A High Throughput Method for Measuring
Cytochrome P450 Inhibition
(Version 4.2: Revised 27 September 2000)
SUMMARY
Summary | Materials
| Solutions
| Instrument
Evaluation | Assay
Procedure | Appendices
This technical bulletin provides a detailed, step-by-step
procedure for the performance of the high throughput cytochrome P450 inhibition
testing method reported in Analytical Biochemistry 248 (1997)
188-190 and further refined by personnel at BD Biosciences. In this document,
we also provide the rationale for some of the recommended procedures and
provide alerts to areas of difficulties which may be encountered. This
bulletin describes the methodology for CYP1A2, CYP2A6, CYP2B6, CYP2C8,
CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 assays.
There are two procedures described in this technical bulletin.
The first procedure is for evaluating the sensitivity of your fluorescent
plate scanner. It is recommended to perform this procedure once for each
enzyme prior to attempting to determine an IC50 for a test
compound. This procedure will determine if an adequate signal to noise
ratio is detected by your fluorescent plate scanner to perform an IC50
by this method. The second procedure is for an IC50 determination
for a test compound.
BD Gentest also performs this assay for clients as
one of our contract
research services.
Final Assay Concentrations and Kinetic Properties
(Based on reaction volume of 200 uL/Well)
| - |
Cytochrome P450 Enzyme |
| REAGENT
|
CYP1A2
|
CYP2A6
|
CYP2B6 |
CYP2C8
|
CYP2C9
|
| NADPH Regenerating
System |
| NADP+ |
1.3 mM |
0.065 mM |
1.3 mM |
1.3 mM |
1.3 mM |
| Glucose-6-Phosphate |
3.3 mM |
3.3 mM |
3.3 mM |
3.3 mM |
3.3 mM |
Magnesium Chloride
Hexahydrate |
3.3 mM |
3.3 mM |
3.3 mM |
3.3 mM |
3.3 mM |
| Glucose-6-Phosphate Dehydrogenase |
0.4 Units/mL |
0.4 Units/mL |
0.4 Units/mL |
0.4 Units/mL |
0.4 Units/mL |
| Other Reagents |
| KPO4 pH 7.4 |
100 mM |
None |
100 mM |
50 mM |
25 mM |
| Tris pH 7.5 |
None |
100 mM |
None |
None |
None |
Positive Control
(highest concentration) |
Furafylline
100 µM |
Tranylcypromine
25 µM |
Tranylcypromine
125 µM |
Quercetin
20 µM |
Sulfaphenazole
10 µM |
| Substrate |
CEC
5 µM |
Coumarin
3 µM |
EFC
2.5 µM |
DBF
1 µM |
MFC
75 µM |
DBF
1 µM
|
| Enzyme* |
0.5 pmol
(456203 (Old P203)) |
1.0 pmol
(456254 (Old P254)) |
1.0 pmol
(456255 (Old P255)) |
4.0 pmol
(456252 (Old P252)) |
1.0 pmol
(456258 (Old P258)) |
2.0 pmol
(456258 (Old P258)) |
| Pluronic F68 |
None |
None |
None |
None |
None |
None |
| Kinetic Properties |
| Apparent Km |
3.5 µM |
1.2 µM |
2.4 µM |
1.0 µM |
78 µM |
0.73 µM |
| Apparent Vmax |
3.4 min-1 |
33 min-1 |
4.5 min-1 |
0.4 min-1 |
2.1 min-1 |
0.19 min-1 |
| Incubation Time |
15 min |
15 min |
30 min |
30 min |
45 min |
30 min |
| - |
Cytochrome P450 Enzyme |
| REAGENT
|
CYP2C19
|
CYP1A1 |
CYP1B1 |
CYP19 |
| NADPH Regenerating
System |
| NADP+ |
1.3 mM |
1.3 mM |
1.3 mM |
1.3 mM |
| Glucose-6-Phosphate |
3.3 mM |
3.3 mM |
3.3 mM |
3.3 mM |
Magnesium Chloride
Hexahydrate |
3.3 mM |
3.3 mM |
3.3 mM |
3.3 mM |
| Glucose-6-Phosphate Dehydrogenase |
0.4 Units/mL |
0.4 Units/mL |
0.4 Units/mL |
0.4 Units/mL |
| Other Reagents |
| KPO4 pH 7.4 |
50 mM |
100 mM |
100 mM |
100 mM |
100 mM |
100 mM |
| Tris pH 7.5 |
None |
None |
None |
None |
Positive Control
(highest concentration) |
Tranylcypromine
500 µM |
a-naphthoflavone 10 µM |
a-naphthoflavone 10 µM |
4-OH
Androstendione
1 µM
|
| Substrate |
CEC
25 µM |
DBF
2 µM |
OMF
2 µM |
BzRes/12.5 µM
|
BzRes/12.5 µM |
DBF/40 µM |
| Enzyme* |
0.5 pmol
(456259 (Old P259)) |
1.0 pmol
(456259 (Old P259)) |
1.0 pmol
(456259 (Old P259)) |
2.5 pmol (456211 old P211) |
