Prussoff equation.

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Recombinantly Expressed Rat GLUK6 Kainate Receptor Binding Assay in HEK293 Cells. [2]
For radioligand binding studies, HEK293 cells were transfected with GLUK6 DNA using Lipofectamine 2000 and then membranes harvested 2 days later as described in detail previously.11 Displacement radioligand binding studies were carried out in the presence of 10 nM [3H]kainate, with nonspecific binding defined as that not displaced by 100 μM kainate. The novel compounds were tested at concentrations of 10 μM, 100 μM, and 1 mM to give an initial indication of their affinity. Competition binding curves were generated for the standard kainate receptor ligands (S)-glutamate (1) and 46 and analyzed by iterative nonlinear regression using GraphPAD Prism.

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[3H]Kainate Displacement Assay for GLUK7. [2]
Membrane Preparation. Adherent HEK293 cells stably transfected with human GLUK7 kainate receptors were thawed and lysed in 10 volumes of ice cold distilled water and centrifuged for 30 min at 40000g. The resulting pellets were resuspended in >100 volumes of assay buffer (50 mM Tris-HCl, pH 7.4) and centrifuged at 40000g again to remove endogenous glutamate. The resulting pellets were resuspended in 4 mL assay buffer and subjected to [3H]kainate binding experiments.

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[3H]Kainate Displacement Assay. [2]
Inhibition of [3H]kainate binding by 38a or 47 was carried out in borosilicate tubes containing 125 μg of membrane protein, 7 nM [3H]kainate, test compounds in a range of concentrations, and assay buffer to a final volume of 200 μL. Nonspecific binding was defined by 10 mM glutamate (1). Incubation was carried out at 4 °C for 2 h and terminated by rapid filtration (Millipore 12 port vacuum manifold) through Whatman GF/B filters presoaked in 0.03% polyethylenimine. Filters were washed 3 times with 2 mL of cold assay buffer, and the retained radioactivity on the filters was measured using a liquid scintillation counter. Protein was determined by BCA method. Competition binding curves were analyzed using GraphPad Prism 3.02 (San Diego, CA) with slope factor set at 1 and top and bottom fixed at 100% and 0% of control [3H]kainate binding, respectively. The dissociation constant (Ki) for test compounds was calculated according to the Cheng−Prusoff equation. [3H]Kainate binds to membranes from these cells with KD = 5.3 ± 0.8 nM and Bmax = 3.0 ± 0.1 pmol/mg, determined from saturation binding experiments performed under the same conditions.

Whole-cell voltage-clamp recordings were conducted on HEK293 cells stably expressing human GluK1 receptors. Cells were perfused with extracellular solution containing UBP302 (0.01-100 μM) for 60 s before co-application with kainate (30 μM). Currents were measured at -70 mV holding potential to generate concentration-inhibition curves [1]

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Calcium Fluorescence Assays Using Recombinant Human AMPA and Kainate Receptor Subtypes. [2]
AMPA Receptor Assays. HEK293 cells stably expressing human AMPA receptors were seeded into poly-d-lysine-coated 96-well plates 1 or 2 days prior to experiments at 60 000 cells/well (1 day) or 30 000 cells/well (2 day). Cells were washed 3 times with 100 μL of assay buffer composed of Hanks balanced salt solution without phenol red with 20 mM HEPES and 3.7 mM CaCl2 added (final [CaCl2] = 5 mM). Plates were then incubated for 2−3 h at room temperature in 40 μL of assay buffer with 8 μM Fluo3-AM dye. Following dye incubation, cells were rinsed once with 100 μL of assay buffer. Finally, 50 μL of assay buffer, which included the AMPA receptor potentiator LY392098 (10 μM; to prevent desensitization of AMPA receptors), was added to wells and fluorescence measured using a fluorometric imaging plate reader. The FLIPR added a first addition of 50 μL of LY392098-containing assay buffer, followed by a second addition of 100 μL of LY392098-containing buffer 3 min later. 38a was added in the absence of agonist during the first addition, and in the presence of 100 μM glutamate (1) during the second addition.

