In vitro UGT assay for inhibition studies of benzodiazepines ... - GTFCh

22
6000
velocity (pmol/min/mg)
20
18
16
14
12
10
8
6
4
2
Menge OG1/Protein (pmol/mg)
5000
4000
3000
2000
1000
0
6 7 8 9 10 11 12
pH
0
0 1 2 3 4 5 6
time (h)
50
14
40
12
velocity (pmol/min/mg)
30
20
velocity (pmol/min/mg)
10
8
6
4
10
2
0
0 20 40 60
temperature [C o ]
0
0 2 4 6 8 10 12 14 16 18
UDPGA (mM)
Fig. 1: The maximum of enzymatic activity of the UGT-enzyme superfamily could be
archived at pH 9 in 50 mM Tris buffer, incubation time of 4 h, 40 °C and 8 mM
and UDPGA.
mAU
Phenacetin
25 Oxazepam
Temazepam
20
15
10
5
R-Oxazepam glucuronide
S-Oxazepam glucuronide
R-Temazepam glucuronide
S-Temazepam glucuronide
0
5 10 15 20 25 30min
Fig. 2: Chromatographic separation of the diastereomeric benzodiazepine-glucuronides
and its aglycons. Phenacetin was used as internal standard.
Chromatographic separation (Fig. 2) of the diastereomeric benzodiazepine
glucuronides was possible using a RP-C 18 column (Spherisorb ODS2, 5 µm, 250 x
4 mm, Trentec), an column oven temperature of 40 °C, and an isocratic mobile
phase (flow rate 1.7 mL/min.) consisting of 0.3 % phosphoric acid (78 %),
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