2.5 pmol (456220 old P220) |
0.4 pmol (456260 old P260) |
| Pluronic F68 |
None |
None |
None |
None |
None |
None |
| Kinetic Properties |
| Apparent Km |
29 µM |
1.6 µM |
1.2 µM |
12.4 µM |
13.0 µM |
0.19 µM |
| Apparent Vmax |
0.016 min-1 |
1.1 min-1 |
2.1 min-1 |
0.72 min-1 |
5.1 min-1 |
3.8 min-1 |
| Incubation Time |
30 min |
30 min |
30 min |
30 min |
15 min |
30 min
|
| -
|
Cytotochrome P450 Enzyme |
| REAGENT
|
CYP2D6
|
CYP2E1
|
CYP3A4
|
CYP3A5 |
CYP3A7 |
| NADPH Regenerating
System |
| NADP+ |
8.2 µM |
1.3 mM |
1.3 mM |
1.3 mM |
1.3 mM |
| Glucose-6-Phosphate |
0.41 mM |
3.3 mM |
3.3 mM |
3.3 mM |
3.3 mM |
Magnesium Chloride
Hexahydrate |
0.41 mM |
3.3 mM |
3.3 mM |
3.3 mM |
3.3 mM |
| Glucose-6-Phosphate Dehydrogenase |
0.4 Units/mL |
0.4 Units/mL |
0.4 Units/mL |
0.4 Units/mL |
0.4 Units/mL |
| Other Reagents |
| KPO4 pH 7.4 |
100 mM |
100 mM |
200 mM |
200 mM |
200 mM |
Positive Control
(highest concentration) |
Quinidine
0.5 µM |
DDTC
100 µM |
Ketoconazole
5 µM |
Ketoconazole
5 µM |
Ketoconazole
5 µM |
| Substrate |
AMMC
1.5 µM |
MAMC
25 µM |
MFC
100 µM |
BzRes
50 µM |
7-BQ
40 µM |
BFC
50 µM |
DBF
1 µM |
BFC
50 µM |
BFC
50 µM |
| Enzyme* |
1.5 pmol
(456217 (Old P217)) |
1.5 pmol
(456217 (Old P217)) |
2.0 pmol
(456206 (Old P206)) |
3.0 pmol
(456202 (Old P202)) |
3.0 pmol
(456202 (Old P202)) |
1.0 pmol
(456202 (Old P202)) |
0.5 pmol
(456202 (Old P202)) |
4.0 pmol
(456235)
|
5.0 pmol
(456237)
|
| Pluronic F68 |
None |
None |
0.01% |
None |
None |
None |
None |
None |
| Kinetic Properties |
| Apparent Km |
1 µM |
8.0 µM |
70 µM |
38 µM |
38 µM |
>200 µM |
1.0 µM |
102 µM |
73 µM
|
| Apparent Vmax |
1 min-1 |
1.4 min-1 |
4.1 min-1 |
0.3 min-1 |
44 min-1 |
1.5 min-1
@40 µM |
22 min-1 |
5.9 min-1 |
1.0 min-1 |
| Incubation Time |
30 min |
60 min |
45 min |
30 min |
30 min |
30 min |
10 min |
30 min |
45 min |
* BD Gentest™ catalog number in parentheses.
|
Fluorescent Detection Parameters
|
|
Enzyme
|
Substrate
|
Product
|
Excitation
(Bandwidth of filter)
|
Emission
(Bandwidth of filter)
|
|
CYP1A2
|
CEC
|
CHC
|
409 nm (20 nm)
|
460 nm (40 nm)
|
|
CYP2A6
|
Coumarin
|
7-HC
|
390 nm (20 nm)
|
460 nm (40 nm)
|
|
CYP2B6
|
EFC
|
HFC
|
409 nm (20 nm)
|
530 nm (25 nm)
|
|
CYP2C8
|
DBF
|
Fluorescein
|
485 nm (20 nm)
|
538 nm (25 nm)
|
|
CYP2C9
|
7-MFC
|
HFC
|
409 nm (20 nm)
|
530 nm (25 nm)
|
| DBF |
Fluorescein
|
485 nm (20 nm)
|
538 nm (25 nm)
|
|
CYP2C19
|
CEC
|
CHC
|
409 nm (20 nm)
|
460 nm (40 nm)
|
| DBF |
Fluorescein
|
485 nm (20 nm)
|
538 nm (25 nm)
|
| OMF |
Fluorescein
|
485 nm (20 nm)
|
538 nm (25 nm)
|
|
CYP2D6
|
AMMC
|
AHMC
|
390 nm (20 nm)
|
460 nm (40 nm)
|
|
MAMC
|
HAMC
|
390 nm (20 nm)
|
460 nm (40 nm)
|
|
CYP2E1
|
7-MFC
|
HFC
|
409 nm (20 nm)
|
530 nm (25 nm)
|
|
CYP3A4
|
7-BQ
|
Quinolinol
|
409 nm (20 nm)
|
530 nm (25 nm)
|
|
BFC
|
HFC
|
409 nm (20 nm)
|
530 nm (25 nm)
|
|
BzRes
|
Resorufin
|
530 nm (25 nm)
|
590 nm (35 nm)
|
|
DBF
|
Fluorescein
|
485 nm (20 nm)
|
538 nm (25 nm)
|
| CYP3A5 |
BFC |
HFC |
410 nm |
538 nm |
| CYP3A7 |
BFC |
HFC |
410 nm |
538 nm |
| CYP1A1 |
BzRes |
Resorufin |
530 nm |
590 nm |
| CYP1B1 |
BzRes |
Resorufin |
530 nm |
590 nm |
| CYP19 |
DBF |
Fluorescein |
485 nm |
538 nm |
Note: These fluorescent parameters
may not be the excitation and emission maxims for these chemicals, however,
BD Biosciences has determined these parameters to be optimal for this
assay using the conditions outlined in this bulletin. For example, interference
from the fluorescence of NADPH (ex = 340, em = 436) has been minimized
by choosing a higher excitation filter for the detection of some products
and using a lower NADPH concentration for some substrates (AMMC).
|
