Preparation of labeled human drug metabolites and drug-drug interaction-probes with fungal peroxygenases.

Enzymatic conversion of a drug can be an efficient alternative for the preparation of a complex metabolite compared with a multi-step chemical synthesis approach. Limitations exist for chemical methods for direct oxygen incorporation into organic molecules often suffering from low yields and unspecific oxidation and also for alternative whole-cell biotransformation processes, which require specific fermentation know-how. Stable oxygen-transferring biocatalysts such as unspecific peroxygenases (UPOs) could be an alternative for the synthesis of human drug metabolites and related stable isotope-labeled analogues. This work shows that UPOs can be used in combination with hydrogen/deuterium exchange for an efficient one-step process for the preparation of 4'-OH-diclofenac-d6. The scope of the reaction was investigated by screening of different peroxygenase subtypes for the transformation of selected deuterium-labeled substrates such as phenacetin-d3 or lidocaine-d3. Experiments with diclofenac-d7 revealed that the deuterium-labeling does not affect the kinetic parameters. By using the latter substrate and H2 (18) O2 as cosubstrate, it was possible to prepare a doubly isotope-labeled metabolite (4'-(18) OH-diclofenac-d6). UPOs offer certain practical advantages compared with P450 enzyme systems in terms of stability and ease of handling. Given these advantages, future work will expand the existing 'monooxygenation toolbox' of different fungal peroxygenases that mimic P450 in vitro reactions.

Exposure of Mytilus trossulus to diclofenac and 4'-hydroxydiclofenac: Uptake, bioconcentration and mass balance for the evaluation of their environmental fate.

Diclofenac (DIC) is one of the most widely consumed drugs in the world, and its presence in the environment as well as potential effects on organisms are the subject of numerous recent scientific works. However, it is becoming clear that the risk posed by pharmaceuticals in the environment needs to be viewed more broadly and their numerous derivatives should also be considered. In fact, already published results confirm that the transformation products of NSAIDs including DIC may cause a variety of potentially negative effects on marine organisms, sometimes showing increased biological activity. To date, however, little is known about bioconcentration of DIC and DIC metabolites and the role of sex in this process. Therefore, the present study for the first time evaluates sex-related differences in DIC bioconcentration and estimates bioconcentration potential of DIC metabolite, 4-OH DIC, in the Mytilus trossulus tissues. In the experiment lasting 7 days, mussels were exposed to DIC and 4-OH DIC at concentrations 68.22 and 20.85 µg/L, respectively. Our study confirms that DIC can be taken up by organisms not only in its native form, but also as a metabolite, and metabolised further. Furthermore, in the present work, mass balance was performed and the stability of both studied compounds under experimental conditions was analysed. Obtained results suggest that DIC is more stable than its derivative under the tested conditions, but further analyses of the environmental fate of these compounds are necessary.