Assay Conditions for Paclitaxel 6(alpha)-Hydroxylation
Paclitaxel 6(alpha)-hydroxylation is a low Km,
relatively high turnover assay for human CYP2C8. The apparent Km
for the CYP2C8-catalyzed reaction is 4-8 uM. When performing kinetic analyses
near the apparent Km, protein concentration and incubation
time should be chosen to avoid excessive metabolism of substrate. GENTEST
supplies 6(alpha)-hydroxypaclitaxel metabolite standard (Cat. No. 451656
(Old B656)) for quantitation of the assay results.
Solutions
I. 5 mM Paclitaxel
in ethanol
II. 20 mg/ml Glucose 6-phosphate,
20 mg/ml NADP, 13.3 mg/ml MgCl2-H2O
III. 40 U/ml Glucose 6-phosphate dehydrogenase
in 5 mM sodium citrate (tribasic)
IV. 0.05 M Potassium Phosphate pH 7.4
V. 100% Acetonitrile
Incubation Conditions (for 1 ml Final
Volume):
50 ul Solution II.
10 ul Solution III.
2 ul Solution
I. (Final concentration 10 uM, near the limit of solubility in aqueous
media)
xx ul Enzyme. (human
liver microsomes or cDNA-expressed)
938 - xx ul Solution IV.
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 300 ul of solution V (per ml)
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 100 ul.
HPLC Conditions
Mobile Phase A: 10% methanol (see Note 1)
Mobile Phase B: 100% methanol
Gradient: Initial conditions: 55% B with a linear gradient to 65% B over
20 minutes then hold at 65% B for 5 minutes
Column: Nucleosil C18, 4.6 x 250 mm, 5 um particle size (see Note 2)
Temperature: 45oC (see Note 3)
Flow Rate: 1 ml/min
Detector: Absorbance at 230 nm
Retention Times: 6(alpha)-Hydroxypaclitaxel, 22 minutes; paclitaxel, 25.5
minutes
Note 1
Mobile phases containing acetonitrile may be used in place of methanol,
however, some adjustment in mobile phase gradient conditions may be desired.
Note 2
6(alpha)-Hydroxypaclitaxel and paclitaxel can be separated
with most C18 columns. However, some adjustment in mobile phase gradient
conditions may be desired.
Note 3
Column temperature can range from room temperature to 50oC.
The use of a controlled, elevated temperature provides greater reproducibility
in retention times and lower column back pressures.
REFERENCES
Cancer Res. (1994) 54, 4026; J. Med. Chem. (1994) 37, 706;
Cancer Res. (1994) 54, 5543; J. Chromatog. B (1995) 674,
261.
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