mGluR2

Eglumegad (LY354740) up-regulates spot 6507 (collapsin response mediator protein 1) and down-regulates spots 1014, 1822 (hypoxia up-regulated protein 1), 4513 (an isoform of protein disulfide isomerase 3), 6204, 6312, 7306 (26S proteasome non-ATPase regulatory subunit 7), and protein spots 1013 and 6005 (destrin)], among other spots in mouse cortical neurons[2].

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Proteomic analysis of cultured cortical neurons treated with LY379268 and LY354740 [2]
Cultured mouse cortical neurons were exposed for 24 h to either LY379268 or LY354740 (both at 1 μM). Control cultures were treated with an equal volume of saline. 2D-electrophoresis allowed to reproducibly separate 916 protein spots (Fig. 1) which were analyzed for differential expression. A total of 17 spots (11 identified by mass spectrometry, as shown in Table 1) were detected with a significant different expression levels (considered as ±1.5 fold-change, p < 0.05) in drug-treated cultures with respect to control cultures. Fold change differences are reported in Table 2, where significant differences between LY354740 and LY379268 treated cells are also shown. Compared to control cultures, differential expression was detected for 9 protein spots in LY354740-treated cultures, and for 14 protein spots in LY379268-treated cultures. Both LY379268 and LY354740 down-regulated spots 1014, 1822 (hypoxia up-regulated protein 1), 4513 (an isoform of protein disulfide isomerase 3), 6204, 6312, and 7306 (26S proteasome non-ATPase regulatory subunit 7), and up-regulated spot 6507 (collapsin response mediator protein 1). Protein spots 1013 and 6005 (destrin) were exclusively down-regulated by LY354740. In contrast, protein spots 3503 (T-complex protein 1 subunit θ), 5106 (isopentenyl-diphosphate Δ-isomerase 1), 05310 (Septin-2), 5321, and 7306 (26S proteasome non-ATPase regulatory subunit 7) were exclusively down-regulated by LY379268. Spot 7518 (T-complex protein 1 subunit ζ) was up-regulated by LY379268. Spots 4417 (Rab GDIβ) and 4418 (unidentified) were down-regulated by LY379268 and showed a trend to an up-regulation in response to LY354740.

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Measurements of Rab GDIα and Rab GDIβ protein levels in cultured cortical and hippocampal neurons treated with LY379268 or LY354740 [2]
We could validate proteomic data using cultured cortical neurons treated with LY379268 or LY354740. Immunoblot analysis with a specific GDIα antibody showed a single band at 50 kDa, corresponding to the deduced molecular size of Rab GDIα. Rab GDIβ could be detected with the GDI2 polyclonal antibody. This antibody labeled two bands between 46 and 50 kDa, with the lower band (which was more faint) corresponding to the deduced molecular size of Rab GDIβ (Fig. 2). A 24-h treatment of cultured cortical neurons with LY379268 (1 μM) reduced Rab GDIβ levels without affecting Rab GDIα levels (Fig. 2A). The lowering action of LY379268 on Rab GDIβ levels was reduced by the preferential mGlu2/3 antagonist, LY341495 (1 μM), and was not mimicked by LY354740 (1 μM) (Fig. 2A).

LY-354740 (15 or 30 mg/kg, i.p.) has no effect on spatial working memory performance. It also has no effect in rewarded alternation testing with a short inter-trial interval when used at a dose of 30 mg/kg in both Gria1^−/− and WT mice. LY354740 (15 or 30 mg/kg, i.p.) decreases spontaneous locomotor activity in wild-type and Gria1^−/− mice. In male naive GluA1-KO and pre-handled GluA1-KO animals, LY354740 (15 mg/kg, i.p.) reduces novelty-induced hyperlocomotion, but not in females. The increased c-Fos expression in GluA1-KO males is significantly reduced to WT male levels by LY354740 (15 mg/kg, i.p.), but not in females[3]. The immobilization stress-induced rise in BDNF mRNA expression in the rat mPFC is attenuated by LY354740 (10 mg/kg, i.p.)[4]. In the rat mPFC, LY354740 (10 mg/kg, i.p.) reduces the rise in BDNF mRNA expression brought on by immobilization stress[4].

