Site‐Specific Dueteration Decreases Cytrochrome P450 Production of 12‐Hydroxy‐Nevirapine through the Kinetic Isotope Effect

Abstract

The non‐nucleoside reverse transcriptase inhibitor nevirapine (NVP), classified as an essential medication by the World Health Organization, has been shown to cause life‐threatening hepatotoxicity and skin rash in patients. The 12‐hydroxyNVP (12‐OHNVP) metabolite of NVP has been implicated in these toxicities and here we aimed to gain a detailed understanding of utility of introducing deuteriums at the 12‐position of NVP as an approach to decreasing the formation of this metabolite. To do so, an ultra‐high performance liquid chromatograph tandem high‐resolution mass spectrometry assay was developed and employed to detect 12‐OHNVP formation following incubation of NVP with human liver microsomes and individual cDNA‐expressed enzymes. Through this work, we found that formation of 12‐OHNVP was most abundant following incubation with cytochrome P450 (P450; CYP) 2C19 although each of the P450s tested, with the exception of CYP3A5, catalyzed the formation of 12‐OHNVP to some extent. In line with this, formation of 12‐OHNVP by human liver microsomes was not abrogated in the presence of any established small molecule inhibitors of P450 activity including ketoconazole (CYP3A inhibitor). In order to test whether the deuterium kinetic isotope effect could be employed to control 12‐OHNVP production by all P450s we investigated the impact of 12th position deuteration of NVP on the formation of this metabolite. Human liver microsomes were incubated with 10 μM NVP or 10 μM 12‐d3NVP for 30 minute and the production of the 12‐OHNVP was measured. The abundance of 12‐OHNVP was 6.8‐fold lower when human liver microsomes were incubated with 12‐d3NVP, as compared to undeuterated NVP incubations. Of note, no difference in the formation of other P450‐dependent metabolites of NVP was observed. In order to test the impact of deuterium kinetic isotope effects on the metabolism of NVP in a whole cell system, we measured metabolite levels in the medium of primary mouse hepatocytes treated with 10 μM NVP and 12‐d3NVP for 24 hours. A 4.7‐fold decrease was observed in 12‐OHNVP production in the medium of cells treated with deuterated NVP as compared to undeuterated NVP. As with the human liver microsome experiments, no significant difference was observed in the production of other P450‐dependent metabolites between treatments. These results demonstrate that dueteration at the 12th position can markedly decrease cytochrome P450 production of 12‐OHNVP through the kinetic isotope effect. The impact of this controlled metabolism on NVP toxicity will be a subject of further research. This work was funded by NSF GRFP DGE‐1232825 awarded to CJSH and NIH R01 GM103853.Support or Funding InformationThis work was funded by NSF GRFP DGE‐1232825 awarded to CJSH and NIH R01 GM103853.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.