β-arrestin-biased dopamine D2 receptor

UNC9975, UNC0006, and UNC9994 Induce D2-Mediated β-Arrestin-2 Translocation as Partial Agonists. [1]
To assess the effects of these compounds on recruiting β-arrestin-2 to D2 receptors, we used a D2-mediated β-arrestin-2 translocation Tango assay that is highly sensitive to β-arrestin-2 recruitment. In this assay, UNC9975, UNC0006, and UNC9994 were potent (EC50 < 10 nM) partial agonists for β-arrestin-2 recruitment to D2 receptors, similar to aripiprazole (Fig. 2B). Whereas UNC9975 (Emax = 43%) and UNC0006 (Emax = 47%) were less efficacious than aripiprazole (Emax = 73%), UNC9994 (Emax = 91%) was more efficacious than aripiprazole and approached the activity of the full agonist quinpirole. Haloperidol, a typical antipsychotic (along with all other typical and atypical antipsychotic drugs tested) did not activate D2-mediated β-arrestin-2 translocation. Because HTLA cells (a HEK293-derived cell line stably expressing a tTA-dependent luciferase reporter gene and a β-arrestin-2-TEV fusion protein) transfected with a D2V2-TCS-tTA construct were used in this assay, we tested these compounds in parallel in HTLA cells transfected with a V2-TCS-tTA construct to investigate the possibility that the observed effect was due to compound acting via the V2 tail or some other nonreceptor-mediated arrestin pathway. As expected, UNC9975, UNC0006, aripiprazole, and quinpirole (a positive control for D2) were inactive in V2 receptor-expressing cells whereas arginine vasopressin (a positive control for V2) was a full agonist (Fig. S2A). These results demonstrate that the effect of UNC9975, UNC0006, UNC9994, and aripiprazole observed in D2V2 receptor-expressing cells is due to the compounds acting via the D2 receptor, not at the V2 tail.

UNC9975 and UNC0006 Induce Catalepsy in β-Arrestin-2 Knockout Mice but Not in Wild-Type Mice.[1]br> To compare potential extrapyramidal side effects of UNC9975, UNC0006, and aripiprazole, we evaluated these compounds along with haloperidol (as a positive control) in a standard drug-induced catalepsy model, using wild-type and β-arrestin-2 knockout mice. As shown in Fig. 5 A and B, UNC9975, UNC0006, or aripiprazole (5.0 mg/kg) failed to significantly induce catalepsy in wild-type mice at either 30 or 60 min after treatment, whereas haloperidol (2.0 mg/kg) induced significant catalepsy at both time points. In notable contrast to the results in wild-type animals, the β-arrestin–biased dopamine D2 ligands UNC9975 and UNC0006 significantly induced catalepsy in β-arrestin-2 knockout mice 60 min after treatment. By comparison, aripiprazole, which activates both Gi-coupled and β-arrestin–mediated pathways, did not induce catalepsy in β-arrestin-2 knockout mice or in wild-type mice. Collectively, these results suggest that β-arrestin recruitment and signaling are protective against motoric side effects. [1] Importantly, UNC9975 displayed potent antipsychotic-like activity without inducing motoric side effects in inbred C57BL/6 mice in vivo. Genetic deletion of β-arrestin-2 simultaneously attenuated the antipsychotic actions of UNC9975 and transformed it into a typical antipsychotic drug with a high propensity to induce catalepsy. Similarly, the antipsychotic-like activity displayed by UNC9994, an extremely β-arrestin-biased D(2)R agonist, in wild-type mice was completely abolished in β-arrestin-2 knockout mice[1].

In Vitro Biochemical Assays. [1]
General procedures. Experimental procedures for the secondary radioligand binding and functional (FLIPR) assays for the GPCRs listed in Table 1 (including D1, D3, D4, D5, 5HT2A, 5HT2B, 5HT2C, 5HT1A, and H1) are available online through the Psychoactive Drug Screening Program (PDSP) website: http://pdsp.med.unc.edu/. The PDSP Assay Protocol book is freely available at http://pdsp.med.unc.edu/UNC-CH% 20Protocol%20Book.pdf. The cAMP biosensor assay for 5HT1A (Table 1) was performed in an analogous manner to the procedure described below, except data were normalized to serotonin instead of quinpirole. Protocols of the dopamine D2 radioligand binding, cAMP biosensor, β-arrestin recruitment Tango, β-arrestin recruitment DiscoveRx, bioluminescent resonance energy transfer (BRET), D2 receptor internalization, and pERK assays are detailed below. Human D2L receptors were used for these assays except the BRET assay, whereas mouse D2L receptors were used.

