Conditions for amodiaquine N-deethylase Assay
The deethylation of amodiaquine is a low Km, high turnover
assay for human CYP2C8, which has recently been recommended as the preferred
assay for CYP2C8 (Walsky and Obach, 2004). The apparent Km for the CYP2C8-catalyzed
reaction is 1-3 mM (Li et.al., 2002, Walsky and Obach, 2004). The turnover
number (per unit cDNA-expressed CYP2C8) is over 30 min-1. When performing
kinetic analyses near the apparent Km, protein concentration and incubation
time should be chosen to avoid excessive metabolism of substrate. BD Gentest
supplies the desethylamodiaquine metabolite standard (Cat.No. 451782)
for quantitation of the assay results.
Solutions
I. 20 mM Amodiaquine
in DMSO or MeOH
II. NADPH Regenerating System Solution
A (451220) (20 mg/ml NADP, 13.3 mg/ml MgCl2·6H2O,
20 mg/ml Glucose 6-phosphate)
III. NADPH Regenerating System Solution
B (451200) (Glucose 6-phosphate dehydrogenase)
IV. 0.1 M KPO4 buffer pH 7.4 (451201)
V. Acetonitrile (ACN)
Incubation Conditions (for 1 ml Final Volume):
| 940 µl |
0.1M KPO4 buffer pH 7.4 |
| 50 µl |
NADPH Regenerating System Solution A |
| 5 µl |
NADPH Regenerating System Solution B |
| xx µl |
Enzyme: (human liver microsomes or cDNA-expressed) (Suggest
0.25mg/mL HLM or 5 pmoles P450) |
| 1 µl |
20mM Amodiaquine (final concentration is 20M (saturating)) |
Mix and prewarm to 37°C all solutions except enzyme. Initiate incubation
with the addition of enzyme. After the desired incubation time, stop the
reaction by the addition of 500 µl of stop solution (ACN) and cool
on ice. Centrifuge 12000 x g for 4 minutes to precipitate protein. Analyze
the supernatant for product formation by HPLC separation with UV detection.
Recommended range of injection volumes - 50 to 150 µl.
HPLC Conditions:
| Mobile Phase A: |
0.1% TFA in deionized water |
| Mobile Phase B: |
0.1% TFA in Acetonitrile |
| Gradient: |
Initial conditions: 100% A with a linear gradient to 17% B over
10 minutes, hold isocratic for 12 minutes (22 min total) and return
to initial conditions (Note 1).
Column: C18, 4.6 x 250mm column |
| Temperature: |
45°C (see Note 2) |
| Flow Rate: |
1 ml/min |
| Detector: |
Absorbance at 340 nm |
| Retention Times: |
Desethylamodiaquine, ~14 minutes; Amodiaquine, ~16 minutes |
Note 1: Desethylamodiaquine and amodiaquine tend to elute together. The
isocratic method presented shows > 1 minutes separation between peaks.
However, some adjustment in mobile phase gradient conditions may be desired.
Note 2: Column temperature can range from room temperature to 50°C.
The use of a controlled, elevated temperature provides greater reproducibility
in retention times and lower column back pressures.
REFERENCES: Li et.al. (2002), JPET 300: 399-407. Walsky and Obach, (2004),
Drug Metab. & Disp. 32 (6), 647-660.
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