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Antimicrobial Agents and Chemotherapy, January 2004, p. 104-109, Vol. 48, No. 1
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.1.104-109.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Stéphane Azoulay,1 Marie-Claire Nevers,2 Christophe Créminon,2 Laurence Heripret,3 Jacques Durant,3 Pierre Dellamonica,3 Jacques Grassi,2 Roger Guedj,1 and Danièle Duval1*

Laboratoire de Chimie Bio-Organique, Université de Nice-Sophia Antipolis,1 Service des Maladies Infectieuses et Tropicales, Hôpital Archet, Nice,3 CEA, Service de Pharmacologie et d'Immunologie, DRM, CEA Saclay, 91191 Gif-sur-Yvette Cedex, France2

Received 12 May 2003/ Returned for modification 18 September 2003/ Accepted 7 October 2003


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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 ~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 µl 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.


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INTRODUCTION
 
Nevirapine (Viramune) (NVP) is a nonnucleoside reverse-transcriptase 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-1) and by fastidious workup, thus excluding their use in the ex vivo monitoring of intracellular levels of the drug.

In this report, we describe the development and application of a competitive enzyme immunoassay (EIA) with a 100-times-better limit of detection. This new assay is based on the use of specific anti-NVP polyclonal antibodies raised in rabbits and an enzyme tracer prepared from a synthetic derivative of NVP. We took advantage of the high sensitivity of the assay to measure and compare NVP levels in the plasma and, for the first time, in PBMC of HIV-infected patients.


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MATERIALS AND METHODS
 
Reagents. Unless otherwise stated, all reagents and solvents were of analytical grade and were from Sigma (St. Louis, Mo.). Keyhole limpet hemocyanin (KLH) was from Pierce (Bezons, France). Acetylcholinesterase (AChE) (EC 3.1.1.7), extracted from the electric organ of the Electrophorus electricus eel, was purified by affinity chromatography as previously reported (1). Ellman's reagent was a solution of 7.5 x 10-4 M acetylthiocholine iodide (enzyme substrate) and 5 x 10-4 M 5,5-dithiobis-2-nitrobenzoic acid (chromogen) in 0.1 M phosphate buffer, pH 7.4.

All reagents used for immunoassays were diluted in EIA buffer (0.1 M potassium phosphate, pH 7.4, containing 0.15 M NaCl, 0.1% bovine serum albumin, and 0.01% sodium azide). The washing buffer was a 10 mM phosphate containing 0.05% Tween 20.

Apparatus. Solid-phase EIA was performed in 96-well microtiter plates (Immunoplate Maxisorb with certificate; Nunc, Roskilde, Denmark) using specialized microtitration equipment, a washer (Atlantis+; ASYSHitech, Engendorf, Austria), and an automatic plate reader (MRX microplate reader; Dynex Technologies, Chantilly, Va.). HPLC experiments were performed with a Waters (St Quentin en Yvelines, France) apparatus, including HPLC 600 pumps, a model 996 photodiode array detector and Millennium chromatographic manager, and a fraction collector (Retriever IV; Isco, Lincoln, Nebr.).

Immunogen preparation and immunization. After chemical modification to introduce an arm spacer bearing a carboxylic function, NVP was coupled to KLH and administered to rabbits in order to induce the synthesis of antibodies as follows.

A solution of methyl 5-bromovalerate (60 µl; 0.36 mmol) in N,N-dimethylformamide (DMF) (1.5 ml) was added dropwise to a mixture of NVP, 5,11-dihydro-11-cyclopropyl-4-methyl-6H-dipyrido[3,2-b:2',3'-e] (1,4)diazepin-6-one (100 mg; 0.36 mmol) and potassium carbonate (78 mg; 0.56 mmol) in DMF (1.5 ml). The reaction mixture was stirred overnight at reflux before the solvent was removed in vacuo. The crude residue was purified by chromatography on a silica gel (hexane-ethyl acetate; 50/50) to generate 5,11-dihydro-5-N-(methylpentanoate)-11-cyclopropyl-4-methyl-6H-dipyrido[3,2-b:2',3'-e] (1,4)diazepin-6-one, an NVP-protected spacer, as a yellow oil (71.4 mg; 50%).

