JAC Advance Access published online on September 21, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm353
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Inhibition of P-glycoprotein and multidrug resistance-associated proteins modulates the intracellular concentration of lopinavir in cultured CD4 T cells and primary human lymphocytes
1 School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, Co., Londonderry BT52 1SA, UK 2 Department of Pharmacology and Therapeutics, The University of Liverpool, 70 Pembroke Place, Block H, First Floor, Liverpool L69 3GF, UK
* Corresponding author. Tel: +44-28-70323125; Fax: +44-28-70324375; E-mail: o.janneh{at}ulster.ac.uk
Received 10 July 2007; returned 20 July 2007; revised 26 July 2007; accepted 17 August 2007
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Abstract |
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Background: HIV protease inhibitors (HPIs) are an important component of highly active antiretroviral therapy. However, the activity of drug efflux transporters, such as P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRP1/MRP2), may limit intracellular drug accumulation. Drugs that inhibit the activity of drug efflux proteins may, in combination with HPIs, enhance the clinical efficacy of the drugs.
Methods: The transport of [14C]lopinavir was evaluated in peripheral blood mononuclear cells (PBMCs) in the absence or presence of known inhibitors: tariquidar (P-gp), MK571 (MRP), frusemide (MRP1/2), dipyridamole (MRP1/P-gp) and probenecid (MRP2/OATP). The effects of ritonavir, amprenavir and atazanavir on the accumulation of lopinavir were also evaluated in cultured CD4+ T-lymphoblastoid cells [CEM (parental), CEMVBL (P-gp-overexpressing) and CEME1000 (MRP1-overexpressing)] and PBMCs. The relative expression of the drug efflux proteins on the PBMCs was assessed by flow cytometric and real-time PCR methods.
Results: Tariquidar, MK571, frusemide and dipyridamole all significantly increased the intracellular accumulation of lopinavir in the PBMCs, whereas probenecid decreased it. The cellular accumulation ratio (CAR) of lopinavir was also increased by ritonavir, amprenavir and atazanavir in a concentration-dependent manner in both cell types. The expression of P-gp, MRP1 and MRP2 mRNA were variable and individually did not correlate with CARs of lopinavir.
Conclusions: We provide evidence that lopinavir is a substrate of P-gp, MRP1 and MRP2. The intracellular concentration of lopinavir is increased when co-incubated with ritonavir, amprenavir and atazanavir in PBMCs. Manipulation of drug efflux transporters may be a useful strategy for increasing the intracellular concentration and thereby enhancing the clinical efficacy of lopinavir.
Key Words: P-gp , MRP1 , MRP2 , antiretroviral transport
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Introduction |
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Although highly active antiretroviral therapy (HAART) has substantially improved HIV therapy by decreasing morbidity and mortality, therapeutic failure is still common. Treatment failure is often due to subtherapeutic drug concentrations that allow the establishment of a sanctuary site for viral replication (and the emergence of virological resistance) during drug therapy.1–3 Therefore, it is important that cells able to support viral replication accumulate enough drugs so that viral replication can be effectively suppressed.4–8
Since a predominant site of HIV replication is within CD4+ T cells, the clinical efficacy of the drugs is dependent on optimum drug concentrations within these cells and the subsequent inhibition in replicating cells of key viral enzymes such as proteases and reverse transcriptases. However, it has been shown that the activity of membrane-bound drug efflux transporters, such as P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs) and organic anion transporting polypeptides (OATPs; influx transporters), may limit the intracellular accumulation of HIV protease inhibitors (HPIs) within cells able to support viral replication.9–13 The overexpression of drug efflux transporters or reduced expression of influx transporters within these cells14,15 could give rise to a potential HIV sanctuary site, which could shield the virus from the therapeutic effects of the drugs and thus contribute to HIV treatment failure. Furthermore, first pass metabolism by the cytochrome P450 enzymes limits the bioavailability of the drugs and hence their therapeutic efficacy. As ritonavir-mediated inhibition of cytochrome P450 (CYP3A4/CYP2D6) and possibly P-gp increases exposure to the co-administered HPI, ritonavir-boosted HPIs are a standard component of antiretroviral regimens in many HIV-infected patients.16
To date, there are limited data on the role of drug efflux transporters in the transport and accumulation of lopinavir in primary human lymphocytes. Furthermore, there is the question of the impact of ritonavir and other HPIs on the intracellular accumulation of lopinavir. Studies measuring intracellular antiretroviral accumulation of lopinavir may provide insights into understanding the evolution of HIV sanctuary sites and provide alternative strategies that could allow the manipulation of its intracellular concentrations. To this end, we investigated the role of drug efflux transporters on the intracellular accumulation of lopinavir in both cultured lymphoblastoid cells and peripheral blood mononuclear cells (PBMCs) in the absence or presence of a panel of inhibitors of drug efflux proteins and other HPIs. We hypothesized that if the HPIs increased the intracellular concentration of lopinavir in the cultured lymphoblastoid cells (expressing varying amounts of P-gp and MRP1), it may allow us to interpret any demonstrable effects on PBMCs (one major site of HIV replication17) in the subsequent experiments.