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CHO-D2 Membrane Preparation and Radioligand Binding Assay. [1]
CHOD2 membrane preparation. Cells stably expressing D2L receptors (CHO-D2L) were plated in 15-cm dishes (in DMEM containing 10% FBS) and grown to 90% confluence. Then, cells were washed with PBS, pH 7.4, and harvested by scraping into PBS, pH 7.4. Harvested cells were centrifuged at 1,000 × g for 10 min and then hypotonically lysed by resuspension into ice-cold 50 mM Hepes, 1% BSA, pH 7.4. Membranes were isolated by centrifugation at 21,000 × g for 20 min. The supernatant was removed and the membrane pellets were stored at −80 °C until used for radioligand binding assays

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Radioligand binding assay. [1]
Membranes prepared as above were resuspended to 1 μg protein/μL (measured by Bradford assay using BSA as standard), and 50 μL was added to each well of a polypropylene 96-well plate containing (per well) 50 μL of buffer (20 mM Hepes, 10 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 100 mM N-methyl-D-gluconate, pH 7.4), 50 μL of 1.5 nM [3 H]Nmethylspiperone (final concentration 0.3 nM), and reference or D2 test ligands at various concentrations ranging from 50 pM to 50 μM (final concentrations ranging from 10 pM to 10 μM, triplicate determinations for each concentration of D2 test ligand). After a 1.5-h incubation in the dark at room temperature, the reactions were harvested onto 0.3% PEI-soaked Filtermax GF/A filters and washed three times with ice-cold 50 mM Tris, pH 7.4, using a Perkin-Elmer Filtermate 96-well harvester. The filters were subsequently dried and placed on a hot plate (100 °C), and Melitilex-A scintillant was applied. The filters were then removed from the hot plate and allowed to cool. The filters were counted on a Wallac TriLux microbeta counter (3 min/well). Residual [3 H]N-methylspiperone binding to filtered membranes was plotted as a function of log [reference] or log [D2 test ligand] and the data were regressed using the one-site competition model built into Prism 4.0.

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D2-Mediated cAMP Assay. [1]
HEK293T cells coexpressing the cAMP biosensor GloSensor-22F and hD2L receptors were seeded (10,000 cells/20 μL/well) into white, clear-bottom, tissue culture plates in HBSS, 10% FBS, 20 mM Hepes, pH 7.4. After a 1- to 2-h recovery, cells were treated with 10 μL of 3× test or reference drug prepared in HBSS, 10% FBS, 20 mM Hepes, pH 7.4. After 30 min, cells were treated with 10 μL of 1,200 nM (4×) isoproterenol in 8% (4×) GloSensor reagent. Luminescence per well per second was read on a Wallac TriLux microbeta plate counter. Data were normalized to the isoproterenol response (100%) and the maximal quinpirole-induced inhibition thereof (0%) and regressed using the sigmoidal dose-response function built into GraphPad Prism 4.0. Notably, HEK293T cells expressing the GloSensor-22F alone (no hD2) were assayed in parallel and displayed no inhibition of isoproterenol-stimulated cAMP, either by quinpirole or by the test compounds, suggesting that the effect observed in hD2L-expressing cells was due to compound acting via the recombinant receptor.

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D2 β-Arrestin Recruitment Assay.[1]
Recruitment of β-arrestin to agonist-stimulated D2L receptors was performed using a previously described “Tango”-type assay. Briefly, HTLA cells stably expressing β-arrestin-TEV protease and a tetracycline transactivator-driven luciferase were plated into 15-cm dishes in DMEM containing 10% FBS. Cells were transfected (calcium phosphate) with 20 μg of a D2V2-TCS-tTA construct. The next day, cells were plated in white, clear-bottom, 384-well plates (10,000 cells/well, 50 μL/well) in DMEM containing 1% dialyzed FBS. The following day, the cells were challenged with 10 μL/well of reference agonist (6 μM) or D2 test ligand (6 μM) ± reference agonist prepared in HBSS, 20 mM Hepes (pH 7.4), and 18% DMSO (final ligand concentrations are 1 μM, final DMSO concentration is 3%). After 18 h, the medium was removed and replaced with 1× BriteGlo reagent, and luminescence per well was read using a TriLux plate reader (1 s/well). Data were normalized to vehicle (0%) and quinpirole (100%) controls and regressed using the sigmoidal dose-response function built into GraphPad Prism 4.0.