The NVP-protected spacer (70 mg; 0.17 mmol) and 0.1 N LiOH (1.95 ml; 0.2 mmol) were stirred for 1 h at room temperature before the solvent was removed in vacuo. The crude residue was purified by chromatography on a silica gel (hexane-ethyl acetate; 10/90) to generate the NVP spacer, 5,11-dihydro-5-N-(carboxypentan)-11-cyclopropyl-4-methyl-6H-dipyrido[3,2-b:2',3'-e] (1,4)diazepin-6-one, as a yellow oil (62 mg; 95%). All products were characterized by nuclear magnetic resonance and mass spectrometry.

The NVP spacer was covalently coupled to KLH by reacting the corresponding activated N-hydroxysuccinimide (NHS) ester with primary amino groups of the carrier. Briefly, NVP spacer (3.83 mg; 10 µmol) was reacted with NHS (5.55 mg; 48 µmol) in the presence of N,N'-dicyclohexylcarbodiimide (2.03 mg; 10 µmol) in anhydrous DMF (360 µl). After overnight incubation at room temperature in the dark, the mixture was incubated for another 6 h with KLH (10 mg), previously dissolved in 5 ml of 0.1 M borate buffer, pH 9.0. The immunogen was then extensively dialyzed against 0.1 M phosphate buffer, pH 7.4; aliquoted; and kept frozen at -20°C until it was used.

Rabbits (Blanc du Bouscat, Evic, France) were immunized with 1 mg of immunogen using complete Freund's adjuvant and multiple subcutaneous injections. Booster injections (1 mg of immunogen in complete Freund's adjuvant) were repeated every 2 months for 8 months. The rabbits were bled from the central ear artery 1 week after each booster injection. The blood was centrifuged, and the sera were stored at 4°C in the presence of sodium azide (0.01% final concentration).

Enzymatic-tracer preparation. The tracer was obtained by covalently coupling the NVP spacer to AChE. NVP spacer (383 µg; 1 µmol) was reacted with NHS (1.15 mg; 10 µmol) in the presence of N,N'-dicyclohexylcarbodiimide (203.6 µg; 1 µmol) in 400 µl of DMF for 18 h at 20°C in the dark; 12.5 nmol (5 µl in DMF) of this NHS-activated ester were reacted with AChE (0.33 nmol; 100 µg) in 750 µl of 0.1 M borate buffer, pH 9.0. After a 2-h reaction at room temperature, the enzymatic tracer was purified by molecular sieve chromatography using a Bio-Gel A 1.5-m column (7) (90 by 1.5 cm; Bio-Rad) and stored at -20°C until it was used.

EIA. Plasma samples were heated at 60°C for 40 min (NVP proved to be stable under these conditions [17]) and centrifuged (12,000 x g for 30 min) before EIA was performed. PBMC were prepared using cell preparation tubes (4-ml Vacutainer CPT tubes [Becton Dickinson]): blood samples were collected and mixed with the anticoagulant by gently inverting the CPT tube 8 to 10 times. After centrifugation of the blood for 30 min at 1,500 x g, PBMC were found in a diffuse layer above the gel. The plasma was removed and collected, and the PBMC were poured into a washing tube and washed three times with cold phosphate-buffered saline (PBS) (5 ml; +4°C) and centrifuged (2,000 x g for 5 min at +4°C). Before the last cell centrifugation, an aliquot was taken to count the cells in Malassez cells. The washing procedure was run as fast as possible (the whole procedure is usually accomplished within 30 min) at +4°C in order to avoid loss of NVP. The dry cell pellet was extracted for at least 2 h in 1 ml of methanol-acetonitrile mixture (60/40 [vol/vol]), samples were centrifuged (12,000 x g for 10 min), and the supernatant was dried under vacuum using a Speed Vac apparatus (Savant, Farmingdale, N.Y.). The dry residue was dissolved in 200 µl of EIA buffer. Assays were performed in 96-well microtiter plates coated with mouse monoclonal anti-rabbit immunoglobulin G antibodies (Jackson, West Grove, Pa.) in order to ensure the separation of bound and free moieties of the enzymatic tracer during the immunological reaction. The coating (200 µl of a 5-µg/ml monoclonal antibody solution in 0.05 M phosphate buffer, pH 7.4/well) was performed for 18 h at room temperature before the plates were washed (300 µl/well; three wash cycles) and saturated for 24 h at 4°C with EIA buffer (300 µl/well). Coated plates covered with an adhesive plastic sheet are stable for at least 3 months when stored at +4°C. After the coated microtiter plate was washed, the assay was performed in a total volume of 150 µl. To each well, 50 µl of calibrator, quality control, buffer, or sample; 50 µl of enzyme tracer (2 Ellman units ml-1); and 50 µl of diluted antiserum were successively added. The working dilutions for the different rabbit antisera were previously determined by serial-dilution experiments. After 24 h of immunoreaction at +4°C, the plates were washed, and 200 µl of Ellman's reagent was added to each well. After the plates were gently shaken for 2 h in the dark at room temperature, the absorbance at 410 nm (reference filter at 570 nm) was measured in each well. The results are expressed in terms of B/B0 x 100 as a function of the concentration (logarithmic scale); B and B0 represent the bound enzymatic activity in the presence and absence of competitor, respectively. A linear log-logit transformation was used to fit the calibration curve. The sensitivity of the assay was characterized by the limit of detection (LOD), taken as the concentration of competitor inducing a significant decrease (3 standard deviations) in B0. Nonspecific binding represented <0.1% of the signal. All measurements for calibrators (78.125, 156.25, 312.5, 625, 1,250, 2,500, 5,000, and 10,000 pg ml-1) and samples were done in duplicate (in quadruplicate for B0 values).

