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 approximately 100 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 micro 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.

FIG. 1.

Typical standard curve for NVP EIA. The experiments were performed as described in Materials and Methods. Duplicates are shown.

FIG. 2.

Effect of temperature on the efflux of NVP at 4 (squares) and 37°C (triangles). The data are expressed as means ± standard deviations (n = 2).

FIG. 3.

Typical immunochromatographic profiles of plasma (A) and PBMC extract (B) from HIV-infected patient taken 12 h after intake of NVP. The elution time of NVP spiked in drug-free plasma was 14 to 16 min.

FIG. 4.

Median plasma concentrations (solid circles) with 95% confidence intervals and plasma concentrations (open circles) versus time curves of NVP in seven HIV-infected patients after oral intake at a dose of 200 mg b.i.d.