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Reagents
[14C]Lopinavir (specific activity 52.1 mCi/mmol) and ritonavir were donated by Abbott Laboratories (North Chicago, USA). Amprenavir and atazanavir were gifts from GlaxoSmithKline (Research Triangle Park, USA) and Bristol-Myers Squibb (Princeton, USA), respectively. Tariquidar (XR9576) was donated by Xenova Group Plc (Berkshire, UK) and MK571 from Alexis Biochemicals (San Diego, CA, USA). PBMCs were isolated from blood buffy coats, which were obtained from the Regional Blood Transfusion Centre (Liverpool, UK). CEM, CEMVBL and CEME1000 cell lines were from Dr R. Davey (Sydney, Australia). Mouse anti-human IgG2A (IgG2A and IgG2A:rPE) and IgG1 isotype control antibodies were purchased from Serotec Ltd (Oxford, UK). Mouse anti-human P-gp antibody (UIC2:rPE) was obtained from Immunotech (Marseilles, France). All other chemicals were supplied by Sigma Chemical Co. (Poole, UK).
The parental cell line was CEM (a CD4 T-cell line). CEMVBL (VBL; P-gp-overexpressing) cells were selected using vinblastine. CEME1000 (E1000; MRP1-overexpressing) cells were selected with epirubicin. The expression of these transporters has been previously validated in our laboratory.11 The cells were maintained at 37°C and 5% CO2 in RPMI 1640 medium supplemented with 10% fetal calf serum.
Twelve PBMC samples were isolated from blood buffy coats using Lymphoprep, following the manufacturer’s instructions. An aliquot of PBMC samples was cryopreserved in fetal calf serum containing 10% DMSO for batch analysis of membrane proteins by flow cytometry and by real-time PCR.
Transport of [14C]lopinavir and the effects of specific inhibitors on transport in PBMCs and in cultured CEM and its variant cells
The transport and accumulation of 0.5 µM [14C]lopinavir was measured by incubating isolated PBMCs (5 x 106 cells/mL) in the absence or presence of fixed concentrations of the inhibitors: tariquidar (1 µM, inhibits P-gp and breast cancer resistance protein18,19), MK571 (50 µM, inhibits MRP and OATP20), frusemide and probenecid (50 µM, inhibit MRP1/2 and MRP/OATP, respectively; http://www.bigfoot.med.unc.edu/watkinsLab/website/intesinfo.htm) and dipyridamole (50 µM, inhibits P-gp/MRP121). As ritonavir was used in combination with lopinavir, we examined the effects of ritonavir and two other HPIs, atazanavir and amprenavir, on the intracellular accumulation of lopinavir in CEMparental, CEMVBL and CEME1000 cells. The transport of [14C]lopinavir in these cells was investigated by incubating the cells in the absence or presence of varying concentrations (0–30 µM) of ritonavir, amprenavir and atazanavir. In the follow-up experiment, the effects of varying concentrations of ritonavir, amprenavir and atazanavir (0–30 µM, concentrations that are clinically relevant22) on the accumulation of lopinavir were investigated in isolated PBMCs as described previously,13 with minor modifications. Briefly, isolated PBMCs were incubated for 15 min at 37°C in the absence or presence of fixed/varying concentrations of the drugs in RPMI medium supplemented with 10% fetal calf serum and containing 0.5 µM [14C]lopinavir. The samples were centrifuged at 15 000 g for 1 min at 4°C. A 100 µL aliquot of the medium was taken for scintillation counting and the pellets were washed three times in ice-cold PBS before solubilization of the pellets for radioactivity counting. Data from the radioactivity counts were expressed as cellular accumulation ratio (CAR), being the amount of [14C]lopinavir associated with the cell pellets to the amount in a similar volume of medium after incubation.
