Raw 264.7 cells (100,000) are grown in 96-well plates. After 24 h, cells are stimulated with vehicle (final concentration of 0.025% DMSO) or with Apremilast at the indicated concentrations. After 30 minutes cells are stimulated with LPS 1 μg/mL for 4 h. When studying CGS21680 , SCH58261, ZM241385, BAY60-6583, or GS6201, the adenosine receptor ligands are added 15 minutes before Apremilast. Methotrexate is added 24 h and 1 h before Apremilast. Supernates are then collected and TNF-α levels are quantified with the Mouse TNF-α Quantikine ELISA Kit. IC₅₀ (EC₅₀) calculations are made using non-linear regression, sigmoidal dose-response, constraining the top to 100 % and bottom to 0 %, allowing variable slope, using GraphPad Prism v6.00^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
Male mice are given weekly intraperitoneal injections of either MTX (1 mg/kg) or vehicle (PBS) for 4 weeks. Air pouches are generated by subcutaneous injection of 3 mL of sterile air and reinflated with 1.5 mL of sterile air 2 days later. Vehicle (0.5 % carboxymethylcellulose and 0.25 % Tween 80) or Apremilast (5 mg/kg) are orally dosed, with a syringe through a blunt-ended curved feeding tube, 24 h and 1 h before inflammation is induced on day 6 by injection of 1 mL of 2 % carrageenan suspension. Four hours later, mice are killed by CO₂ narcosis, and exudates harvested with 2 mL PBS. Leukocytes are counted in a hemocytometer chamber and concentrations of cytokines are measured by ELISA or by the Luminex platform.
Rats^[2]
Male Sprague Dawley rats (180-220 g) are used to study the pharmacokinetics of Apremilast. Diet is prohibited for 12 h before the experiment, but water is freely available. Blood samples (0.3 mL) are collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8 and 12 h after oral administration of Apremilast (6.0 mg/kg). The samples are immediately centrifuged at 4,000 g for 8 min. The plasma obtained (100 μL) is stored at ?20°C until analysis. Plasma Apremilast concentration versus time data for each rat is analyzed by DAS (Drug and statistics) software.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Perez-Aso M, et al. Apremilast, a novel phosphodiesterase 4 (PDE4) inhibitor, regulates inflammation through multiple cAMP downstream effectors. Arthritis Res Ther. 2015 Sep 15;17:249.  [Content Brief]

  [2]. Chen LG, et al. Determination of Apremilast in Rat Plasma by UPLC-MS-MS and Its Application to a Pharmacokinetic Study. J Chromatogr Sci. 2016 Sep;54(8):1336-40.  [Content Brief]