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UNC9994 HCl

Alias: UNC 9994 hydrochloride; 1354030-51-5; 2108826-33-9; UNC9994 (hydrochloride); UNC9994hydrochloride; UNC9994 hydrochloride; 5-[3-[4-(2,3-dichlorophenyl)piperidin-1-yl]propoxy]-1,3-benzothiazole;hydrochloride;
Cat No.:V44438 Purity: ≥98%
UNC9994 HCl is a functionally selective β-arrestin-biased dopamine D2 receptor (D2R) agonist that selectively activates β-arrestin recruitment and signaling.
UNC9994 HCl
UNC9994 HCl Chemical Structure CAS No.: 2108826-33-9
Product category: New3
This product is for research use only, not for human use. We do not sell to patients.
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Other Forms of UNC9994 HCl:

  • UNC9994
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Top Publications Citing lnvivochem Products
Product Description
UNC9994 HCl is a functionally selective β-arrestin-biased dopamine D2 receptor (D2R) agonist that selectively activates β-arrestin recruitment and signaling. The Ki for D2R is 79 nM. UNC9994 HCl is also an antagonist of Gi-regulated cAMP production and a partial agonist of the D2R/β-arrestin-2 interaction. UNC9994 HCl has psychoactive effects.
Biological Activity I Assay Protocols (From Reference)
Targets
β-arrestin-biased dopamine D2 receptor; D3 Receptor (Ki = 17 nM); 5-HT2B Receptor (Ki = 25 nM); 5-HT1A Receptor (Ki = 26 nM); D2 Receptor (Ki = 79 nM); D4 Receptor (Ki = 138 nM); 5-HT2A Receptor (Ki = 140 nM); 5-HT2C Receptor (Ki = 512 nM); D1 Receptor (Ki = 4000 nM);
ln Vitro
UNC9994 Hydrochloride causes D2-mediated β-arrestin-2 translocation with EC50 values of 6.1 nM and 448 nM in Tango assay and DiscoveRx assay, respectively [1]. In contrast to being an agonist of 5HT2C and 5HT1A, UNC9994 hydrochloride is an antagonist of 5HT2A and 5HT2B [1].
ln Vivo
UNC9994 (2.0 mg/kg; intraperitoneal; once) hydrochloride displays antipsychotic action that is reduced in β-arrestin-2 knockout mice [1].
Enzyme Assay
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.
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
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.
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.
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.
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.
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.
Cell Assay
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.
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%).
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.
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.
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.
Animal Protocol
Animal/Disease Models: C57BL/6J wild-type and β-arrestin-2 knockout mice [1]
Doses: 2.0 mg/kg, followed 30 minutes later by 6 mg/kg phencyclidine (PCP, intraperitoneal (ip) injection) Method: intraperitoneal (ip) injection, one time.
Experimental Results: Dramatically inhibited PCP-induced excessive locomotion in wild-type mice, and this activity was completely eliminated in β-arrestin-2 knockout mice.
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 (An analog of UNC9994); 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.
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 (An analog of UNC9994) (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.
ADME/Pharmacokinetics
In mouse pharmacokinetic (PK) studies, both UNC9975 (An analog of UNC9994) 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.
References

[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.

Additional Infomation
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]
These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C21H23CL3N2OS
Molecular Weight
457.84
Exact Mass
456.059
CAS #
2108826-33-9
Related CAS #
UNC9994;1354030-51-5
PubChem CID
121230971
Appearance
White to off-white solid powder
Hydrogen Bond Donor Count
1
Hydrogen Bond Acceptor Count
4
Rotatable Bond Count
6
Heavy Atom Count
28
Complexity
464
Defined Atom Stereocenter Count
0
SMILES
C1(OCCCN2CCC(CC2)C2=C(C(Cl)=CC=C2)Cl)=CC2=C(C=C1)SC=N2.Cl
InChi Key
MTDQOQYYKZUEEK-UHFFFAOYSA-N
InChi Code
InChI=1S/C21H22Cl2N2OS.ClH/c22-18-4-1-3-17(21(18)23)15-7-10-25(11-8-15)9-2-12-26-16-5-6-20-19(13-16)24-14-27-20;/h1,3-6,13-15H,2,7-12H2;1H
Chemical Name
5-[3-[4-(2,3-dichlorophenyl)piperidin-1-yl]propoxy]-1,3-benzothiazole;hydrochloride
Synonyms
UNC 9994 hydrochloride; 1354030-51-5; 2108826-33-9; UNC9994 (hydrochloride); UNC9994hydrochloride; UNC9994 hydrochloride; 5-[3-[4-(2,3-dichlorophenyl)piperidin-1-yl]propoxy]-1,3-benzothiazole;hydrochloride;
HS Tariff Code
2934.99.9001
Storage

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.
Shipping Condition
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
Solubility Data
Solubility (In Vitro)
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
Solubility (In Vivo)
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.

Injection Formulations
(e.g. IP/IV/IM/SC)
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 : PEG300Tween 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 : PEG300Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH 400 μLPEG300 50 μL Tween 80 450 μL Saline)


Oral Formulations
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


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.

 (Please use freshly prepared in vivo formulations for optimal results.)
Preparing Stock Solutions 1 mg 5 mg 10 mg
1 mM 2.1842 mL 10.9208 mL 21.8417 mL
5 mM 0.4368 mL 2.1842 mL 4.3683 mL
10 mM 0.2184 mL 1.0921 mL 2.1842 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

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Method for preparing DMSO stock solution mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.

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