Validation studies. The specificity of the NVP assay was checked by testing its capacity to detect compounds likely to be present in NVP-treated subjects by establishing the corresponding calibration curve for each of them. The results were expressed in terms of the percentage of cross-reactivity, defined as the ratio (percent) of the concentration of NVP and compounds at a B/B0 ratio of 50%. The repeatability and reproducibility were estimated by calculating the coefficients of variation (CV) of calibrator samples plotted the same day and of the quality controls (100, 500, 1,000, and 10,000 pg ml-1) added in drug-free plasma samples and cell pellets assayed in six independent assays. The accuracies for the quality controls (100, 500, 1,000, and 10,000 pg ml-1) added to drug-free plasma samples and cell pellets were calculated as the ratio between measured and theoretical concentrations multiplied by 100.

HPLC was combined with competitive EIA to further validate the nature of the immunoreactivity present in plasma or cell extracts and to evaluate the possible recognition of NVP metabolites and/or endogenous compounds by the antibodies. Sixty microliters of plasma from HIV patients receiving NVP were injected into a reversed-phase C18 column (Hypersil HS C18; 5-µm; 250 by 4.6 mm; Thermohypersil, Les Ulis, France). Chromatographic elution was achieved at a flow rate of 1 ml min-1 using a linear gradient from 30 to 100% of solvent B in 30 min (solvent A, H2O-0.1% trifluoroacetic acid; solvent B, acetonitrile). One-milliliter fractions were collected, dried under vacuum, and resuspended for 1 h in 150 µl of EIA buffer before NVP was assayed. The same experiment was performed using 60 µl of cellular extracts.

Cell cultures in the presence of nevirapine. Cultured human T-lymphoblastoid (CEM) CCL-19 cells (American Type Culture Collection, Manassas, Va.) were grown at 37°C in RPMI 1640 medium supplemented with 0.2 mM sodium pyruvate, 0.2 mM glutamine, 10% heat-inactivated fetal calf serum, and antibiotics in a 5% CO2 humidified atmosphere. The cell density was adjusted to 1.3 x 106 ml-1, and 15 ml of the suspension was incubated with 0 to 20 µg of nevirapine ml-1 for 12 h at 37°C. The cell suspensions were then centrifuged (2,000 x g for 5 min) and washed as described above. The dry pellet was extracted with a methanol-acetonitrile mixture (60/40 [vol/vol]) for at least 2 h at -20°C. The insoluble material was removed by centrifugation (10,000 x g for 10 min), and the supernatant phase was dried under vacuum before being dissolved in 200 µl of EIA buffer for 1 h and assayed for NVP content.

Drug efflux was assessed at 4 and 37°C. Cell suspensions were incubated with 20 µg of NVP ml-1 for 12 h at 37°C. The cell suspensions were centrifuged (2,000 x g for 5 min at +4°C) and resuspended in a drug-free medium at 4 and 37°C. At baseline and then 5, 20, 40, 60, and 90 min after resuspension, the cells were centrifuged (2,000 x g for 5 min at +4°C) and treated as described above before NVP quantification.

Intracellular concentrations were calculated assuming a 1-pl volume for a single CEM cell and PBMC.