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Group II metabotropic glutamate receptor agonists have been suggested as potential anti-psychotics, at least in part, based on the observation that the agonist LY354740 appeared to rescue the cognitive deficits caused by non-competitive N-methyl-d-aspartate receptor (NMDAR) antagonists, including spatial working memory deficits in rodents. Here, we tested the ability of LY354740 to rescue spatial working memory performance in mice that lack the GluA1 subunit of the AMPA glutamate receptor, encoded by Gria1, a gene recently implicated in schizophrenia by genome-wide association studies. We found that LY354740 failed to rescue the spatial working memory deficit in Gria1-/- mice during rewarded alternation performance in the T-maze. In contrast, LY354740 did reduce the locomotor hyperactivity in these animals to a level that was similar to controls. A similar pattern was found with the dopamine receptor antagonist haloperidol, with no amelioration of the spatial working memory deficit in Gria1-/- mice, even though the same dose of haloperidol reduced their locomotor hyperactivity. These results with LY354740 contrast with the rescue of spatial working memory in models of glutamatergic hypofunction using non-competitive NMDAR antagonists. Future studies should determine whether group II mGluR agonists can rescue spatial working memory deficits with other NMDAR manipulations, including genetic models and other pharmacological manipulations of NMDAR function.[1]

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LY379268 and LY354740, two agonists of mGlu2/3 metabotropic glutamate receptors, display different potencies in mouse models of schizophrenia. This differential effect of the two drugs remains unexplained. We performed a proteomic analysis in cultured cortical neurons challenged with either LY379268 or LY354740. Among the few proteins that were differentially influenced by the two drugs, Rab GDP dissociation inhibitor-β (Rab GDIβ) was down-regulated by LY379268 and showed a trend to an up-regulation in response to LY354740. In cultured hippocampal neurons, LY379268 selectively down-regulated the α isoform of Rab GDI. Rab GDI inhibits the activity of the synaptic vesicle-associated protein, Rab3A, and is reduced in the brain of schizophrenic patients. We examined the expression of Rab GDI in mice exposed to prenatal stress ("PRS mice"), which have been described as a putative model of schizophrenia. Rab GDIα protein levels were increased in the hippocampus of PRS mice at postnatal days (PND)1 and 21, but not at PND60. At PND21, PRS mice also showed a reduced depolarization-evoked [(3)H]d-aspartate release in hippocampal synaptosomes. The increase in Rab GDIα levels in the hippocampus of PRS mice was reversed by a 7-days treatment with LY379268 (1 or 10 mg/kg, i.p.), but not by treatment with equal doses of LY354740. These data strengthen the validity of PRS mice as a model of schizophrenia, and show for the first time a pharmacodynamic difference between LY379268 and LY354740 which might be taken into account in an attempt to explain the differential effect of the two drugs across mouse models [2].

Protein identification by MALDI-ToF mass spectrometry [2]
Protein spots of interest were manually excised from the gel, washed with high-purity water and 50% acetonitrile/water and dehydrated with 100% acetonitrile. Gel slices were swollen at room temperature in 20 μL of 40 mM NH4HCO3/10% acetonitrile containing 25 ng/μL trypsin (Trypsin Gold, mass spectrometry grade). After 1 h, 50 μL of 40 mM·NH4HCO3/10% acetonitrile were added and digestion proceeded overnight at 37 °C. The generated peptides were extracted with 50% acetonitrile/5% trifluoroacetic acid (TFA, 2 steps, 20 min at room temperature each), dried by vacuum centrifugation, suspended in 0.1% TFA, passed through micro ZipTip C18 pipette tips and directly eluted with the spectrometer matrix solution (10 mg/ml α-cyano-4-hydroxycinnamic acid in 50% acetonitrile/1% TFA). Mass spectra of the tryptic peptides were obtained using a Voyager-DE MALDI-ToF mass spectrometer. Peptide mass fingerprinting database searching was performed using MASCOT searching engine (http://www.matrixscience.com) in the NCBInr/Swiss-Prot databases. Parameters were set to allow one missed cleavage per peptide, a mass tolerance of 0.5 Da, and considering carbamido-methylation of cysteines as a fixed modification and oxidation of methionines as a variable modification. The criteria used to accept identifications included the extent of sequence coverage, number of matched peptides and probabilistic score, as detailed in Table 1.

