Application Data (P450)

Application Data

- Examination of inhibition on CYP3A4, one of P450 molecular species -

Outline

IC₅₀ in CYP3A4 is examined when Ketoconazole is applied as inhibitor.

Procedure of measurement
Reagent
	Substrate	:	BFC (7-Benzyloxy-4-(trifluoromethyl)-coumarin) Final Concentration: 50µM
	Inhibitor	:	Kotoconazole (3-fold Serial dilution from Final Conc.:5µM to 0.00229µM)
	Expression system	:	Human CYP3A4 (Nosan Corporation) : 1 pmol/well
	NADPH Production system	:	Final Conc.3.3mM G-6-P, 3.3mM MgCl₂•6H₂O, 1.3mM NADP, 0.4U/ml G-6-PDH
	Buffer	:	200mM K-PO4 , 0.1mM EDTA (pH7.4)
	Metabolite	:	HFC (7-Hydroxy-4-(trifluoromethyl)-coumarin)

Procedure

150µ of Phosphoric bufferwhich contains NADPH-producing-system-containing dissolved in acetonitrile into A1 and B1 well
Phosphoric buffer which contains NADPH production system of specified conc. of BFC desolved in acetonitrile shall be dispensed into wells of C1, D1 and E1, each 150µl.
NADPH producing Phosphoric buffer which contains specified conc. of acetonitrile shall be dispensed into each well among A2 to A10, B2 to B10, C2 to C10, D2 to D10 and E2 to E10, each 100µl.
50µL of sample shall be flactionated from 1st line, and it shall be dispensed dispensed into2nd line. After mixing the content of the well by pipetting, 50µL content in 2nd line shall be dispensed into 3rd line. 3-fold serial dilution shall be done until 8th line. 50µL of reagent flactionated from the 8th line shall be discarded.
Preincubation under 37ºC and mixing for 10 minutes
100µL Specified Conc. of CYP3A4 shall be dispensed into 1st to 9th line.
Incubation under 37ºC and mixing for 30 minutes
30 minutes after, 75µL of quencher acetonitrile shall be added.
During mixing,100µL of CYP3A4 with specified Conc. shall be each well among 10th line.

Reading setting

Result
Data 1 : Dilute series of Metabolite


Data 2 : Detection of IC 50

Conclusion
IC ₅₀ of Ketoconazole in human CYP3A4 is worked out to 0.05µM.