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D2 β-Arrestin Recruitment DiscoveRx Assay. [1]
Assays using DiscoveRx D2L CHO-K1 PathHunter Express cells were conducted exactly as instructed by the manufacturer. Briefly, cells were thawed, resuspended in the supplied medium, and plated in the furnished plates. Two days later, the cells were challenged with 10× dilutions of agonist (prepared in PBS) for 90 min or 20 h. Next, the detection reagents were reconstituted, mixed at the appropriate ratio, and added to the cells. After 60 min, luminescence per well was measured on a TriLux plate counter. Data were normalized to vehicle (0%) and quinpirole (100%) controls and regressed using the sigmoidal dose-response function built into GraphPad Prism 4.0.

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BRET Assay. [1]
BRET assays were performed as previously described with minor modifications. Briefly, HEK293T cells were transfected by calcium phosphate with D2LR-RLuc alone (for basal BRET measurements) at the same fixed concentration of D2LR-RLuc with a saturating concentration of β-arrestin 2-YFP9 and D2LR-RLuc/β-arrestin 2 YFP with GRK2 overexpressed. Twenty-four hours posttransfection, the cells were plated into poly-D-lysine–coated white 96-well plates at a density of 100,000 cells/well in phenol-red free MEM with 2% FBS. BRET assays were performed 24 h after plating. The compounds were dissolved in DMSO and serially diluted in PBS supplemented with calcium and magnesium. A final concentration of 5 μM coelenterazine h was added to the cells in PBS followed by the compounds. After 10 min incubation, the ratio of YFP (515–555 nm) to RLuc (465–505 nm) was measured by the Mithras LB940 instrument. Basal BRET was subtracted and all results were normalized to the maximum response of quinpirole (10−6 M). All data presented are mean ± SEM of six to nine independent experiments. Graphpad Prism 5 was used to determine EC50 and Emax values.

Flow Cytometric Analysis of D2 Receptor Internalization. [1]
HEK293 cells were triple transfected with FLAG-tagged D2R (SF-D2R), GRK2, and β-arrestin-2 (Arr3). A final concentration of 1 μg/ mL tetracycline was added to the cells to induce expression of GRK2 and Arr3 24 h before final drug treatment. D2R internalization was induced by incubating the cells with a final concentration of 1 μM of each drug for 60 min at 37 °C. Control cells were treated with vehicle. After treatment, the cells were chilled on ice and washed with ice-cold PBS. The cells were first stained with mouse anti-FLAG M2 antibody and then stained with Alexa Fluor-647 rabbit anti-mouse antibody. The expression of D2R on the cell surface was quantified using an Accuri C6 flow cytometer. D2R internalization was defined as the decreased D2R surface expression of the drug-treated cells compared with control cells. Results were analyzed for statistical significance using a one-way ANOVA followed by Tukey’s multiple-comparison posttest, using Graphpad Prism 5.0. Quinpirole induced 33% internalization. Data were normalized to the internalization induced by quinpirole.

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D2 p-ERK Reporter Assay. [1]
HEK293T cells were transfected in 10-cm dishes (4 million cells/dish) with a plasmid encoding SRE-luc2P to monitor MAPK pathways and with expression vectors encoding the indicated proteins. On the next day, the cells were split into white, glass-bottom, poly-Llysine–coated 384-well plates using DMEM containing 1% FBS (15,000 cells/well). The medium was replaced with serum-free DMEM after 24 h. The cells were incubated for 2–4 h in serumfree medium and then stimulated with reference (quinpirole) and test compounds at the indicated concentrations. After a 4-h incubation, the medium was removed and 1× BriteGlo reagent was added. The luminescence was measured on a TriLux plate reader. Notably, HEKT cells transfected with SRE-luciferase and pcDNA3.0 displayed no response to quinpirole or the test compounds. Concentration-response curves were regressed using the three-parameter logistic equation built into GraphPad Prism 4.0. For each condition (transfection group), the best-fit (global) bottom and top for quinpirole were used to normalize the data (scale: 0–100%).

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pERK Immunofluorescence Assay. [1]
Cell culture. [1]
Chinese Hamster Ovary (CHO) cells stably expressing the hD2L dopamine receptor were maintained in Ham’s F-12 medium supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.5 μg/mL G418. On day 1 of the assay, cells were seeded into black clear-bottom tissue culture-treated 96-well plates. On day 2 of the assay, cells were washed with serumfree medium (Ham’s F-12, penicillin, and streptomycin) and incubated in 100 μL serum-free medium overnight.

