Darstellung verschiedener Benzodiazepin-Glucuronide sowie die Testung der Benzodiazepine hinsichtlich ihres Interaktionspotentials im Phase-II-Metabolismus mit Opiaten

An enzyme-assisted synthesis for glucuronides of (R,S)-oxazepam, (R,S)-temazepam and (R,S)-lorazepam was developed by using swine liver microsomes. The resulted diastereomeric pairs of glucuronides of the benzodiazepines were separated by preparative high performance liquid chromatography (HPLC). It was possible to obtain the benzodiazepine glucuronides with a yield of 10-28 %. The synthesis products were characterized by LC/MS and 1H-NMR spectroscopy. In comparison to the theoretical mass of the synthesized glucuronides, the obtained LC/MS data showed with a minimum accuracy of 0.7 ppm identical mass values for the precursor ion and characteristic fragments. 1H-NMR measurements were necessary to show differences in the stereochemistry of R- and S-glucuronides. S-glucuronides displayed similar behaviour in the chemical shifts of the two relevant protons on G1 and the 3-carbon. Otherwise, all R-glucuronides showed also similiar behaviour. The availability of reference standards provided the determination of pharmacokinetic drug interactions during phase II metabolism of the benzodiazepines with opiates. An in vitro UGT assay using human liver microsomes (HLMs) was developed and optimized. Km and Vmax values have been evaluated for both enantiomers of temazepam and oxazepam. R- and S-oxazepam showed higher affinity to the UDP-glucuronosyltransferases (UGTs) as the comparable enantiomers of temazepam. Experiments showed that the Km values for S-oxazepam and S-temazepam were lower than for R-enantiomers. S-oxazepam and S-temazepam glucuronidation were best described by a single enzyme kinetic model with substrate inhibition, whereas the simple Michaelis-Menten model best described data for R-enantiomers. For a better understanding of the results of inhibition studies all available UGT isoforms were screened for R- and S-temazepam glucuronidation activities, as it is already known of R- and S-oxazepam. Of 12 different UGT isoforms evaluated, both stereoisomers of temazepam were glucuronidated by UGT2B7. In contrast, UGT2B15 only catalyzed the glucuronidation of S-temazepam. The predominant glucuronidating isoform of S-temazepam could not be identified by screening. For inhibition studies morphine or codeine was incubated with oxazepam or temazepam with the optimized in vitro UGT assay. By detection and quantification of the opiate glucuronides as well as the benzodiazepine glucuronides by HPLC it was possible to determine the opiates and benzodiazepines as substrates and inhibitiors. The inhibition studies demonstrated a competitive inhibition of benzodiazepine glucuronidation by opiates and vice versa. Codeine was a more potent inhibitor of oxazepam and temazepam glucuronidation compared to morphine with lower Ki values. The glucuronidation of the S-enantiomers of oxazepam and temazepam was more inhibited by morphine and codeine as the compared R-enantiomers. Inhibition constants showed that the glucuronidation of codeine was more inhibited by oxazepam and temazepam than morphine 3- and morphine 6-glucuronidation. For morphine glucuronidation the Ki value for oxazepam was lower for postition 3 than for position 6 and temazepam inhibited the glucuronidation on both positions in an equal fashion with similar Ki values. Furthermore the diastereomeric pure temazepam glucuronides provided the possibility of in vitro pharmcological testings of these metabolites. Therefore a novel biosensor based on neocortical rat neurons, cultivated on a planar microelectrode array (MEA), has been used. Temazepam did amplify the inhibitory effect of GABA which resulted in a concentration dependent suppression of the burst rate. In case of temazepam glucuronides the cell potential of the neurons was not significantly affected. It could be shown that neuronal networks growing on a MEA can be used for pharmacological and toxicological testing of benzodiazepines and their phase II metabolites.