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

In vitro UGT assay for inhibition studies
of benzodiazepines and opiates during
phase II-metabolism
T. Pallmann, K. Bender, G. Sticht, M.A. Rothschild, H. Käferstein
Introduction
Hydroxy-benzodiazepines and opiates are metabolized through glucuronidation
which is the predominant pathway in the clearance mechanism of exogenous
and endogenous substances during phase II-metabolism. The reaction is
catalyzed by uridine-diphosphoglucuronyltransferases (UGTs) [1]. The presented
work is motivated by the fact that the combination of benzodiazepines and opiates
is common both in clinical practice and as combined abuse. Inhibition of the glucuronidation
can lead to a decelerated elimination of the phase I-metabolites,
respectively of substances which are subjects of the phase II-metabolism. In the
case of pharmacological and toxicological active substances, like the hydroxybenzodiazepines
and opiates, this can result in a prolonged pharmacological
activity and in a higher risk of side effects of these substances [2, 3]. Therefore an
in vitro UGT-assay for inhibition studies between hydroxy-benzodiazepines and
opiates has been developed and optimized using pooled human liver microsomes
(HLMs).
Methods
For the investigation of the interaction capabilities of some benzodiazepines
and opiates during phase II-metabolism an in vitro assay was developed
for oxazepam and temazepam (10-1000 µmol), morphine and codeine (2.5, 5 and
10 mM) using pooled human liver microsomes (HLMs, BD Bioscience) with a
total protein concentration of 1.0 mg/ml. As shown in Fig. 1, the maximum of the
enzymatic activity of the UGTs was achieved at a pH of 9 using 50 mM Tris
buffer, 40 °C, 8 mM UDPGA, and an incubation time of 240 min. It is striking,
that the pH- and temperature-values do not reflect physiological conditions.
Therefore, the incubations were performed at 37 o C in 50 mM Tris buffer (pH 7.4)
with 5 mM MgCl 2 , 8 mM UDPGA, and alamethicin (50 µg/mg microsomal proteins)
in a total volume of 100 µl for 240 min. After quenching of the enzymatic
activity and centrifugation, the supernatant was analyzed by HPLC/DAD.
165

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 %),
166

acetonitrile (16 %), and isopropanol (6 %). For morphine-3- and morphine-6-
glucuronide a mobile phase consisting of 98 % 10 mM phosphoric acid (pH 2.7)
and 2 % acetonitrile was used and for codeine-6-glucuronidethe concentration
was 93 % 10 mM phosphoric acid (pH 2.7) and 7 % acetonitrile.
Results
K m and V max values have been evaluated for both enantiomers of
temazepam and oxazepam with two batches of HLMs (Fig. 3).
50
velocity (pmol/min/mg)
40
30
20
10
S-Oxazepam: Substrate inhibition model
K m = 28.8 µM
V max = 54.9 pmol/min/mg
R-Oxazepam: Michaelis Menten model
K m = 90.7 µM
V max = 25.1 pmol/min/mg
(A)
(B)
0
0 200 400 600 800 1000 1200
Oxazepam (µM)
100
velocity (pmol/min/mg)
80
60
40
20
S-Temazepam: Substrate inhibition model
K m = 82.9 µM
V max = 83.7 pmol/min/mg
R-Temazepam: Michaelis Menten model
K m = 370.2 µM
V max = 110.2 pmol/min/mg
(C)
(D)
0
0 200 400 600 800 1000 1200
Temazepam (µM)
Fig. 3: Enzyme kinetic data of S-oxazepam (A), R-oxazepam (B), R-temazepam (C),
and S-temazepam using HLMs.
167