Pharmacokinetics of NVP in patients. The study protocol was approved by the Institutional Ethics Committee, and written informed consent was obtained from all participants. Each subject underwent a 12-h NVP pharmacokinetic study. Seven patients (four male and three female) were included. The median age of the patients was 42 years (range, 30 to 52 years). The combination regimen included stavudine and lamivudine or zidovudine and lamivudine. The patients had a median CD4-cell count of 720 x 106 cells liter-1 (range, 321 x 106 to 1,020 x 106 cells liter-1), and one subject had an undetectable viral load in plasma (<20 copies ml-1; Roche [Basel, Switzerland] Monitor 20). The median viral load among the remaining patients was 20 copies ml-1 (range, 20 to 490 copies ml-1). After the patients fasted overnight, blood samples were collected in heparinized tubes at baseline and after 1, 2, 3, 4, 6, and 8 h from patients on the twice-daily regime. At 12 h, blood samples were collected in 4-ml Vacutainer CPT tubes and treated as described above. To minimize drug efflux out of the cells and to obtain accurate results, all blood samples were treated within 2 h. Plasma and dry cell pellets were stored at -80°C until they were analyzed. The CD4 cell count and plasmatic HIV RNA were measured by routine laboratory testing. NVP levels in plasma and PBMC were measured by EIA, and the area under the concentration-time curve (AUC) was calculated for each patient. The trapezoidal AUC was determined between the first and last (12-h) sampling times.

Pharmacokinetic analysis was performed using Prism software (version 3.03; GraphPad Software, San Diego, Calif.).


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RESULTS
 
The carboxylic function added to the synthetic NVP derivative (see Materials and Methods) provides a useful way to couple NVP to KLH, which is an obligatory step toward specific antibody production and AChE. Very good antibody titers (above 1/100,000) was measured by EIA for the two immunized rabbits from the second booster injection onward. Using the NVP-AChE tracer, we selected the best antiserum and optimized the dilution for the assay. A typical routine calibration curve for the NVP assay using antiserum (L1667-S5) at an initial dilution of 1/250,000 is shown in Fig. 1. The sensitivity was characterized by an LOD close to 100 pg ml-1 (7 fmol/well) in EIA buffer. The precision was also very satisfactory, since the CV for the standard curves from six independent experiments ranged from 3.3 to 12%, while the errors ranged from -2 to 6% in reproducibility experiments. There was no obvious bias, since the errors were both positive and negative.



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  		FIG. 1. Typical standard curve for NVP EIA. The experiments were performed as described in Materials and Methods. Duplicates are shown.

When assaying pure plasma samples, we observed a matrix effect, leading to changes in the sensitivity and precision characteristics of the assay. A fivefold dilution of these samples in EIA buffer totally eliminated these interferences but resulted in a lower absolute sensitivity (an LOD close to 500 pg ml-1) linked to the dilution of plasma samples. However, the original sensitivity of the assay could be recovered by performing a simple extraction procedure with a mixture of methanol-acetonitrile (60/40 [vol/vol; 1 volume of plasma/3 volumes of methanol-acetonitrile]), which allows the precipitation of most of the plasma proteins and their elimination by centrifugation. The supernatant phase can thus be treated like those of the cell pellet (data not shown). For intracellular quantification, the LOD is dependent on the number of cells from which the cellular extract is obtained. Based on an average of 2 x 106 cells (the minimum number of cells that allows good precision and accuracy), the LOD is close to 100 pg ml-1.

The assay precision and accuracy were evaluated with normal human plasma and CEM cell extracts spiked with known concentrations of NVP. As shown in Table 1, recovery of NVP ranged from 87 to 104% in both samples, with a CV between 5.5 and 10.8%.


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  		TABLE 1. Validation parameters for EIA of NVP in plasma and cells

Drug efflux experiments showed that efflux of NVP at 37°C was rapid (Fig. 2), with a loss of nearly 80% within 20 min. However as demonstrated previously for protease inhibitors (13), the use of ice-cold PBS and centrifugation at 4°C resulted in the retention of >80% of the intracellular drug at 60 min. Drug efflux from cells was therefore critically time and, more importantly, temperature dependent.



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  		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).

The specificity of the polyclonal anti-NVP antibodies was first analyzed with antiviral agents commonly used during HIV treatment. No interference, as characterized by cross-reactivity of <0.01%, occurred with drugs structurally unrelated to NVP, such as zidovudine, lamivudine, dideoxyadenosine, dideoxyinosine, abacavir, lopinavir, ritonavir, nelfinavir, indinavir, and saquinavir, while low cross-reactivity (<2%) was observed for efavirenz (EFZ), which has some structural similarities to NVP.