Primary cultures of mouse cortical neurons [2]
Primary cultures of cortical neurons (three preparations) were obtained from CD1 mice at embryonic day 15 as described previously (Di Menna et al., 2013). Briefly, cortices from 14–16 embryos per preparation were dissected in a Ca2+/Mg2+-free buffer and mechanically dissociated. Cortical cells were plated at a density of 2 × 106/dish on 35-mm dishes precoated with 0.1 mg/ml poly-d-lysine (molecular weight 70,000–150,000, 0.1 mg/ml) and 0.002 mg/ml laminin in Neurobasal-A B27 medium supplemented with the following components: bovine serum albumin (10 mg/ml), insulin (10 μg/ml), transferrin (100 μg/ml), putrescine (100 μM), progesterone (20 nM), selenium (30 nM), glutamine (2 mM), glucose (6 mg/ml), penicillin/streptomycin (100 U/ml to 100 μg/ml). Cytosine-d-arabinofuranoside (10 μM) was added to the cultures 18 h after plating to avoid the proliferation of non-neuronal elements and was kept for 3 days before medium replacement. This method yields >99% pure neuronal cultures (Di Menna et al., 2013). Cultures at 10–11 days in vitro (DIV), containing mature neurons, were exposed for 24 h to either LY379268 or LY354740 combined or not with LY341495 (all at 1 μM). Control cultures were treated with an equal volume of saline.

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Primary cultures enriched of hippocampal neurons [2]
Cultures (two preparations) were obtained from the hippocampus of 12–13 CD1 mice at postnatal day (PND) 1 and 2. Hippocampi were dissected in Ca2+/Mg2+ free buffer, mechanically dissociated, and then triturated with a fire polished Pasteur pipette to dissociate the tissue into single cells. After centrifugation (1500 × g, 6 min), the cell pellet was resuspended in Neurobasal-B27 medium supplemented 500 mM l-glutamine, and plated at a density of 4 × 105–7 × 105 cells/ml in 24-well multiplates precoated with 0.1 mg/ml poly-d-lysine. Cytosine-d-arabinofuranoside (10 μM) was added to the hippocampal cultures 24 h after plating to avoid the proliferation of non-neuronal elements and was kept for 3 days before medium replacement. This method yields hippocampal neuronal cultures with >85% neurons and about 10–12% of glial cell. Hippocampal cultures were grown in a humidified atmosphere containing 5% CO2 at 37 °C. At 12 DIV, cultures were exposed for 24 h to either LY379268 or LY354740 combined or not with LY341495 (all at 1 μM), while control cultures were treated with an equal volume of saline.

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Differential analysis of gel images and statistics [2]
Protein samples from cortical neurons were obtained from three different culture dishes for each condition (treatment with saline, LY354740 or LY379268). Each protein sample was then analyzed in three technical replicates, obtaining 9 gels for each condition. Image analysis was performed using the Bio-Rad PDQuest software, version 7.1.0. Spot quantity was normalized to the total density in valid spots, defined as spots with a coefficient of variation ≤5% inside each technical replicate group. Normalized spot quantities were then imported in the NCSS software for statistical analysis. A one-way ANOVA test (p ≤ 0.05) was used to test differences in protein expression among the three groups (saline, LY354740 and LY379268), followed by post hoc comparisons made between groups using the Tukey–Kramer multiple comparison test. Statistical significance was set at p < 0.05. Proteins with at least 1.5 fold expression increase or decrease and a p < 0.05 were defined as differentially expressed.