The results show that the K m values for S-oxazepam (28.8+/-5.3 and
37.1+/-9.6 µmol) and S-temazepam (77.4+/-12.6 and 82.9+/-7.7 µmol) were
lower than for R-enantiomers (R-oxazepam (90.7+/-12.6 and 104.9+/-10.6 µmol),
R-temazepam (336.4+/-37.6 and 370.2+/-94.6 µmol)). The determined K m -values
of R- and S-oxazepam indicating a higher affinity to the UGT-enzyme
superfamily as the enantiomers of temazepam. Furthermore the measured
K m -values are indicating a higher UGT-affinity of the S-enantiomers unlike the R-
enantiomers of the benzodiazepines oxazepam and temazepam. Inhibition studies
between the benzodiazepines oxazepam, temazepam and the opiates morphine and
codeine as inhibitors showed that the K i -values for S-enantiomers are smaller than
for R-enantiomers, which is exemplarily shown in Fig. 4.
velocity(pmol/min/mg)
25
20
15
10
5
(A)
1/velocity(pmol/min/mg)
0,8
0,6
0,4
0,2
(B)
velocity(pmol/min/mg)
0
0 50 100 150 200 250 300
40
35
30
25
20
15
10
5
(C)
(R,S) Oxazepam (µM)
-0,010 -0,005 0,000 0,005 0,010 0,015 0,020 0,025
1/velocity(pmol/min/mg)
0,12
0,10
0,08
0,06
0,04
0,02
1/(R,S) Oxazepam (µM)
(D)
0
0 50 100 150 200 250 300
(R,S) Oxazepam (µM)
-0,02 -0,01 0,00 0,01 0,02 0,03
1/(R,S) Oxazepam(µM)
Fig. 4: Michaelis-Menten plot (left) and Lineweaver-Burk plot (right) of R-oxazepam (A/B) and
S-oxazepam (C/D) inhibited by 0 µM (-●-), 2.5 µM (-○-), 5.0 µM (-▼-), and 10.0 µM (-∇-)
morphine.
By using the benzodiazepines as inhibitors, temazepam showed the highest
inhibitory effect on the glucuronidation of morphine, whereas oxazepam inhibited
the codeine glucuronidation most. In addition, the glucuronidation of codeine was
much more affected by the benzodiazepines than it was observable for morphine.
168

Conclusion
With the developed and optimized in vitro assay, consisting of pooled
human liver microsomes, it was possible to analyze the interaction potentials
between the hydroxy-benzodiazepines oxazepam and temazepam and the opiates
morphine and codeine. Furthermore it was possible to consider the enantiomer
specific differences of the benzodiazepine-glucuronidation. The results show that
S-enantiomers of the benzodiazepines exhibit a higher affinity to the UGTs than
the R-enantiomers do. This can be deduced from the lower K m -values of the
S-enantiomers compared to the R-enantiomers. The glucuronidation of morphine
was more negatively affected by temazepam than by oxazepam. In addition to
these findings the benzodiazepines exhibits a higher inhibitory effect on the
codeine glucuronidation as on the morphine glucuronidation. Furthermore, the
codeine itself is a stronger inhibitor of the benzodiazepine-glucuronidation
compared to morphine. In particular, the glucuronidation of the S-enantiomers are
more affected by the opiates than the R-enantiomers. This is feasible by the fact
that the S-enantiomers exhibits a higher UGT-affinity as mentioned before. It is
thought, that S-oxazepam is more active on the GABA A -receptor than R-
oxazepam [4]. Due to this, it can be derived that the inhibition of the S-
enantiomer glucuronidation of the benzodiazepines by morphine and codeine can
contribute to the side effects of the benzodiazepines.
In fact, UGT-catalyzed metabolic reactions account for about 35 % of all
drugs which undergo phase II-metabolism [5]. Assuming, there is a high number
of other substances which can at least partly inhibit the glucuronidation, drug
interactions based on inhibitory effects during the phase II-metabolism, becomes
obvious [2, 3].
The presented work was financially supported by the Koeln Fortune
Program / Faculty of Medicine, University of Cologne, Grants 51/2005 and
68/2006. Full paper version will be published in an additional journal.
References
[1] King, C. D.; Rios, G. R.; Green, M. D.; Tephly, T. R., UDP-glucuronosyltransferases.
Current Drug Metabolism 2000, 1, (2), 143-161.
[2] Kiang, T. K. L.; Ensom, M. H. H.; Chang, T. K. H., UDP-glucuronosyltransferases and
clinical drug-drug interactions. Pharmacology & Therapeutics 2005, 106, (1), 97-132.
[3] Lin, J. H.; Wong, B. K., Complexities of glucuronidation affecting in vitro-in vivo
extrapolation. Current Drug Metabolism 2002, 3, (6), 623-646.
[4] Mohler, H.; Okada, T.; Heitz, P.; Ulrich, J., Biochemical Identification Of Site Of Action Of
Benzodiazepines In Human-Brain By Diazepam-H-3 Binding. Life Sciences 1978, 22, (11),
985-995.
169

[5] Evans, W. E.; Relling, M. V., Pharmacogenomics: translating functional genomics into
rational therapeutics. Science 1999, 286, 487-491.
Dr. Katja Bender
Institut für Rechtsmedizin
Klinikum der Universität zu Köln
E-Mail: katja.bender@uk-koeln.de
170