Objectives: To investigate the intracellular accumulation of HIV protease inhibitors (PI) and to assess the effect of active transport on this accumulation.
Methods: CEM cells were incubated with a PI for 18 h and the intracellular concentration determined using cell number and radioactivity. The effect of active transport was investigated using cells expressing P-glycoprotein (CEMVBL) and cells expressing multidrug resistance-associated protein 1 (MRP1; CEME1000). Incubations were also carried out at 4°C and in the presence of 2-deoxyglucose plus rotenone to examine the effect of inhibiting active transport.
Results: Nelfinavir (NFV) accumulated to the greatest extent (> 80-fold) followed by saquinavir (SQV; ∼ 30-fold), ritonavir (RTV; 3–7-fold) and finally indinavir (IDV; extracellular equivalent to intracellular). In CEMVBL cells there was a significant reduction in the intracellular accumulation of NFV, SQV and RTV and in CEME1000 cells there was reduced accumulation of SQV and RTV. Inhibition of active transport processes caused a reduction in SQV and RTV accumulation but had no effect on IDV accumulation in all cell types. NFV accumulation was increased in CEMVBLcells as a result of inhibition of active transport.
Conclusions: Marked differences can be detected in the intracellular accumulation of HIV PI drugs in vitro. Both P-glycoprotein and MRP1 may play a role in limiting the intracellular concentration of the PI and active influx mechanisms may contribute to drug accumulation.
From the aDepartment of Pharmacology and Therapeutics, University of Liverpool, Liverpool, UK, and the bClinical Oncology Department, Royal North Shore Hospital, St. Leonards 2065, Australia.
Received: 17 August 2000;
revised: 17 November 2001; accepted: 30 January 2001.
Requests for reprints to Dr K. Jones, Department of Pharmacology and Therapeutics, University of Liverpool, New Medical Building, Ashton Street, Liverpool L69 3GE, UK.
The introduction of protease inhibitors (PI) into treatment regimens resulted in a marked increase in potency of antiretroviral therapy and, consequently, improved survival and decreased mortality . However, many patients still fail to achieve maximal virological suppression despite antiretroviral therapy .
The bulk of HIV is contained and replicates within cells. Antiviral drugs need to penetrate into these cells at concentrations high enough to inhibit viral replication in order to be effective. Failure to do so potentially results in the establishment of a sanctuary site where virus may replicate unhindered by drug action. These sanctuary sites may evolve as a result of expression of efflux proteins on the surface of cell membranes that reduce the accumulation of drugs within cells by pumping the drug out.
While the relationship between PI and influx transporters is unknown, PI drugs are substrates for the efflux transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP1) [3,4]. P-gp is a 170 kDa transmembrane efflux pump encoded by the gene MDR1 and is expressed in the intestinal lumen, at the blood–brain barrier , the blood–testis barrier  and on normal peripheral blood mononuclear cells (PBMC) . In MDR1 knockout mice, oral administration of the PI drugs nelfinavir (NFV), indinavir (IDV) and saquinavir (SQV) resulted in a 2–5-fold increase in plasma concentration while intravenous administration of these drugs resulted in elevated brain concentrations of 7–36-fold .
MRP1 is a 190 kDa transmembrane efflux pump that has been shown to be present on a number of drug-resistant cancers that have little or no expression of P-gp . MRP1 is expressed on the choroid plexus epithelium and contributes to the blood–cerebrospinal fluid barrier . It is also expressed on PBMC .
In this study, differences in intracellular accumulation of PI [NFV, SQV, ritonavir (RTV) and IDV] were examined in vitro in CEM cells (a T-lymphoblastoid cell line) and in cells with increased expression of P-gp (CEMVBL) and MRP1 (CEME1000). The effect was also assessed of inhibiting active transport (influx and efflux) processes, by incubating at 4°C or with 2-deoxyglucose plus rotenone (D + R), and of HIV infection in vitro.