Since the main known metabolites of NVP (18) were not available to us as pure chemical compounds, potential recognition by the antibodies of NVP metabolites or endogenous compounds in plasma and PBMC samples from HIV patients was evaluated through HPLC fractionation coupled to EIA detection. As shown in Fig. 3, we observed that immunoreactive materials in both types of sample eluted as a single homogeneous peak corresponding to the elution volume of true NVP.



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  		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.

The assay was first used in a 12-h plasma pharmacokinetic study in seven HIV patients given NVP at a dose of 200 mg b.i.d. The median plasma pharmacokinetic profile is shown in Fig. 4. The median total AUC was 52,510 ng · h ml-1 (range, 46,340 to 72,910 ng · h ml-1). The median values for the maximum concentration of drug in serum and plasma drug concentration at 12 h (Ctrough) were 6,085 ng ml-1 (range, 5,820 to 8,910 ng ml-1; CV, 28%) and 3,735 (range, 2,838 to 5,287 ng ml-1; CV, 10%), respectively.



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  		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.

The immunoassay proved to be sensitive enough to measure the intracellular concentrations of NVP. This was observed in CEM cells cultured in the presence of three different concentrations of NVP (200, 2,000, and 20,000 ng ml-1) for 24 h. The results, summarized in Table 2, demonstrated a concentration-dependent uptake of NVP into CEM cells. However, the intracellular NVP concentrations represented only 2 to 20% of the extracellular concentrations, showing that CEM cells did not accumulate NVP.


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  		TABLE 2. Intracellular/extracellular ratio of NVP

At each step of washing, the PBS medium was assayed for NVP content. No NVP was detected in the third wash, confirming that three washes with ice-cold PBS were required to remove any contaminating medium containing NVP. Furthermore, all of the CEM cell pellets were extracted twice, and no NVP was detected in the second extraction.

The intracellular NVP content was further assayed in PBMC from seven HIV patients taken at Ctrough, presenting a median NVP concentration close to 517 ng ml-1 (range, 81 to 2,045 ng ml-1; CV, 81%) (Table 2). The median plasma value was sevenfold higher at 3,735 ng ml-1 (range, 2,838 to 5,287 ng ml-1; CV, 10%). It is worth noting that the intracellular concentrations reported in Table 2 are derived from measurements made of cellular extracts (currently, 200-µl volume) and were calculated assuming a 1-pl intracellular volume for both a CEM cell and a PBMC. Clearly, the concentrations in cellular extracts are at least 80-fold lower, which explains why they cannot be determined by methods with a lower sensitivity. The calculated intracellular/extracellular concentration ratios for these seven patients ranged from 0.02 to 0.50, with the median at 0.134. Taken together, these data indicated that the intracellular NVP concentrations and the intracellular/extracellular concentration ratios measured for HIV patients vary more than the plasma levels at Ctrough. On the other hand, low absolute intracellular NVP concentrations were observed in both CEM cells and PBMC from HIV patients.


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DISCUSSION
 
This paper reports the development of a new competitive EIA of NVP allowing measurements in biological fluids and cell extracts. The polyclonal anti-NVP antibodies raised in rabbits are specific for NVP, since no interference due to other anti-HIV drugs or endogenous plasma or cell compounds was detected. The low cross-reactivity observed with EFZ is not surprising, since NVP and EFZ are structurally related compounds. However, this is not of major concern, since the combination of the two drugs is not currently prescribed for HIV treatment. Furthermore, a recent trial (F. van Leth, E. Hassink, P. Phanuphak, S. Miller, B. Gazzard, P. Cahn, R. Wood, K. Spires, C. Katlama, B. Santos, P. Robinson, R. van Leenven, F. Wit, and J. Lange, Abstr. 10th Conf. Retroviruses Opportunistic Infect., abstr. 176, 2003) showed that the combination of NVP and EFZ did not provide additive benefit and, moreover, increased the adverse side effects. The present assay was also highly sensitive, with a detection limit of 100 pg ml-1, which compares favorably with those previously reported for UV and tandem mass spectrometry methods: close to 50 (6, 10, 22) and 10 (14) ng ml-1, respectively. The accuracy and precision of the EIA were also good, so the assay is useful for pharmacokinetic studies. No extraction step is required to assay plasma samples (in this case, the LOD is decreased to 500 pg ml-1), and the analytical method can be carried out in <24 h in any conventional laboratory.