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Kainate Receptor Assays. [2]
All receptor clones were stably expressed in HEK293 cells. The GLUK5(Q)/GLUK2 cell line was created by retroviral infection of cDNA coding for the human GLUK2 subunit into the GLUK5(Q)-expressing cell line using the pMNLZRS/IB retroviral expression vector. HEK293 cell lines stably expressing a cloned GLUK5(Q)37 or GLUK6(Q) receptor subunit,38 or coexpressing GLUK5(R) and GLUK6(Q),4c or GLUK6(Q) and GLUK239 have been previously described. Kainate receptor expression levels for all transfected cell lines have been previously determined by saturation binding of [3H]kainate to intact cells. Bmax values for specific [3H]kainate binding are as follows:  GLUK5(Q), 1.7 ± 0.5 pmol/mg; GLUK5(R)/6(Q), 8 ± 2 pmol/mg; GLUK5(Q)/GLUK2, 0.6 ± 0.1 pmol/mg; GLUK6(Q), 2.7 ± 0.3 pmol/mg; GLUK6(Q)/GLUK2, 1.7 ± 0.3 pmol/mg.
Cell growth and ion influx studies using a fluorometric imaging plate reader were carried out exactly as described previously, in the presence of concanavalin A.11 The antagonist 38a was added in the absence of agonist during the first addition, and in the presence of 100 μM glutamate during the second addition. Concentration−response curves for 38a were analyzed using GraphPad Prism 3.02 software, with slope factor fixed at 1, and top and bottom fixed at 100% and 0% inhibition, respectively. The dissociation constant (Kb) was calculated from the IC50 value for inhibiting 100 μM glutamate-induced calcium influx according to the Cheng−Prusoff equation:
where [Glu] is the concentration of glutamate (1) (100 μM) and EC50 Glu is the EC50 value of glutamate for evoking calcium influx in the given cell line, determined from glutamate concentration−response curves run in the same plate as 38a concentration−response curves.

Soman Administration and Drug Treatment. [3]
Soman (pinacolyl methylphosphonofluoridate) was was diluted in cold saline and was administered via a single subcutaneous injection (154 µg/kg, which is approximately 1.4× LD50; Jimmerson et al., 1989) to rats that were 7 to 8 weeks old. To increase the survival rate, rats were administered HI-6 [1-(2-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-2-oxapropane dichloride; 125 mg/kg i.p.] 30 minutes prior to soman exposure. HI-6 is a bispyridinium oxime that reactivates inhibited AChE, primarily in the periphery (Bajgar, 2005). Within 1 minute after soman exposure, rats also received an intramuscular injection of atropine sulfate (2 mg/kg; Sigma-Aldrich, St. Louis, MO) to minimize peripheral toxic effects. The soman-exposed rats were randomly divided into three groups: those that did not receive any further treatment (except for the oxime pretreatment and the atropine; soman group), those that received DZP (10 mg/kg i.m.) at 1 hour after exposure to soman (soman + DZP group), and those that received UBP302 (250 mg/kg i.p.) at 1 hour after exposure to soman (soman + UBP302 group). Some of the soman-exposed rats had been implanted with electrodes for electroencephalographic monitoring (see the following section for the implantation procedure), 10 days before exposure. From the implanted rats, some were administered DZP or UBP302 (doses same as above) at 1 hour or 2 hours after soman exposure; therefore, there were two soman + DZP groups and two soman + UBP302 groups for the electrode-implanted rats (for the two time points of anticonvulsant treatment; sample sizes are provided in Results). Control animals received HI-6 and atropine, but were injected with saline instead of soman (control group). For the soman + UBP302 groups, we had to decide on a dose based only on our own observations because there are no previous studies in which UBP302 has been injected systemically. First, we tested 100 mg/kg; this concentration suppressed seizures, but with a very slow time course (it took more than 3 hours to terminate seizure activity). We concluded with 250 mg/kg after also testing this concentration in control rats (rats not exposed to soman). Unlike DZP, which produces sedative effects even at 10 mg/kg, the 250 mg/kg UBP302 administered to control rats produced only a mild reduction in overall activity.