Materials and methods
CEM cells were used as a control cell line. CEMVBL cells were derived from CEM cells treated with vinblastine and have increased expression of the efflux transporter P-gp. CEME1000 cells were derived from CEM cells treated with epirubicin and have increased expression of the efflux transport protein MRP1. HIV-1 virus (strain U-455) was obtained from the Medical Research Council. [14C]-SQV (specific activity 26.5 μCi/mg; > 99% purity), [3H]-NFV (specific activity 11.3 μCi/mg; 99% purity) were donated by Roche Pharmaceuticals (Welwyn Garden City, UK); [3H]-RTV (specific activity 1.1 Ci/mmol; 99.9% purity) and [3H]-IDV (specific activity 1.1 Ci/mmol; 99% purity) were obtained from Moravek Biochemicals (Brea, California, USA); RTV was a gift from Abbot Laboratories (North Chicago, Illinois, USA) and IDV was donated by Merck (West Point, Pennsylvania, USA). All other chemicals were purchased from Sigma Chemical Co. (Poole, UK).
All cells were routinely maintained in 75 cm2 flasks containing RPMI growth media (RPMI 1640 media supplemented with 10% fetal calf serum and 2 mmol/l l-glutamine) at 37°C in a humidified 5% CO2 gassed incubator. These cells were maintained as a suspension (1 × 105 to 1 × 106 cells/ml), doubling approximately every 24 h.
Cell viability was assessed by the Trypan blue dye exclusion assay. Briefly, 4 μl Trypan blue (0.5% w/v) was added to 20 μl of cells and cell viability was determined by counting the number of cells unable to exclude the dye. A threshold of 98% viability was used.
Cells (10 × 106 in 10 ml) were incubated for 18 h at 37°C, at 4°C or with 2-deoxyglucose (2 g/l) and rotenone (100 nmol/l) (D + R) at 37°C in the presence of extracellular 1 or 10 μmol/l of the PI drugs NFV, SQV, RTV or IDV (0.05, 0.1, 0.135 and 0.135 μCi, respectively; n ≥ 4). In addition, cells chronically infected with HIV were incubated at 37°C with drug as detailed above. For the D + R experiments, the cells were washed twice in RPMI growth media containing D + R minus glucose. The cells were then pre-incubated in this media for 20 min to deplete ATP levels.
Following incubation, the cell suspensions were counted, centrifuged (2772 × g for 4 min at 4°C) and four 100 μl samples of the supernatant fraction were removed to determine the extracellular concentration. The excess supernatant fraction was then discarded and the resulting cell pellet was washed three times in 5 ml phosphate-buffered saline and centrifuged (2772 × g; 4 min; 4°C) to remove any excess radioactivity. The cell pellet was mixed in a vortex with 5 ml 60% methanol and extracted for at least 3 h. After extraction, the cells were again centrifuged (2772 × g; 4 min; 4°C) and the supernatant fraction was decanted into a glass tube and evaporated to dryness. The resulting residue was reconstituted in 150 μl of 60% methanol and 100 μl taken to determine the intracellular concentration by liquid scintillation counting.
Isolation of total RNA from cells
Total RNA was isolated from cells using ultraPURE TRIzol reagent (10 × 106 cells/ml TRIzol), according to the manufacturer's instructions (Gibco BRL, Life Technologies, Paisley, UK).
Measurement of MRP1 mRNA in cells
The mRNA encoding MRP1 and P-gp were measured by reverse transcription-polymerase chain reaction (RT-PCR). Before reverse transcription, total RNA was visualized by electrophoresis on a 1% agarose gel stained with ethidium bromide to determine RNA integrity. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the Promega Reverse Transcription System (Promega, Southampton, UK). Briefly, reactions contained 0.5 μg oligo(dT)15 primer, 15 U avian myeloblastosis virus reverse transcriptase, 1 mmol/l of each dNTP, 20 U ribonuclease inhibitor, 4 μl 5× reverse transcription buffer and 5 mmol/l MgCl2, in a total volume of 20 μl nuclease-free water. The reaction was carried out at 42°C for 1 h.
