Intracellular and Plasma Pharmacokinetics of Nelfinavir and M8 in HIV-Infected Patients: Relationship with P-Glycoprotein Expression

Abstract

One of the targets of antiretroviral therapy is within cells infected with HIV. In order to improve therapeutic efficacy, it is therefore important that the intracellular pharmacokinetics of drugs, such as nelfinavir mesylate and its active metabolite M8, are studied in addition to plasma pharmacokinetics. Previously, the intracellular accumulation of protease inhibitors has been reported in vivo, displaying the following hierarchy: nelfinavir > saquinavir > ritonavir > indinavir. Multidrug resistance transporters, such as P-glycoprotein (P-gp), may result in a lower intracellular concentration of drug via an efflux mechanism, thus contributing to sanctuary site formation. The objective of this study was to determine concentrations of nelfinavir and M8 in plasma and peripheral blood mononuclear cells from HIV-infected patients, and to ascertain the relationship between intracellular accumulation and lymphocyte P-gp expression. Venous blood samples from 12 HIV-infected patients (viral load <50 copies/ml) receiving nelfinavir (1250 mg twice daily) and dual nucleoside reverse transcriptase inhibitor therapy were collected over a full dosage interval (0, 2, 4, 8 and 12 h). Plasma and intracellular (cell-associated) drug concentrations were measured by HPLC-MS/MS. Drug exposure in plasma and cells was expressed as the area under the concentration–time curve (AUC0-12h), derived from non-compartmental modelling. The ratio of intracellular AUC0-12h/total plasma AUC0-12h was calculated to determine cellular drug accumulation. P-gp expression on lymphocytes was determined by flow cytometry. The median (range) AUC0-12h of nelfinavir in plasma and cellular compartments was 21.8 mg.h.l-1 (5.64–50.8) and 104.6 mg.h.l-1 (23.1–265.7), respectively. Corresponding values for M8 in plasma and cells were 6.60 mg.h.l–1 (2.16–17.3) and 19.6 mg.h.l–1 (5.14–60.8). A ratio of plasma M8/plasma nelfinavir (AUC0–12h) and intracellular M8/intracellular nelfinavir (AUC0–12h) gave median values of 0.32 and 0.17, respectively. The cellular accumulations [median; (range)] of nelfinavir and M8 were 5.30 (2.28–16.2) and 2.32 (1.01–10.7), respectively. A significant correlation between plasma and intracellular nelfinavir minimum concentration (Cmin) (r2=0.34; P=0.049), but not between plasma and intracellular M8 Cmin was observed. C0h concentrations were higher than C12h for both nelfinavir and M8. No relationship was observed between nelfinavir or M8 accumulation and lymphocyte cell surface expression of P-gp. This study illustrates that intracellular concentrations were higher than plasma concentrations for both nelfinavir and M8, suggesting lymphocyte accumulation. The mechanism of differential intracellular accumulation of nelfinavir and M8 remains to be elucidated. It may be that affinities for influx transporters or fundamental drug characteristics play a major role in the greater accumulation of nelfinavir than M8.

Correction: Ultra-Fast Analysis of Plasma and Intracellular Levels of HIV Protease Inhibitors in Children: A Clinical Application of MALDI Mass Spectrometry

“…Runtime of that assay was 20 minutes, and accuracies were still below the recommended 15% when a variable amount of input material, i.e. between 1.4610 6 PBMCs and 9.6610 6 PBMCs, was used for the assay. Pe `lerin et al described an LC-ESI-MS assay with an LLOQ of 70 femtomole per mL intracellular volume for ritonavir and 80 femtomole per mL intracellular volume for lopinavir [30].…”

“…Runtime of the assay was 20 minutes. Ter Heine et al described an LC-ESI-MS assay with LLOQ of 1.9 femtomole/3610 6 PBMCs for lopinavir and an LLOQ of 1.7 femtomole/3610 6 PBMCs for ritonavir [31]. Runtime of the assay was 10 minutes.…”

“…All stock solutions for MALDI-triple quadrupole MS analysis were prepared in methanol by serial dilution. As internal standards we used: 20 mM nelfinavir (quantitation of lopinavir and ritonavir in plasma), 5 mM methotrexate (quantitation of saquinavir, nelfinavir, and indinavir in plasma), 2 mM nelfinavir (quantitation of lopinavir and ritonavir in 1610 6 PBMCs). To investigate the effect of co-medication on the quantitation performance, the following drug mixtures were added to the quality control samples: drug mixture A: 10 mM carbamazepine, metoprolol, metronidazole, amoxicillin, piroxicam, nevirapine, saquinavir, efavirenz, indinavir, and tipranavir (quantitation of lopinavir and ritonavir in plasma).…”

“…HIV protease inhibitors exert their action inside cells that are susceptible to HIV infection. The intracellular pharmacokinetics of these drugs, its relation to the plasma pharmacokinetics, and its relation to drug efficacy and toxicity is therefore an active area of research [1][2][3][4][5][6][7][8][9][10]. However, determination of the intracellular levels of antiretroviral drugs is an analytical challenge, because of the small amount of target cells, e.g.…”

Quantitative Analysis of HIV-1 Protease Inhibitors in Cell Lysates Using MALDI-FTICR Mass Spectrometry

“…The “biological” LLOQs are thus 40 fmol for saquinavir, 160 fmol for lopinavir, 310 fmol for ritonavir, and 1 pmol for indinavir and nelfinavir per million PBMCs. The reported minimum intracellular concentrations ( C min or C predose ) of HIV-1 protease inhibitors in HIV-1 infected adults are 6.4 pmol/(1 × 10 6 PBMC) for lopinavir, 850 fmol/(1 × 10 6 PBMC) for ritonavir, 450 fmol/(1 × 10 6 PBMC) for saquinavir, 87 fmol/(1 × 10 6 PBMC) for indinavir, and 2.1 pmol/(1 × 10 6 PBMC) for nelfinavir . Except for indinavir, the MALDI-FTICR assay can thus be used for quantitative analysis of HIV-1 protease inhibitors in 1 000 000 PBMCs.…”