Fenofibrate 

The half-maximal inhibitory concentrations (IC₅₀s) of Fenofibrate, statins (atorvastatin, lovastatin, pravastatin, simvastatin and simvastatin acid, the active form of simvastatin) and glipizide for recombinant human CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 are determined using fluorometric CYP450 inhibition assays. Briefly, the drugs are dissolved in methanol or acetonitrile. In 96 well assay plates, the drugs are diluted to a series of concentrations in a solution containing cofactors including NADP⁺ (final concentration 1.3 mM), MgCl₂ (final concentration 3.3 m M), glucose-6-phosphate (G6P, final concentration 3.3 mM) and glucose 6-phosphate dehydrogenase (final concentration 0.4 U/mL). The mixture is pre-incubated at 37°C for 10 min. The enzymes and fluorogenic substrates are diluted to desired concentrations in sodium phosphate reaction buffer (pH 7.4, final concentration 200 mM) and mixed. Reactions are initiated with addition of the enzyme and substrate mixture to the cofactor and drug mixture. The final reaction volume of all assays is 200 μL. After incubating at 37°C for a pre-specified period of time (15 to 45 min), the reactions are stopped with addition of 75 μL quenching solution (0.5 M Tris base or 2N NaOH). Fluorescence is determined using a BioTek Synergy 2 fluorescence reader. Each of the drugs is tested at eight concentrations in duplicate. To estimate IC₅₀s, percent of inhibition is calculated using net fluorescence that is corrected for the background. The values of percent of inhibition are then fitted to a three or four parameter log-logistic model^[1].

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

Mice^[2]
The mouse oxygen-induced retinopathy (OIR) model is used. Briefly to induce retinal neovascularization, mouse pups and their nursing mother are exposed to 75±3% oxygen from P7 to P12. For the higher dose Fenofibrate (F6020) treatment (100 mg/kg/day). Fenofibrate is dissolved in corn oil to make 100mg/mL solution and pure corn oil is used as vehicle control. For the lower dose treatment (10 mg/kg/day), Fenofibrate is dissolved in 10% DMSO, D2650 to make a 10 mg/mL solution and 10% DMSO is used as vehicle control. After return to room air, mice are orally gavaged with Fenofibrate (100 or 10 mg/kg) or vehicle control daily from P12 to P16. At P17, eyes are enucleated immediately after euthanasia and fixed in 4% paraformaldehyde in PBS for 1 h at room temperature. Retinas are then dissected and stained overnight with Alexa Fluor 594 conjugated isolectin GS-IB4 (10 μg/mL) at room temperature. After washing with PBS, retinas are mounted onto microscope slides with photoreceptor side down and embedded in SlowFade antifade mounting medium. Retinal images are taken using a fluorescence microscope with image software. Retinal neovascularization is analyzed.

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

• [1]. Schelleman H, et al. Pharmacoepidemiologic and in vitro evaluation of potential drug-drug interactions of sulfonylureas with fibrates and statins. Br J Clin Pharmacol. 2014 Sep;78(3):639-48.  [Content Brief]

  [2]. Gong Y, et al. Fenofibrate Inhibits Cytochrome P450 Epoxygenase 2C Activity to Suppress Pathological Ocular Angiogenesis. EBioMedicine. 2016 Sep 30. pii: S2352-3964(16)30448-0.  [Content Brief]