Abstract

Antiretroviral drugs such as efavirenz (EFV) are essential to combat HIV infection in the brain, but little is known about how these drugs are metabolized locally. In this study, the cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT)-dependent metabolism of EFV was probed in brain microsomes from mice, cynomolgus macaques, and humans as well as primary neural cells from C57BL/6N mice. Utilizing ultra-high performance liquid chromatography high resolution mass spectrometry (uHPLC-HRMS), the formation of 8-hydroxyefavirenz (8-OHEFV) from EFV and the glucuronidation of P450-dependent metabolites 8-OHEFV and 8,14-dihydroxyefavirenz (8,14-diOHEFV) was observed in brain microsomes from all three species. The direct glucuronidation of EFV, however, was only detected in cynomolgus macaque brain microsomes. In primary neural cells treated with EFV, microglia were the only cell type to exhibit metabolism, forming 8-OHEFV only. In cells treated with the P450-dependent metabolites of EFV, glucuronidation was detected only in cortical neurons and astrocytes, revealing that certain aspects of EFV metabolism are cell-type specific. Untargeted and targeted proteomics experiments were used to identify the P450s and UGTs present in brain microsomes. Eleven P450s and 11 UGTs were detected in human brain microsomes, while 10 P450s and 15 UGTs were identified in mouse brain microsomes and 17 and 6 P450s and UGTs, respectively, were observed in macaque brain microsomes. This was the first time many of these enzymes have been noted in brain microsomes at the protein level. This study indicates the potential for brain metabolism to contribute to pharmacological and toxicological outcomes of EFV in the brain.

Significance Statement Metabolism in the brain is understudied, and the persistence of HIV infection in the brain warrants the evaluation of how antiretroviral drugs, such as efavirenz, are metabolized in the brain. Using brain microsomes, the metabolism of efavirenz by both P450s and UGTs is established. Additionally, proteomics of brain microsomes characterizes P450s and UGTs in the brain, of which many have not yet been noted in the literature at the protein level.