TABLE 5

Determination of NADPH dependence and irreversibility for P450 enzyme activity inhibition with irosustat and 667-coumarin

For irreversibility testing (dilution test), test compounds were preincubated with HLMs with 25-fold increased microsomal protein concentrations and with NADPH. Incubates were diluted 25-fold before the addition of P450 substrates. During incubations, the actual concentrations of irosustat and 667-coumarin were 2 μM irosustat and 0.2 μM 667-coumarin for CYP2B6 and CYP3A4/5 and 0.2 μM irosustat and 0.02 μM 667-coumarin for CYP1A2. Results are expressed as mean percentages of remaining P450 enzyme activity (n = 3).

P450	Test Compound	+NADPH			−NADPH			With Dilution
P450	Test Compound	0 min	15 min	30 min	0 min	15 min	30 min	0 min	15 min	30 min
		% remaining
CYP1A2	5 μM Irosustat	73	48	31	69	53	43	99	94	94
	0.5 μM 667-Coumarin	65	44	37	61	55	58	104	96	95
	2 μM Furafylline	53	21	14	56	49	52	99	65	55
CYP2B6	50 μM Irosustat	77	69	62	N.D.	62	58	102	98	90
	5 μM 667-Coumarin	82	77	79	N.D.	75	76	113	102	91
	4 μM Thio-TEPA	70	47	<31	N.D.	79	74	90	47	39
CYP3A4/5 (midazolam 1′-hydroxylation)	50 μM Irosustat	86	76	67	N.D.	76	69	121	101	95
	5 μM 667-Coumarin	99	98	88	N.D.	101	104	111	104	103
	50 μM Mifepristone	30	11	10	N.D.	29	33	67	22	14
CYP3A4/5 (testosterone 6β-hydroxylation)	50 μM Irosustat	48	40	36	N.D.	46	38	89	98	86
	5 μM 667-Coumarin	74	69	67	N.D.	80	76	87	107	102
	50 μM Mifepristone	15	<11	<11	N.D.	17	13	73	12	<6

Thio-TEPA, N,N′,N′-triethylenethiophosphoramide; N.D., not done (samples not prepared). The same values as for samples with NADPH (0 min) were assumed.