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Immunofluorescence. [1]
Automatic multichannel pipetters were used for liquid handling and multichannel vacuum manifolds for aspirations. Each tested concentration was typically measured in triplicate or quadruplicate. For concentration curves, half-log dilutions were used. Drug dilutions were prepared in stimulation medium (serum-free medium, 100 mg/L ascorbic acid). Dopamine (100 μM) and 1 μM phorbol 12-myristate 13-acetate (PMA) were used as positive controls. Medium only was used as a negative control. Specificity of the response was confirmed by pretreatment for 5 min with 10 μM of the antagonist spiperone and in experiments using wild-type CHO cells. Cells were stimulated on day 3 by fast addition of 50 μL equilibrated 3× drug dilutions in a tissue culture incubator for 5 min. Plates were placed on ice, the medium was aspirated, and 100 μL/well of freshly prepared ice-cold fixing buffer (4% formaldehyde and 0.5 mM CaCl2 in PBS) solution was added. After 30 min fixing at room temperature, the plates were washed with 350 μL/well PBS/Ca (0.5 mM CaCl2 in PBS) and permeabilized for 20 min in 100 μL/well 0.3% Triton X-100 in PBS/Ca. Plates were incubated in 100 μL/well blocking buffer (PBS/ Ca, 5% goat serum, 0.1% Triton X-100) for 1 h at room temperature and then in blocking buffer containing rabbit phosphoThr202/p-Tyr204-ERK antibody (Cell Signaling 9101) at 1:1,000 at 4 °C overnight. Cells were washed three times for 5–10 min with 250 μL/well wash buffer (PBS/Ca, 0.03% Triton X-100). Plates were incubated with 50 μL/well Alexa-594–coupled goat anti-rabbit IgG at 1:250, 5 μg/mL Hoechst 33342 nuclear stain, and 25 μg/mL concanavaline-Alexa488 conjugate (ConA; Invitrogen) in blocking buffer for 2 h at room temperature. Plates were washed three times with 250 μL/well washing buffer, postfixed for 10 min in fixing buffer, washed with 250 μL PBS/Ca, filled with 200 μL/well PBS/Ca, sealed with transparent adhesive plate seals, and stored at 4 °C.

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Microscopy and Image Analysis. [1]
Plates were scanned with a highcontent automated microscopic system, using a 20× objective and a 2 × 2 image montage setting. The Alexa594 light path was used for the target signal, Alexa488 for whole-cell staining, and Hoechst for the nuclear staining. Images were analyzed using CellProfiler software. Well-averaged individual cellbased measurements were exported to Excel and cell-free background intensity was subtracted from the whole-cell intensity. Concentration curves were analyzed in GraphPad Prism by fitting against a sigmoidal dose-response model and normalization to the dopamine curve.

Catalepsy testing. [1]
In this paradigm, mice were initially injected (i.p.) with vehicle (0.9% saline/0.2% acetic acid); with 5 mg/kg each of aripiprazole, UNC0006, or UNC9975; or with 2 mg/kg haloperidol and returned to their home cage. The latency of movement was assessed 30, 60, 90, and 120 min after drug injection and the maximal latency to move in the mice was observed 60 min after drug treatments. Therefore, we focused all drug catalepsy studies on this 60-min time point. Mice were placed upright on a 45° angled screen. The time required for the animal to move all four paws was scored in seconds (maximum of 5 min) and is reported as the latency to movement. An extended delay to voluntarily move on the inclined screen test is indicative of drug-induced catalepsy. The data are displayed as mean ± SEM. Catalepsy data were analyzed using a one-way ANOVA followed by a Newman–Keuls multiple-comparison test, using Graphpad Prism 5.0.

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In Vivo Studies in Mice. [1]
C57BL/6J wild-type and β-arrestin-2 knockout mice were housed under standard conditions: 12-h light/dark cycle with food and water provided ad libitum. Adult, age-matched male and female wild-type and β-arrestin-2 knockout drug-naive mice were used for all behavioral testing. Locomotor activity was assessed under standardized environmental conditions in 21 × 21-cm Plexiglas chambers with photobeams spaced at 2.5 cm s previously described. Mice were injected (i.p.) with vehicle (0.9% saline/0.2% acetic acid), aripiprazole (0.1, 0.25, 0.50, or 2.0 mg/kg), or UNC9975 (0.25, 0.50, or 2.0 mg/kg) and placed into the open field. For the studies with SR46349B and clozapine, mice were injected (i.p.) with vehicle (0.9% saline/50 mM tartaric acid), SR46349B (1.0 mg/kg), or clozapine (1.0 mg/kg) and placed into the open field. Thirty minutes later d-amphetamine (3 mg/kg) or phencyclidine (6.0 mg/kg) was administered and mice were immediately returned to the open field. Activity was monitored throughout this entire period. Horizontal activity was measured as the total distance traveled in centimeters. The means ± SEMs of the locomotor responses were analyzed using Graphpad Prism 5.0. To estimate the half-maximal inhibitory concentration (ED50), dose responses of total locomotor activity during the 90-min period after d-amphetamine or phencyclidine administration were plotted and best-fit decay curves were determined using a nonlinear regression one-phase decay equation.