As a first application of the assay, we analyzed a limited number of plasma samples from HIV patients to define the plasma pharmacokinetic parameters. The median AUC, observed here as the maximum concentration of drug in serum and Ctrough, is 52,510 ng · h ml-1 (6,085 and 3,735 ng ml-1, respectively), which is in accordance with published data (55,950 ng · h ml-1 and 5,860 and 3,720 ng ml-1) (20, 23). To better illustrate the value of the method, we measured the intracellular uptake of NVP. PBMC are considered a major site of virus-host interaction (15, 26), and knowledge of intracellular penetration is crucial to a better understanding of the development of drug resistance and the failure of antiretroviral therapy. How PBMC accumulate NVP is not presently known, since the available techniques are not sensitive enough to estimate intracellular concentrations of NVP. This is the first intracellular study of NVP. The first experiments performed with CEM cells demonstrated the capacity of the present immunoassay to accurately measure the intracellular NVP content and also indicated that cells take up NVP. Moreover, we observed that cells do not accumulate NVP, since the measured intracellular NVP concentrations corresponded to only 2 to 20% of the NVP concentration present in the culture medium. These results could suggest that the uptake of NVP was very slow and that equilibrium had not been reached after 24 h of contact with the drug. However, this hypothesis seems unlikely, since longer incubation times did not result in a larger accumulation ratio (data not shown). Alternatively, it can be hypothesized that NVP is secreted via an active process or degraded by the cells. Cell-washing experiments and study of the efflux kinetics of NVP emphasize once again the importance of following stringent laboratory procedures when intracellular measurements are performed.

Finally, intracellular NVP concentrations were also determined in PBMC from HIV patients at Ctrough. These data have to be interpreted cautiously, because they have been calculated assuming that the intracellular volume is precisely 1 pl, which is not clearly demonstrated. In addition, taking into account the difficult problem of preparing cell extract under conditions that avoid any loss of intracellular drug or contamination with extracellular drug, these data have to be considered preliminary. Despite these reservations, the results indicated a median intracellular concentration (517 ng ml-1) that was seven times lower than the median concentration in plasma. The intracellular/extracellular concentration ratios observed with PBMC (0.02 to 0.5) are in the same range as those calculated for cultured CEM cells (0.016 to 0.23). As a comparison, intracellular NVP concentrations in PBMC are lower than those generally observed for protease inhibitors (12). Moreover, we observed a large interindividual variation in the intracellular NVP concentration at Ctrough (CV, 81%) that was greater than the fluctuation of the corresponding plasma NVP concentration (CV, 10%). Although NVP did not appear to be a P-glycoprotein substrate in cell culture (21), these variations may be due to other transport proteins, such as multidrug resistance-associated protein, whose expression can vary greatly among patients. Several publications have reported that transport proteins show variable expression in the general population and play an important role in the intracellular concentration (3, 11, 25). Moreover, binding to plasma protein reduces the fraction of drug available for penetration into cells. In vitro studies have shown that plasma protein binding of NVP was 62% (4), and fluctuations in the concentration of plasma protein in HIV patients may also account for differences (2).

It is worth noting that conflicting results have been reported relating to the correlation between the plasma NVP concentration and virological response (8; G. Peytavin, J. L. Meynard, C. Lamotte, F. Brun-Vénizet, and D. Costagliola, Abstr. 4th Int. Workshop Clin. Pharmacol. HIV Ther., abstr. 17, 2003). Moreover, resistance to NVP develops rapidly when the drug is administered in suboptimal regimens, and side effects such as rash are common (5). These observations and the results described above emphasize the need for complementary studies of intracellular quantification. This assay provides the opportunity to further explore the relationship between the intracellular concentration of NVP and its efficacy and toxicity in HIV-infected patients. In addition to the potential advantages of NVP EIA in intracellular quantification studies, as demonstrated here, the technique should also be useful in the clinical laboratory for monitoring patients during antiretroviral therapy.


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ACKNOWLEDGMENTS
 
This work was supported in part by the French National Agency for AIDS Research (ANRS), Ensemble contre le Sida (SIDACTION), and CAMPLP. S. Azoulay is the recipient of a Ph.D. fellowship from the CNRS and region PACA.

We gratefully acknowledge Catherine Frelin for the CEM culture and Christian Frelin for critical reading of the manuscript and for helpful discussions. We are also indebted to M. A. Serini for her help during this work.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Chimie Bio-Organique UMR 6001, Faculté des Sciences, Université de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice Cedex 2, France. Phone: 33 4 92 07 61 15. Fax: 33 4 92 07 61 51. E-mail: duvald{at}unice.fr. Back


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Antimicrobial Agents and Chemotherapy, January 2004, p. 104-109, Vol. 48, No. 1
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.1.104-109.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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