PCR amplification of MRP1 and P-gp cDNA was carried out in accordance with the method of Endo et al., with small variations in reaction conditions. Newly synthesized cDNA (1 μl of the reverse transcription reaction) was added to 49 μl of PCR mix containing 10× reaction buffer, 1.5 mmol/l MgCl2, 50 μmol/l of each dNTP, either MRP1 or P-gp primer, β2-microglobulin (β2-m; internal control) primer and 0.5 U recombinant Taq DNA polymerase (MBI Fermentas, Helena Biosciences, Sunderland, UK). Amplification was for 30 cycles of denaturing at 94°C for 1 min, annealing at 57°C for 1 min and extension at 72°C for 1 min, followed by a final polymerization step at 72 C for 9 min. The PCR products were separated using 3% agarose gel/ethidium bromide electrophoresis, scanned with a computed densitometer (Speedlight Platinum Gel Documentation System, Lightools Research, Encinitas, California, USA) and the integrated optical density of the MRP and P-gp bands were normalised against the β2-m internal control (Gel-Pro Analyser, Media Cybernetics, Silver Spring, Maryland, USA).
Results have been calculated using cell counts at the conclusion of each experiment. Statistical analysis was performed using a Mann–Whitney U-Test (ARCUS software). Intracellular concentrations were calculated using the volume of a single CEM, CEMVBLand CEME1000 cell to be 1 pl. This is based on fluorescence assisted cell sorting analysis in our laboratory (data not shown).
RT-PCR confirmed increased expression of P-gp mRNA in the CEMVBL cells compared with control CEM and CEME1000 cells (data not shown). There was also increased expression of MRP1 mRNA in CEME1000 cells compared with control CEM and CEMVBL cells (data not shown). These data are in accordance with those previously published .
Marked differences were observed in intracellular accumulation within CEM cells. For incubations using an extracellular concentration of 1 μmol/l drug, the intracellular concentrations were 84.5 ± 9.67, 33.4 ± 7.0, 7.1 ± 0.8 and 0.8 ± 0.1, respectively, for NFV, SQV, RTV and IDV (Tables 1–4; Fig. 1). Similar changes were seen at incubations of 10 μmol/l extracellular drug (Tables 1–4).
CEMVBL cells expressing high levels of P-gp had a reduced intracellular concentration of all four PI compared with control CEM cells at 37°C (extracellular concentration 1 μmol/l). NFV accumulation was reduced to 29.44 ± 4.88 μmol/l, SQV accumulation to 10.3 ± 1.5 μmol/l, RTV accumulation to 4.7 ± 0.4 μmol/l and IDV accumulation to 0.5 ± 0.1 μmol/l (Tables 1–4). These changes corresponded to falls of 65, 69, 34 and 37%, respectively. A similar pattern was observed at 10 μmol/l SQV, RTV and IDV; however, NFV accumulation was unchanged (Tables 1–4).
CEME1000 cells expressing high levels of MRP1 also showed reduced intracellular PI concentrations. At 1 μmol/l incubations, SQV accumulation was reduced to 15.4 ± 1.3 μmol/l and RTV accumulation was reduced to 5.3 ± 0.5 μmol/l. These changes corresponded to falls of 54% and 25% for SQV and RTV, respectively. However, NFV and IDV accumulation remained unchanged (Tables 1–3).
The effect of inhibiting active transport (influx and efflux) was examined by incubating the cells at 4°C. CEM cells incubated at 4°C (extracellular drug concentration 1 μmol/l) had reduced accumulation of SQV (by 77%) and RTV (by 80%) but there was no change in NFV or IDV accumulation (Tables 1–4). SQV accumulation was reduced by 45% in both CEMVBL and CEME1000 cells while RTV accumulation was reduced by 30% and 60% in CEMVBL and CEME1000 cells, respectively (Tables 1–4). NFV accumulation was restored to control (CEM) levels when incubated at 4°C in CEMVBL cells. No other differences were observed.
Similar findings were observed in all three cell types when D + R was used as a more potent inhibitor of active transport processes. At 1 μmol/l incubations in CEM cells, SQV accumulation was reduced to 1.9 ± 0.1 μmol/l (a reduction of 94%) and RTV accumulation was reduced to 0.8 ± 0.2 μmol/l (89%). Significant reductions were also seen with SQV (60%) and RTV (36%) in CEMVBL cells and in CEME1000 cells (SQV 86% and RTV 70%). D + R reduced the accumulation of NFV in CEM cells, increased accumulation in CEMVBL cells and had no effect in CEME1000 cells. All incubations performed with an extracellular drug concentration of 10 μmol/l produced similar results (Tables 1–4).