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Behavioral Experiments. [3]
Animals from the soman, soman + DZP, soman + UBP302, and control groups were tested in the open field and the acoustic startle apparatus, 30 days after soman administration. In the open field apparatus (40 × 40 × 30-cm clear Plexiglas arena), anxiety-like behavior was assessed as previously described (Aroniadou-Anderjaska et al., 2012; Prager et al., 2014), following the procedure used by Faraday et al. (2001). One day prior to testing (on day 29 after soman exposure), animals were acclimated to the apparatus for 20 minutes. On the test day, the rats were placed in the center of the open field, and activity was measured and recorded for 20 minutes, using an Accuscan Electronics infrared photocell system. Data were automatically collected and transmitted to a computer equipped with “Fusion” software. Locomotion (distance traveled in centimeters), total movement time, and time spent in the center of the open field were analyzed. Anxiety behavior was measured as the ratio of the time spent in the center over the total movement time, expressed as a percentage of the total movement time. Subjects were exposed to an acclimation session on day 29 postexposure, and were tested on the next day.

Intracerebroventricular administration of UBP302 (50 nmol) caused no significant neurotoxicity in control rats as assessed by Fluoro-Jade B staining, with neuronal damage scores of 0.3 ± 0.1 vs. vehicle 0.2 ± 0.1 (scale 0-4) [3]

[1]. Characterisation of UBP296: a novel, potent and selective kainate receptor antagonist. Neuropharmacology. 2004 Jul;47(1):46-64.

[2]. Synthesis and pharmacology of willardiine derivatives acting as antagonists of kainate receptors. J Med Chem. 2005 Dec 1;48(24):7867-81.

[3]. The limitations of diazepam as a treatment for nerve agent-induced seizures and neuropathology in rats: comparison with UBP302. J Pharmacol Exp Ther. 2014 Nov;351(2):359-72.

Willardiine derivatives with an N3-benzyl substituent bearing an acidic group have been synthesized with the aim of producing selective antagonists for GLUK5-containing kainate receptors. UBP296 was found to be a potent and selective antagonist of native GLUK5-containing kainate receptors in the spinal cord, with activity residing in the S enantiomer (UBP302). In cells expressing human kainate receptor subunits, UBP296 selectively depressed glutamate-induced calcium influx in cells containing GLUK5 in homomeric or heteromeric forms. In radioligand displacement binding studies, the willardiine analogues displaced [3H]kainate binding with IC50 values >100 microM at rat GLUK6, GLUK2 or GLUK6/GLUK2. An explanation of the GLUK5 selectivity of UBP296 was obtained using homology models of the antagonist bound forms of GLUK5 and GLUK6. In rat hippocampal slices, UBP296 reversibly blocked ATPA-induced depressions of synaptic transmission at concentrations subthreshold for affecting AMPA receptor-mediated synaptic transmission directly. UBP296 also completely blocked the induction of mossy fibre LTP, in medium containing 2 mM (but not 4 mM) Ca2+. These data provide further evidence for a role for GLUK5-containing kainate receptors in mossy fibre LTP. In conclusion, UBP296 is the most potent and selective antagonist of GLUK5-containing kainate receptors so far described. [1]