Mice: Experiment 1A involves 30 drug-free testing trials (five trials per day for six days) for wild-type (female: N = 6; male: N = 5) and Gria1^−/− (female: N = 7; male: N = 8) mice. Each animal is then tested on rewarded alternation following an injection of either Eglumegad (LY354740) (15 mg/kg) or vehicle. Animals are given injections, and then they are kept in their home cage for 30 minutes before behavioral testing starts. In the T-maze, ten trials of rewarded alternation are given to each animal. Mice are allowed a maximum of 120 seconds to finish a trial. The animals are retested without receiving any medication at least 24 hours after the initial round of drug testing to make sure the drug has no long-term effects and the mice continue to perform at a high level during alternation. After retesting for twenty-four hours, mice are given ten more trials of rewarded alternation testing, but this time they are administered a drug that they have not previously received. Considering the quantity of mice, every effort is made to counterbalance the order of drug exposure within genotype and sex. All trials in which the animal alternated are counted, along with the amount of time it took to run (sample latency) from the start arm to the food well and the amount of time it took to make a decision (choice latency) during the choice run. Utilizing a stopwatch, the researcher determines latencies. For the duration of the experiment, the researcher is blind to the animals' genotype and drug allocations. A higher dose of Eglumegad (LY354740) (30 mg/kg) or a vehicle is given to distinct groups of male wild-type (N = 7) and Gria1^−/− mice (N = 7) after they undergo the same procedure as in Experiment 1A. The process used in Experiment 1B is then repeated in Experiment 1C to examine the possible effects of increased proactive interference. The drug dose (30 mg/kg) and the mice are used, but a modified testing protocol is used this time, reducing the interval between trials to 20 s. Rats: Two experiments are conducted. In the first experiment, rats kept in their home cages or rats exposed to two hours of immobilization stress in plastic cones are compared for the effects of LY354740 (10 mg/kg, i.p., neutralized to a pH ~ 7.4) or vehicle (0.9% saline neutralized to pH ~ 7.4) (n = 7, cage control/vehicle; n = 7, cage control/LY354740; n = 6, stress/vehicle; n = 6, stress/Eglumegad (LY354740)). In the second experiment, rats given a 2-hour immobilization stress are compared to rats given vehicle in their home cages, or two lower doses of LY354740 (1 and 3 mg/kg, i.p.) or vehicle (n = 4 for all groups). Fifteen minutes before they are placed in plastic cones with the open end tightly closed, all animals receive an injection of either Eglumegad (LY354740) or a vehicle. Every immobilized rat is taken to a quiet room away from the animal colony, put in a plexiglass chamber with bedding on the bottom, and then put right into a plastic cone. The rats are beheaded and placed in the plastic containers; this is done two hours later. After removal, the brains are preserved at -80°C by freezing them on dry ice.

[1]. The group II metabotropic glutamate receptor agonist LY354740 and the D2 receptor antagonist haloperidol reduce locomotor hyperactivity but fail to rescue spatial working memory in GluA1 knockout mice. Eur J Neurosci. 2017 Apr;45(7):912-921.

[2]. Levels of the Rab GDP dissociation inhibitor (GDI) are altered in the prenatal restrain stress mouse model of schizophrenia and are differentially regulated by the mGlu2/3 receptor agonists, LY379268 and LY354740. Neuropharmacology. 2014 Nov:86:133-44.

[3]. Reversal of novelty-induced hyperlocomotion and hippocampal c-Fos expression in GluA1 knockout male mice by the mGluR2/3 agonist LY354740. Neuroscience. 2013 Oct 10:250:189-200.

[4]. The mGlu2/3 receptor agonist LY354740 suppresses immobilization stress-induced increase in rat prefrontal cortical BDNF mRNA expression. Neurosci Lett. 2006 May 8;398(3):328-32.

Eglumetad is a L-alpha-amino acid.
Eglumetad is a L-alpha-amino acid.

