SphK2 ( IC50 = 60 μM )

ABC294640 (100 mg/kg, p.o.) significantly reduces tumor growth in mice with mammary adenocarcinoma xenografts; this effect is linked to S1P depletion. ABC294640 increases autophagy markers and delays tumor growth in mice with severe combined immunodeficiency carrying A-498 xenografts. ABC294640 enhances liver function and survival by preventing inflammation brought on by liver transplantation and the interaction between innate and adaptive immunity, two key processes that cause and exacerbate graft injury.

An HPLC-based SK activity assay that was recently developed is used to determine the IC50 values for ABC294640 and DMS. The test compounds are, in short, incubated with recombinant SK1 or SK2 and NBD-Sph in the isozyme-selective assay buffers described below, containing 400 μM MgCl2, 100 μM ATP, and 1 mg/ml fatty acid-free bovine serum albumin. The following is how HPLC separates the product, or NBD-S1P, from NBD-Sph: Utilizing a Waters 2495 fluorescence detector, a C8 Chromolith RP-8e column (100 × 4.6 mm) and a 1 ml/min mobile phase (pH 2.5 sodium phosphate buffer with acetonitrile/20 mM) at 45:55 make up the Waters 2795 HPLC system. Fluorescence is observed with excitation at 465 nm and emission at 531 nm. The NBD-S1P/(NBD-Sph + NBD-S1P) ratio is used to calculate the level of SK activity. 20 mM Tris, pH7.4, 5 mM EDTA, 5 mM EGTA, 3 mM β-mercaptoethanol, 5% glycerol, 1× protease inhibitors, and 1× phosphatase inhibitors were all present in each SK-isozyme selective assay buffer. 0.25% (final) Triton X-100 is added to the SK1 assay buffer, and 1 M (final) KCl is added to the SK2 buffer. The kinase reaction is stopped by adding 1.5 volumes of methanol after the assays have been running for two hours at room temperature. The samples are centrifuged at 20,000 g for 10 minutes to remove the precipitated protein, and the supernatants are then subjected to HPLC analysis. The ADP Quest assay system is used to measure kinase activity in the presence of different concentrations of sphingosine and ABC294640 in experiments to determine the Ki for inhibition of SK2 by ABC294640. In order to ascertain the impact of ABC294640 on cellular SK activity, near-confluent MDA-MB-231 cells undergo an overnight serum starvation protocol followed by exposure to different concentrations of ABC294640. Next, [3H]sphingosine is added to the cells at a final concentration of 1 μM. The cells take up the exogenous sphingosine, which is converted to S1P via SK activity, and [3H]S1P is separated from [3H]sphingosine by extraction and quantified by scintillation counting.

In order to ascertain the impact of the test compounds on proliferation, 96-well microtiter plates are seeded with cells (1025LU, Hep-G2, A-498, MCF-7, Caco-2, MDA-MB-231, HT-29, Panc-1, DU145, T24, and SK-OV-3 cell lines) and left to adhere for a full day. Separate wells are filled with varying concentrations of ABC294640, and the cells are incubated for a further 72 hours. Using the sulforhodamine-binding assay, the number of viable cells is ascertained at the conclusion of this time. As a percentage of sulforhodamine-binding compared to control cultures, the percentage of cells killed is computed. GraphPad Prism is used to perform regression analyses of inhibition curves.

[1]. J Pharmacol Exp Ther . 2010 Apr;333(1):129-39.

[2]. J Pharmacol Exp Ther . 2010 May;333(2):454-64.

C23H25CLN2O

380.91

380.17

915385-81-8

1185157-59-8 (HCl); 915385-81-8

White solid powder

C1C2CC3(CC1CC(C2)(C3)C(=O)NCC4=CC=NC=C4)C5=CC=C(C=C5)Cl

CAOTVXGYTWCKQE-UHFFFAOYSA-N

InChI=1S/C23H25ClN2O/c24-20-3-1-19(2-4-20)22-10-17-9-18(11-22)13-23(12-17,15-22)21(27)26-14-16-5-7-25-8-6-16/h1-8,17-18H,9-15H2,(H,26,27)

3-(4-chlorophenyl)-N-(pyridin-4-ylmethyl)adamantane-1-carboxamide

ABC294640; ABC 294640; ABC-294640; Trade name Yeliva

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)

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.)