Efavirenz Biotransformation in Brain Microsomes and Neural Cells

Abstract

Efavirenz (EFV) is a non‐nucleoside reverse transcriptase inhibitor that is metabolized by both the cytochromes P450 and UDP‐glucuronosyltransferases (UGTs). EFV is one of several anti‐HIV medications that can cross the blood‐brain barrier and is essential to combatting the viral reservoir in the brain that can eventually lead to HIV‐associated neurocognitive disorder (HAND) in nearly 50% of patients. EFV and 8‐hydroxyefavirenz (8‐OHEFV), the primary P450‐dependent metabolite of EFV, have both been linked to neurological adverse events and neurotoxicity. However, very little is known about the fate of EFV in the brain, particularly how it’s metabolized or the precise mechanism of neurotoxicity. To investigate EFV biotransformation in the brain, we first performed microsomal metabolism assays with brain microsomes obtained from mice, cynomolgus macaques, and humans. EFV was incubated with the brain microsomes and NADPH to characterize P450‐dependent metabolism, while EFV or one of its P450‐dependent metabolites, 8‐OHEFV or 8‐,14‐dihydroxyefavirenz (8,14‐diOHEFV), was incubated with brain microsomes and UDPGA to investigate UGT‐catalyzed metabolism. Ultra‐high‐performance liquid chromatography tandem high resolution mass spectrometry (UHPLC‐HRMS) was employed to detect metabolites. Formation of 8‐OHEFV was observed in all three species, as was glucuronidation of 8‐OHEFV and 8,14‐diOHEFV. Interestingly, direct glucuronidation of EFV was only found in the cynomolgus macaque brain microsomes. When comparing brain microsome metabolism to liver microsome metabolism, we see that the liver has a greater capacity to transform EFV and its P450‐dependent metabolites. We tested cell‐type specific metabolism using primary cortical neurons, striatal neurons, astrocytes, and microglia from mice. Cells were cultured and treated with EFV, 8‐OHEFV, or 8,14‐diOHEFV for approximately 24 hours, with DMSO as a vehicle control. In the microglia and astrocytes, we observed P450‐mediated formation of 8‐OHEFV in the EFV‐treated cells. In the cortical neurons, striatal neurons, and astrocytes, both 8‐OHEFV and 8,14‐diOHEFV were glucuronidated. These results shed additional light on the microsome data, indicating which cell‐types may be contributing to EFV metabolism. In male mice dosed with EFV for 5 days in drinking water, UHPLC‐HRMS analysis of brain tissue homogenate and serum revealed 8‐OHEFV in brain tissue in conjunction with serum concentrations in the range of 15‐20 ng/mL. EFV and other metabolites were below the limit of detection in both brain tissue and serum. These data indicate that 8‐OHEFV has the potential to be formed and/or further metabolized in mouse brain. Additionally, inter‐species differences in EFV glucuronidation were observed in brain microsomes. The characterization of EFV metabolism in the brain improves our understanding of tissue‐specific EFV disposition as well as brain glucuronidation more broadly.