At 1 μmol/l drug incubations, CEM cells chronically infected with HIV showed reduced intracellular accumulation of RTV, to 4.2 ± 0.3 μmol/l (Table 2), and NFV, to 66.29 ± 6.06 μmol/l (Table 4); no effect was seen with SQV or IDV. The intracellular accumulation of SQV, NFV, RTV and IDV was reduced in chronically HIV-infected CEMVBL cells (Tables 1–4). Only NFV accumulation was not reduced in chronically HIV-infected CEME1000 cells.
In this study, we have demonstrated in vitro marked differences in the intracellular accumulation of PI, with NFV achieving intracellular concentrations of approximately 90-fold, SQV approximately 30-fold, RTV 3–7-fold and IDV demonstrating no appreciable intracellular accumulation. These data are in keeping with those of Nascimbeni et al., who found that differences in the antiviral kinetics of the PI could be a result of differences in the rates of cellular clearance.
Significantly reduced accumulation of NFV, SQV and RTV was observed in CEMVBL cells (increased P-gp expression) and of SQV and RTV in CEME1000 cells (increased MRP1 expression) but intracellular accumulation was not abolished, suggesting that there are other processes at work. These could either be intracellular trapping of drug (e.g., by binding onto protein) or the existence of active influx processes.
The inhibition of active transport by incubating at 4°C or with D + R resulted in a marked reduction in the intracellular accumulation of both SQV and RTV. D + R effectively abolished intracellular accumulation of SQV and RTV. This provides further evidence that active influx processes may mediate the intracellular accumulation of these drugs. These influx transporters are as yet uncharacterized.
HIV infection in vitro appears to reduce intracellular accumulation of the PI in cells expressing an efflux transporter. The effect of HIV infection on P-gp expression and function is unclear. Some reports have demonstrated that HIV infection increased P-gp expression in vitro and in vivo but the protein may be functionally defective. Other reports [18–20] showed decreased P-gp expression and also rearrangement of P-gp in HIV-infected patients. The effect of HIV infection on MRP1 has yet to be investigated. Our findings are not related to direct cytotoxicity induced by viral infection of the cells, as assessed by Trypan blue dye exclusion (data not shown).
Intracellular accumulation of drugs is a dynamic process influenced by cellular influx and efflux, plasma protein binding and intracellular trapping. PI drugs vary in their degree of plasma protein binding (> 90% for NFV, SQV, RTV and ∼ 60% for IDV) and in their affinity for P-gp. We have found that PI accumulate within cells in the rank order NFV > SQV > RTV > IDV. This finding may relate to differences in binding to intracellular proteins or to preferential concentration within some cellular subcompartments. However, the marked reduction in intracellular accumulation of SQV and RTV in the presence of D + R provides indirect evidence that ATP-dependent transport of these drugs into cells is important. Variability in plasma protein binding between the PI might be expected to reduce the observed differences between NFV, SQV and IDV. However, caution must be exercised when interpreting our findings since these may not directly translate into differences in clinical efficacy. Antiviral activity data in cell lines provide conflicting evidence in the presence of efflux pumps. Srinivas et al.  demonstrated no decline in antiviral efficacy in the presence of these pumps. In contrast a significant decrease in antiviral activity was observed in similar experiments .
Accumulation was assessed in the present study using radioactive labelling; consequently, some intracellular metabolic products containing the label may also be assessed and not just parent drug. It must be noted that the concentrations of extracellular PI used in this study, particularly 10 μmol/l, are generally significantly higher than usual unbound PI achieved in vivo and that cell exposure in vitro does not necessarily resemble the situation in vivo. The clinical relevance of these findings is unknown because of the cell types used.
We have previously shown that the addition of α1-acid glycoprotein decreases intracellular accumulation of SQV, RTV and IDV . This study highlights further important differences between the PI in intracellular accumulation and underlines the urgent need for studies assessing the intracellular pharmacokinetics of PI in patients, as well as the need to evaluate the degree of drug binding to intracellular proteins.
We are grateful to AVERT for providing us with financial support.
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Keywords:© 2001 Lippincott Williams & Wilkins, Inc.
protease inhibitors; antiretroviral therapy; accumulation; P-glycoprotein; multidrug resistance-associated protein