Compartmentalization and antiviral effect of efavirenz metabolites in blood plasma, seminal plasma, and cerebrospinal fluid

Abstract

Efavirenz (EFV) is one of the most commonly prescribed antiretrovirals for use in the treatment of human immunodeficiency virus (HIV) infection. EFV is extensively metabolized by cytochrome P450 to a number of oxygenated products; however, the pharmacologic activity and distribution of these metabolites in anatomic compartments have yet to be explored. The systemic distribution of EFV oxidative metabolites was examined in blood plasma, seminal plasma, and cerebrospinal fluid from subjects on an EFV-based regimen. The 8-hydroxy EFV metabolite was detected in blood plasma, seminal plasma, and cerebrospinal fluid, with median concentrations of 314.5 ng/ml, 358.5 ng/ml, and 3.37 ng/ml, respectively. In contrast, 7-hydroxy and 8,14-hydroxy EFV were only detected in blood plasma and seminal plasma with median concentrations of 8.84 ng/ml and 10.23 ng/ml, and 5.63 ng/ml and 5.43 ng/ml, respectively. Interestingly, protein-free concentrations of metabolites were only detectable in seminal plasma, where a novel dihdyroxylated metabolite of EFV was also detected. This accumulation of protein-free EFV metabolites was demonstrated to be the result of differential protein binding in seminal plasma compared with that of blood plasma. In addition, the oxidative metabolites of EFV did not present with any significant pharmacologic activity toward HIV-1 as measured using an HIV green fluorescent protein single-round infectivity assay. This study is the first to report the physiologic distribution of metabolites of an antiretroviral into biologic compartments that the virus is known to distribute and to examine their anti-HIV activity. These data suggest that the male genital tract may be a novel compartment that should be considered in the evaluation of drug metabolite exposure.

Figures

Fig. 1.

Distribution of EFV and hydroxylated EFV metabolites into blood plasma, seminal plasma, and cerebrospinal fluid. Total concentrations of 8-OH EFV, 7-OH EFV, and 8,14-OH EFV were analyzed in subjects on a regular EFV-based regimen and were found to be detectable in blood plasma (A, ▾), seminal plasma (B, ●), and cerebrospinal fluid (C, ▴). Protein-free (○) concentrations of 8-OH EFV, 7-OH EFV, and 8,14-OH EFV were only detectable in seminal plasma (B).

Fig. 2.

Physiologic formation of dihydoxylated EFV metabolites. The production of a second di-OH EFV metabolite (1.73-minute retention time), distinct from 8,14-OH EFV (1.56-minute retention time) was determined in the clinical matrices from subjects on a steady-state EFV-containing regimen. The panels depict the MRM transition of m/z 346 > 262 for: (A) blood plasma, (B) seminal plasma, and (C) cerebrospinal fluid from an individual subject.

Fig. 3.

Formation of dihydoxylated EFV metabolites from human liver microsomes. Production of dihydroxylated EFV metabolite after substrate incubation with human liver microsomes. The panels depict the common MRM transition of m/z 346 > 262 for human liver microsomes incubated with (A) 20 μM EFV, (B) 20 μM 8-OH EFV, and (C) 20 μM 7-OH EFV.

Fig. 4.

Fragmentation patterns of dihydroxylated metabolites of EFV. The MS/MS fragmentation patterns were determined for each distinct dihydroxylated EFV metabolite with retention times of (A) 1.57 minutes, (B) 1.73 minutes, and (C) 1.92 minutes.

Fig. 5.

Contribution of individual CYPs to the formation of EFV metabolites. Individual cDNA-expressed CYPs were incubated with 5 μM EFV, 8-OH EFV, or 7-OH EFV and analyzed by UPLC-MS/MS for the formation of dihdyroxylated products of EFV at (A) 1.57 minutes, (B) 1.74 minutes, and (C) 1.92 minutes. The data are presented as mean ± standard deviation (SD) of n = 3.

Fig. 6.

Proposed schematic of EFV mono-oxidative and dioxidative metabolism. EFV metabolism to 8-hydroxy-EFV catalyzed by CYP2B6 was previously established by Ward et al. (2003). EFV metabolism to 7-hydroxy-EFV catalyzed by CYP2A6 and to 8-14-dihydroxy-EFV catalyzed by CYP2B6 was previously established by Ogburn et al. (2010).