In mouse pharmacokinetic (PK) studies, both UNC9975 and aripiprazole displayed high exposure levels in brain and excellent CNS penetration. Although the brain exposure level of UNC9975 was about threefold lower, UNC9975 had a longer half-life in brain and a higher brain/plasma ratio over 24 h compared with aripiprazole. The excellent in vivo PK parameters of UNC9975 make it a suitable tool for in vivo pharmacodynamic studies.

[1]. Discovery of β-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy. Proc Natl Acad Sci U S A. 2011 Nov 8;108(45):18488-93.

Elucidating the key signal transduction pathways essential for both antipsychotic efficacy and side-effect profiles is essential for developing safer and more effective therapies. Recent work has highlighted noncanonical modes of dopamine D(2) receptor (D(2)R) signaling via β-arrestins as being important for the therapeutic actions of both antipsychotic and antimanic agents. We thus sought to create unique D(2)R agonists that display signaling bias via β-arrestin-ergic signaling. Through a robust diversity-oriented modification of the scaffold represented by aripiprazole (1), we discovered UNC9975 (2), UNC0006 (3), and UNC9994 (4) as unprecedented β-arrestin-biased D(2)R ligands. These compounds also represent unprecedented β-arrestin-biased ligands for a G(i)-coupled G protein-coupled receptor (GPCR). Significantly, UNC9975, UNC0006, and UNC9994 are simultaneously antagonists of G(i)-regulated cAMP production and partial agonists for D(2)R/β-arrestin-2 interactions. Importantly, UNC9975 displayed potent antipsychotic-like activity without inducing motoric side effects in inbred C57BL/6 mice in vivo. Genetic deletion of β-arrestin-2 simultaneously attenuated the antipsychotic actions of UNC9975 and transformed it into a typical antipsychotic drug with a high propensity to induce catalepsy. Similarly, the antipsychotic-like activity displayed by UNC9994, an extremely β-arrestin-biased D(2)R agonist, in wild-type mice was completely abolished in β-arrestin-2 knockout mice. Taken together, our results suggest that β-arrestin signaling and recruitment can be simultaneously a significant contributor to antipsychotic efficacy and protective against motoric side effects. These functionally selective, β-arrestin-biased D(2)R ligands represent valuable chemical probes for further investigations of D(2)R signaling in health and disease.[1]

C23H28CL2N4O2

463.403

462.1589

C, 59.61; H, 6.09; Cl, 15.30; N, 12.09; O, 6.91

1354030-19-5

56593482

Typically exists as solid at room temperature

4.6

O=C1NC2=NC(OCCCCN3CCN(C4=CC=CC(Cl)=C4Cl)CCC3)=CC=C2CC1

JQSRFMXTGAVHIR-UHFFFAOYSA-N

InChI=1S/C23H28Cl2N4O2/c24-18-5-3-6-19(22(18)25)29-13-4-12-28(14-15-29)11-1-2-16-31-21-10-8-17-7-9-20(30)26-23(17)27-21/h3,5-6,8,10H,1-2,4,7,9,11-16H2,(H,26,27,30)

7-[4-[4-(2,3-dichlorophenyl)-1,4-diazepan-1-yl]butoxy]-3,4-dihydro-1H-1,8-naphthyridin-2-one

UNC9975; CHEMBL2165119; 1354030-19-5; 7-(4-(4-(2,3-dichlorophenyl)-1,4-diazepan-1-yl)butoxy)-3,4-dihydro-1,8-naphthyridin-2(1h)-one; 7-{4-[4-(2,3-dichlorophenyl)-1,4-diazepan-1-yl]butoxy}-1,2,3,4-tetrahydro-1,8-naphthyridin-2-one; 7-(4-(4-(2,3-dichlorophenyl)-1,4-diazepan-1-yl)butoxy)-1,2,3,4-tetrahydro-1,8-naphthyridin-2-one; SCHEMBL252358;

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.)