Challenges regarding analysis of unbound fraction of highly bound protein antiretroviral drugs in several biological matrices: lack of harmonisation and guidelines.

The unbound drug concentration in plasma is usually considered the only active fraction; thus the binding of a drug to a protein limits its pharmacological actions. This is of special importance for those highly bound drugs. Therefore, binding studies can be of great utility for those drugs where relationship between free and total drug concentration is variable among patients, or it can be altered by some condition or disease, or even by interactions with other drugs. However, there is a lack of validation guidelines for the determination of unbound concentrations. Antiretroviral drugs (ARVs), protease inhibitors (PIs), efavirenz and nevirapine are highly bound to proteins. Here, we present a review on the overall methods for the study of unbound fractions of highly bound plasma protein ARVs. We also provide a critical evaluation of the methods applied, their differences and the main points to be controlled and validated.

CYP2B6 genetic variants are associated with nevirapine pharmacokinetics and clinical response in HIV-1-infected children.

BACKGROUND: Cytochrome P450 2B6 (CYP2B6)-G516T genotype is associated with altered activity of hepatic CYP2B6 and efavirenz pharmacokinetics, but the relationship between the CYP2B6-G516T genotype and nevirapine (NVP) pharmacokinetics in plasma and cerebrospinal fluid (CSF) is limited. METHODS: In 126 children who received NVP and protease inhibitors from PACTG 366 and 377 cohorts, CYP2B6 and ATP-binding cassette, sub-family B, member 1 (ABCB1) gene polymorphisms were analyzed using real-time PCR. Plasma NVP pharmacokinetics and clinical data were collected and levels of NVP in CSF were evaluated in children with HIV-related neurologic diseases. RESULTS: NVP oral clearance in children with the CYP2B6-516-T/T genotype (homozygous variant, n = 14) was 1.6 l/h per m2, which was significantly decreased compared to 2.3 l/h per m2 in those with the -G/G (wild type, n = 49, P = 0.002) and 2.1 l/h per m2 in those with the -G/T genotype (heterozygous variants, n = 63, P = 0.008). Furthermore, children with the -T/T genotype had a significant increase in CD4+ T-cell percentage (+9.0%) compared with those with the -G/G (+3.2%, P = 0.01) and -G/T genotype (+5.0%, P = 0.04) from baseline to week 12. The same trend continued at week 24. Although ABCB1-C3435T genotypes did not affect plasma NVP pharmacokinetics (P = 0.39), the NVP CSF: plasma ratios were significantly higher in children with the ABCB1-3435-C/T or -T/T genotypes (0.62, n = 9) in comparison with those with the ABCB1-3435-C/C genotype (0.43, n = 5) (P = 0.01). CONCLUSIONS: The CYP2B6-G516T genotype alters NVP pharmacokinetics and the immunologic response to NVP-containing HAART regimens in children. These data suggest that the CYP2B6-G516T is an important genetic variant that alters the pharmacokinetics and response to HAART regimens containing NVP.

Nevirapine uptake into the central nervous system of the Guinea pig: an in situ brain perfusion study.

The presence of human immunodeficiency virus (HIV) in the central nervous system (CNS) is associated with the development of HIV-1-associated dementia (HAD), a major cause of HIV-related mortality. To eradicate HIV in the CNS, anti-HIV drugs need to reach the brain and cerebrospinal fluid (CSF) in therapeutic concentrations. This involves passage through the blood-brain and blood-CSF barriers. Using a well established guinea pig in situ brain perfusion model, this study investigated whether nevirapine [6H-dipyrido(3,2-b:2',3'-e)(1,4)diazepin-6-one,11-cyclopropyl-5,11-dihydro-4-methyl], a non-nucleoside reverse transcriptase inhibitor (NNRTI), could effectively accumulate in the CNS. [(3)H]Nevirapine was coperfused with [(14)C]mannitol (a vascular/paracellular permeability marker) through the carotid arteries for up to 30 min, and accumulation in the brain, CSF, and choroid plexus was measured. [(3)H]Nevirapine uptake into the cerebrum was greater than uptake of [(14)C]mannitol, indicating significant passage across the blood-brain barrier and accumulation into the brain (this was further confirmed with capillary depletion and high-performance liquid chromatography analyses). Likewise, [(3)H]nevirapine showed a great ability to cross the blood-CSF barrier and accumulate in the CSF, compared with [(14)C]mannitol. The CNS accumulation of [(3)H]nevirapine was unaffected by 100 muM nevirapine, suggesting that passage across the blood-brain barrier can occur by diffusion. Furthermore, coperfusion with 100 muM efavirenz [2H-3,1-benzoxazin-2-one, 6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-, (4S)-; another NNRTI] did not significantly alter CNS accumulation of [(3)H]nevirapine, indicating that the efficacy of nevirapine in the CNS would not be altered by the addition of this drug to a combination therapy. Together, these data indicate that this anti-HIV drug should be beneficial in the eradication of HIV within the CNS and the subsequent treatment of HAD.