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Both a 5-hydroxytryptamine2A (5-HT2A) agonist and immobilization stress previously have been shown to differentially alter brain-derived neurotrophic factor (BDNF) mRNA expression in the neocortex and hippocampus. Both 5-HT2A receptor activation and immobilization stress also increase glutamate release in the rat prefrontal cortex. Given that the metabotropic glutamate2/3 receptor (mGluR2/3) agonist (1S,2S,5R,6S)-2-aminobicyclo[3.1.0] hexane-2,6-dicarboxylate monohydrate (LY354740) suppressed electrophysiological, behavioral and biochemical effects of 5-HT2A receptor activation in the medial prefrontal cortex (mPFC), we assessed the efficacy of the mGluR2/3 agonist in suppressing the stress-induced increase in BDNF mRNA expression. LY35740 (10 mg/kg, i.p.) attenuated the immobilization stress-induced increase in BDNF mRNA expression in the rat mPFC. This result is consistent with the hypothesis that mGlu2/3 agonists may be an efficacious treatment for stress-induced neuropsychiatric syndromes.[4]

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Dysfunctional glutamatergic neurotransmission has been implicated in schizophrenia and mood disorders. As a putative model for these disorders, a mouse line lacking the GluA1 subunit (GluA1-KO) of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor displays a robust novelty-induced hyperlocomotion associated with excessive neuronal activation in the hippocampus. Agonists of metabotropic glutamate 2/3 receptors (mGluR2/3) inhibit glutamate release in various brain regions and they have been shown to inhibit neuronal activation in the hippocampus. Here, we tested a hypothesis that novelty-induced hyperlocomotion in the GluA1-KO mice is mediated via excessive hippocampal neuronal activation by analyzing whether an mGluR2/3 agonist inhibits this phenotypic feature. GluA1-KO mice and littermate wildtype (WT) controls were administered with (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740) (15 mg/kg, i.p.) 30 min before a 2-h exposure to novel arenas after which c-Fos immunopositive cells were analyzed in the hippocampus. LY354740 (15 mg/kg) decreased hyperactivity in male GluA1-KO mice, with only a minimal effect in WT controls. This was observed in two cohorts of animals, one naïve to handling and injections, another pre-handled and accustomed to injections. LY354740 (15 mg/kg) also reduced the excessive c-Fos expression in the dorsal hippocampal CA1 pyramidal cell layer in maleGluA1-KO mice, while not affecting c-Fos levels in WT mice. In female mice, no significant effect for LY354740 (15 mg/kg) on hyperactive behavior or hippocampal c-Fos was observed in either genotype or treatment cohort. A higher dose of LY354740 (30 mg/kg) alleviated hyperlocomotion of GluA1-KO males, but not that of GluA1-KO females. In conclusion, the excessive behavioral hyperactivity of GluA1-KO mice can be partly prevented by reducing neuronal excitability in the hippocampus with the mGluR2/3 agonist suggesting that the hippocampal reactivity is strongly involved in the behavioral phenotype of GluA1-KO mice.[3]

C8H11NO4

185.18

185.068

C, 51.89; H, 5.99; N, 7.56; O, 34.56

176027-90-0

Eglumegad;176199-48-7;Eglumegad hydrochloride

213056

White to off-white solid powder

1.6±0.1 g/cm3

376.4±32.0 °C at 760 mmHg

181.4±25.1 °C

0.0±1.8 mmHg at 25°C

1.619

-1.22

3

5

2

13

290

4

C1C[C@]([C@H]2[C@@H]1[C@@H]2C(=O)O)(C(=O)O)N

VTAARTQTOOYTES-RGDLXGNYSA-N

InChI=1S/C8H11NO4/c9-8(7(12)13)2-1-3-4(5(3)8)6(10)11/h3-5H,1-2,9H2,(H,10,11)(H,12,13)/t3-,4-,5-,8-/m0/s1

(1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid

2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid; 177317-28-1; 2-Aminobicyclo(3.1.0)hexane-2,6-dicarboxylic acid; 176027-90-0; LY366563;

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.)