| In Vitro | In vitro activity: Fenofibrate is a relatively potent inhibitor of CYP2B6 (IC50=0.7±0.2 μM) and CYP2C19 (IC50=0.2±0.1 μM). Fenofibrate is also a moderate inhibitor of CYP2C8 (IC50=4.8±1.7 μM) and CYP2C9 (IC50=9.7 μM).
Fenofibrate binds to and inhibits cytochrome P450 epoxygenase (CYP)2C
with higher affinity than to PPARα. Fenofibrate is a well-known PPARα
agonist, but an in vitro assessment of 209 frequently prescribed drugs
and related xenobiotics suggests that Fenofibrate is also a potent
inhibitor of cytochrome P450 epoxygenase (CYP)2C. The affinity of
Fenofibrate to CYP2C is>10 times higher (EC50=2.39±0.4 μM) than to PPARα (EC50=30 μM). Fenofibrate at a low dose inhibits CYP2C8 activity without PPARα activation.
Kinase Assay: 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
Cell Assay: |
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| Formulation & Dosage | 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. |
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