Cytochrome P450-dependent metabolism of the anti-HIV drug nevirapine (NVP) to 12-hydroxy-NVP (12-OHNVP) has been implicated in NVP toxicities. We investigated the impact of twelfth-position trideuteration (12-D 3 NVP) on the hepatic metabolism of and response to NVP. Formation of 12-OHNVP decreased in human (10.6-fold) and mouse (4.6-fold) hepatocytes incubated with 10 μM 12-D 3 NVP vs NVP. An observed kinetic isotope effect of 10.1 was measured in human liver microsomes. During mouse hepatocyte treatment (400 μM) with NVP or 12-D 3 NVP, cell death was reduced 30% with 12-D 3 NVP vs NVP, while glucuronidated and glutathione-conjugated metabolites increased with 12-D 3 NVP vs NVP. Using mass spectrometry proteomics, changes in hepatocyte protein expression, including an increase in stress marker insulin-like growth factor-binding protein 1 (IGFBP-1), were observed with 12-D 3 NVP vs NVP. These results demonstrate that while deuteration can reduce P450 metabolite formation, impacts on phase II metabolism and hepatocyte protein expression should be considered when employing deuteration to reduce P450 metabolite-related hepatotoxicity.

Emtricitabine (FTC), tenofovir (TFV), efavirenz (EFV), and rilpivirine (RPV) are currently used as components of HIV combination therapy. Although these drugs are widely used in antiretroviral therapy, several organ toxicities related to TFV and EFV have been observed clinically. TFV is associated with nephrotoxicity, whereas EFV-related hepatotoxicity and neurotoxicity have been reported. While the precise molecular mechanisms related to the above-mentioned clinically observed toxicities have yet to be elucidated, understanding the local tissue distribution profiles of these drugs could yield insights into their safety profiles. To date, the distributions of these drugs in tissue following in vivo exposure are poorly understood. Therefore, in this study, we employed a matrix-assisted laser desorption/ionization mass spectrometry imaging method to generate spatial distribution profiles of FTC, TFV, EFV, and RPV in mouse tissues following in vivo dosing of following drug regimens: TFV–FTC–EFV and TFV–FTC–RPV. For this study, liver, brain, kidney, spleen, and heart tissues were obtained from mice ( n = 3) following separate oral administration of the above-mentioned drug regimens. Interestingly, EFV was detected in liver, brain, and heart following TFV–FTC–EFV treatment. Additionally, hydroxylated EFV, which encompasses the cytochrome P450-dependent monooxygenated metabolites of EFV, was detected in liver, brain, spleen, and heart tissue sections. Notably, the tissue distribution profiles of RPV and hydroxylated RPV following in vivo dosing of TFV–FTC–RPV were different from EFV/hydroxylated EFV despite RPV belonging to the same drug class as EFV. In conclusion, the observed spatial distribution profiles of the study drugs are in agreement with their safety profiles in humans.

Efavirenz (EFV), an antiretroviral that interacts clinically with co-administered drugs via activation of the pregnane X receptor (PXR), is extensively metabolized by the cytochromes P450. We tested whether its primary metabolite, 8-hydroxyEFV (8-OHEFV) can activate PXR and potentially contribute to PXR-mediated drug-drug interactions attributed to EFV. Luciferase reporter assays revealed that despite only differing from EFV by an oxygen atom, 8-OHEFV does not activate PXR. Corroborating this, treatment with EFV for 72 h elevated the mRNA abundance of the PXR target gene, Cyp3a11, by approximately 28-fold in primary hepatocytes isolated from PXR-humanized mice, whereas treatment with 8-OHEFV did not result in a change in Cyp3A11 mRNA levels. FRET-based competitive binding assays and isothermal calorimetry demonstrated that even with the lack of ability to activate PXR, 8-OHEFV displays an affinity for PXR (IC 12.1 μm; K 7.9 μm) nearly identical to that of EFV (IC 18.7 μm; K 12.5 μm). The use of 16 EFV analogues suggest that other discreet changes to the EFV structure beyond the 8-position are well tolerated. Molecular docking simulations implicate an 8-OHEFV binding mode that may underlie its divergence in PXR activation from EFV.