Sensitive Enzyme Immunoassay for Measuring Plasma and Intracellular Nevirapine Levels in Human Immunodeficiency Virus-Infected Patients

Abstract

We have developed an enzyme immunoassay to measure nevirapine (NVP) in plasma and peripheral blood mononuclear cells. Anti-NVP polyclonal antibodies were raised in rabbits by using a synthetic NVP derivative coupled to keyhole limpet hemocyanin as the immunogen, and the enzyme tracer was prepared by chemically coupling the NVP derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates with a 100-pg ml ؊1 limit of detection and thus ϳ100 times more sensitive than previously published techniques. The plasma assay was performed directly without extraction (in this case, a 500-pg ml ؊1 limit of detection was observed) on a minimum of 30 l of plasma. This assay shows good precision and efficiency, since recovery from human plasma and cell extracts spiked with NVP ranged between 87 and 104%, with coefficients of variation of <10%. A pharmacokinetic analysis of plasma NVP was performed for seven patients infected with human immunodeficiency virus (HIV), and it gave results similar to published findings. Intracellular concentrations of NVP were measured in cultured human T-lymphoblastoid cells and peripheral blood mononuclear cells from HIV-infected patients. The results indicated a very low intracellular/extracellular concentration ratio (0.134), thus demonstrating the absence of intracellular drug accumulation. This is the first intracellular assay of a nonnucleoside reverse-transcriptase inhibitor, and this method could be useful in monitoring plasma and intracellular NVP levels in HIV-infected patients.Nevirapine (Viramune) (NVP) is a nonnucleoside reversetranscriptase inhibitor indicated for the treatment of human immunodeficiency virus (HIV) type 1 infection. It represents an attractive option for patients who prefer a protease-sparing regimen, because it can be taken twice daily (200 mg b.i.d.) and ingested without food restrictions. The drug binds to viral reverse transcriptase and blocks polymerase activity by disrupting the catalytic site (16). Consequently, nevirapine must enter cells to inhibit viral replication, and it is important to consider the intracellular drug concentration in peripheral blood mononuclear cells (PBMC) and other compartments, as the distribution of antiviral drugs from the plasma into cells and tissues is dependent on many complex factors, including affinities for cells versus plasma components or drug transporters (9, 19). As a consequence, the intracellular levels may be very different from those recorded in plasma. Knowledge of the intracellular distribution may help in understanding the mechanisms that are involved in the evolution of drug resistance and the development of sanctuary sites.Several high-performance liquid chromatographic (HPLC) assays combined with UV detection (6, 10, 22) or tandem mass spectrometry (14,24) for the quantitative determination of NVP in plasma have been described. However, these methods are characterized by a relatively high limit of quantification (10 ng ml Ϫ...

Human Immunodeficiency Virus Protease Inhibitors Accumulate into Cultured Human Adipocytes and Alter Expression of Adipocytokines

“…Competitive ELISA-RTV, NVP, and LPV were quantified by competitive ELISA as previously described (15,16). Results were expressed as nanomole of HIV drugs/10 6 cells.…”

“…This result strongly suggested that accumulation of the drugs into preadipose cells could be regulated via MDR transporters. We have previously shown that the antibodies used in the ELISAs are specific for the native form of the drugs and do not recognize metabolic forms (15,16). In addition, HPLC-UV analysis indicated that the PIs stored into adipose cells displayed a chemical profile identical to the native form of the PIs (not shown).…”

“…For that purpose, intracellular APV-dansyl and IDV-MANT have been recovered from adipocytes 2 days after incubation and analyzed by HPLC-UV. No uncoupled PIs and no free fluorophore were detected, indicating that the labeling was stable and that the fluorescence reflected the PI localization.Competitive ELISA-RTV, NVP, and LPV were quantified by competitive ELISA as previously described (15,16). Results were expressed as nanomole of HIV drugs/10 6 cells.…”

Development of sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in plasma

“…Assays were performed in 96-well microtiter plates coated with mouse monoclonal anti-rabbit IgG antibodies (Bertin Pharma, Montigny le Bretonneux, France) as previously described [16]. After washing the coated microtiter plate, the assay was performed in a total volume of 150 μL.…”

Quantitative Immunoassay To Measure Plasma and Intracellular Atazanavir Levels: Analysis of Drug Accumulation in Cultured T Cells

“…Enzyme immunoassays were performed in 96-well microtiter plates coated with mouse monoclonal anti-rabbit immunoglobulin G antibodies (Jackson, West Grove, PA), as described previously (3). After the coated microtiter plate was washed, the assay was performed in a total volume of 150 l. The following were successively added to each well: 50 l of calibrator, quality control, buffer, or sample; 50 l of enzyme tracer (2 Ellman units/ml); and 50 l of diluted antiserum.…”