CYTOCHROME P450 METABOLISM INVOLVEMENT IN THE DEVELOPMENT OF HEPATOTOXICITY WITH FIRST-GENERATION NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS
Embargo until
2023-08-01
Date
2019-06-25
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Publisher
Johns Hopkins University
Abstract
The cytochrome P450 monooxygenase enzymes have been demonstrated to play a role in the metabolism of ~75% of FDA-approved drugs. Though generally thought to facilitate excretion, the metabolism of drugs by P450s generates new molecular entities, which can have differential effects from those of the parent drugs, including off-target, cytotoxic effects. Two hepatotoxic drugs used to treat HIV, efavirenz (EFV) and nevirapine (NVP), are so extensively metabolized by cytochrome P450 enzymes that their main P450 metabolites, 8-hydroxy-EFV (8-OHEFV) and 12-hydroxy-NVP (12-OHNVP), respectively, exist at micromolar concentrations in patient plasma. Previous work suggests that these metabolites play a role in the respective hepatotoxic events observed with these drugs. We probed whether EFV and 8-OHEFV activate hepatic IRE1α-XBP1 signaling, which has been previously demonstrated to activate hepatocyte death under select stimuli. Interestingly, we observed that EFV treatment resulted in a greater magnitude of IRE1α-XBP1 signaling activation in primary hepatocytes than 8-OHEFV. With this, we surveyed other structural changes to EFV and two other compounds (analog 3 and 14) with only single-atom changes from EFV that demonstrated greater activation of IRE1α-XBP1 than EFV itself. Despite all being activators of IRE1α-XBP1, only EFV and analog 3 induced hepatocyte cell death, and only cell death with EFV was inhibited by co-treatment with the IRE1α-XBP1 inhibitor STF083010. Taken together these results suggest that EFV activates IRE1α-XBP1 and that this activation plays a role in the hepatocyte death observed with EFV. In addition, while other very similar compounds, analogs 3 and 14, are activators of IRE1α-XBP1, they demonstrate variable toxicity or toxicity mechanisms different from that of EFV. We also investigated whether twelfth position deuteration on NVP, generating 12-D3NVP is an effective strategy in controlling hepatic P450 metabolism to 12-OHNVP and ultimately NVP toxicity. A 10.6-fold reduction in production of 12-OHNVP was observed in human hepatocytes during treatments with clinically relevant concentrations of 12-D3NVP (as compared to NVP) with no impact on the formation of other detectable P450 metabolites of NVP. In addition, a kinetic isotope effect of 10.1-fold was measured in incubations with human liver microsomes. Despite this large change in 12-OHNVP production, hepatocyte death was only modestly reduced with 12-D3NVP in primary mouse hepatocytes, as compared to NVP. To investigate potential differences in cell signaling with these compounds we performed relative quantitation proteomics analysis on hepatocytes treated with NVP or 12-D3NVP and observed differential protein expression with these two treatments. These results suggest that deuterium substitution, while an effective strategy for controlling 12-OHNVP production does not drastically reduce NVP hepatotoxicity and that this compound can induce differential protein expression from that observed with NVP.
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Keywords
Drug Metabolism, Cytochrome P450, Hepatotoxicity, HIV, NNRTI, Kinetic Isotope Effect