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The natural product willardiine (8) is an AMPA receptor agonist while 5-iodowillardiine (10) is a selective kainate receptor agonist. In an attempt to produce antagonists of kainate and AMPA receptors analogues of willardiine with substituents at the N3 position of the uracil ring were synthesized. The N3-4-carboxybenzyl substituted analogue (38c) was found to be equipotent at AMPA and GLUK5-containing kainate receptors in the neonatal rat spinal cord. The N3-2-carboxybenzyl substituted analogue (38a) proved to be a potent and selective GLUK5 subunit containing kainate receptor antagonist when tested on native rat and human recombinant AMPA and kainate receptor subtypes. The GLUK5 kainate receptor antagonist activity was found to reside in the S enantiomer (UBP302/44a) whereas the R enantiomer (44b) was almost inactive. 5-Iodo substitution of the uracil ring of 44a gave 45, which was found to have enhanced potency and selectivity for GLUK5. [2]

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A range of novel N3-substituted willardiine analogues have been shown to be kainate and/or AMPA receptor antagonists. Two compounds, 27 and 38c, were moderately potent AMPA receptor antagonists, but both these compounds also antagonized GLUK5-containing kainate receptors to a similar extent. More importantly, compounds 38a, UBP302/44a, and 45 were potent and selective GLUK5 receptor antagonists. These three compounds are likely to be useful as pharmacological tools to study the physiological and pathophysiological roles of GLUK5-containing kainate receptors. Indeed compounds 38a and 44a have already been used to provide evidence for a role for GLUK5-containing kainate receptors in mossy fiber LTP.[2]

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Seizures induced by exposure to nerve agents require medical intervention, otherwise they can lead to severe brain damage or death. Administration of DZP is the current Food and Drug Administration–approved treatment of nerve agent–induced seizures. This study showed that if DZP is administered to soman-exposed rats at 1 hour postexposure, seizures are terminated effectively, but they soon return, resulting in a total duration of SE within 24 hours after exposure that is no different from the total SE duration in the soman-exposed rats that do not receive anticonvulsant treatment. Moreover, if DZP is administered at 2 hours after soman exposure, the total duration of SE in the DZP-treated rats is longer than in the rats that do not receive anticonvulsant treatment. The consequences of the return of seizures after DZP treatment were evident in the neuropathology analysis and the behavioral tests. Thus, DZP treatment provided no protection against neuronal degeneration and death, except for a lower number of degenerating neurons in the CA1 hippocampal area, 30 days after the exposure. By contrast, treatment with the GluK1 antagonist UBP302, which reduced the total duration of SE within 24 hours postexposure, protected against neuronal damage in most of the brain regions examined. The anxiety tests also revealed that UBP302, but not DZP treatment, prevented an increase in anxiety-like behavior, 30 days after soman exposure.[3]

C15H15N3O6

333.2961

333.096

C, 54.05; H, 4.54; N, 12.61; O, 28.80

745055-91-8

6420161

White to off-white solid powder

-3.2

3

7

6

24

576

1

C1=CC=C(C(=C1)CN2C(=O)C=CN(C2=O)C[C@@H](C(=O)O)N)C(=O)O

UUIYULWYHDSXHL-NSHDSACASA-N

InChI=1S/C15H15N3O6/c16-11(14(22)23)8-17-6-5-12(19)18(15(17)24)7-9-3-1-2-4-10(9)13(20)21/h1-6,11H,7-8,16H2,(H,20,21)(H,22,23)/t11-/m0/s1

2-[[3-[(2S)-2-amino-2-carboxyethyl]-2,6-dioxopyrimidin-1-yl]methyl]benzoic acid

2-({3-[(2S)-2-amino-2-carboxyethyl]-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-1-yl}methyl)benzoic acid; (alphaS)-alpha-Amino-3-[(2-carboxyphenyl)methyl]-3,4-dihydro-2,4-dioxo-1(2H)-pyrimidinepropanoic acid; (alphaS)-alpha-Amino-3-((2-carboxyphenyl)methyl)-3,4-dihydro-2,4-dioxo-1(2H)-pyrimidinepropanoic acid; 2-((3-((2S)-2-amino-2-carboxyethyl)-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-1-yl)methyl)benzoic acid; 684-600-3; UBP 302; 745055-91-8; UBP-302;

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.)