Flow cytometry and real-time PCR to determine drug efflux transporters
The relative expression of P-gp, MRP1 and MRP2 mRNA was assessed as described previously.13 Briefly, flow cytometric analysis of P-gp was carried out by fixing the cells (1:10 CellFIX; Becton–Dickinson, Oxford, UK). The cells were then resuspended in Hanks balanced salt solution (HBSS) and 400 000 cells were incubated in duplicate with either P-gp-specific UIC2 antibody (Coulter Immunotech, Marseilles, France) at 50 µg/mL or isotype control IgG2a at 100 µg/mL, for 30 min at 4°C in the dark. Cells were then washed twice in HBSS and incubated with an r-phycoerythrin (rPE)-conjugated IgG2a secondary antibody (Serotec) at 100 µg/mL. Following incubation in the dark for 30 min at 4°C, the cells were washed twice in HBSS and fixed (1:10 CellFIX). For MRP1 detection, the MRP1-specific mouse anti-human primary antibody QCRL-1 (Merck, Nottingham, UK), at 25 µg/mL, recognizes an internal epitope of MRP1. It requires cell permeabilization with saponin (0.1 mg/mL, 30 min) after fixing the cells and it is present throughout all the washing steps. As this treatment causes an increase in FL2 background detection, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG secondary antibody (IgG–FITC; 50 µL, 5 µg/mL) was used in place of the rPE-conjugated antibody. Samples were then analysed by flow cytometry (Coulter epics XL-MCL, Coulter, UK). The surface expression was determined by subtracting the median fluorescence of isotype control antibody from that of the test antibody. All data are presented in relative fluorescence units for each protein.
MRP2 mRNA expression levels in PBMCs relative to that in a human liver biopsy were measured by standard real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase was quantified as an internal control.23 MRP2 primers and probes were obtained from Applied Biosystems assays on demand range (http://www.home.appliedbiosystems.com). PCR amplification was performed by standard methodology. Standard methods were also used to verify linearity and product size.
Data are expressed as means ± SD. For the results presented in Table 1, top section, four observations were made for each PBMC sample. Therefore, to assess statistical significance on each PBMC sample, drug-treated samples were compared with control by one-way ANOVA followed by Bonferroni’s method. To assess the overall effects of the inhibitors on the PBMC samples, the Shapiro–Wilk test was used to assess the distribution of the data, followed by the Kruskal–Wallis test to allow multiple comparisons of drug-treated samples with respective controls. Analyses were performed using Statsdirect statistical software, version 2.3.1, 2003 StatsDirect Ltd (Cheshire, UK). In each case, P value of <0.05 between control and drug-treated means was considered statistically significant.
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Transport of [14C]lopinavir and the effects of inhibitors of drug efflux proteins on the accumulation
We determined the effects of a range of inhibitors of drug efflux transporters on the accumulation of lopinavir in PBMCs isolated from healthy volunteers. As shown in Table 1, the baseline CAR of lopinavir in the PBMCs tested ex vivo ranged from 22.2 ± 1.2 to 40.9 ± 0.6 (mean ± SD CAR of 30.2 ± 6.2). Tariquidar increased the CAR in 2 of 12 samples. MK571 and frusemide significantly increased the CAR in 11 of 12 and in 10 of the 12 samples, respectively. Co-incubation with dipyridamole significantly (P < 0.01) increased the CAR of lopinavir in all the PBMC samples. In contrast, probenecid decreased the CAR in 5 out of 12 PBMC samples.
The overall effects of the drugs are shown in Figure 1(a). Tariquidar, MK571, frusemide and dipyridamole significantly increased the CAR of lopinavir in the PBMC samples (P < 0.01), whereas probenecid significantly decreased the CAR of lopinavir in the PBMC samples (P < 0.05).
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Relative expression of P-gp, MRP1 and MRP2 mRNA on the PBMC samples
Aliquots of the PMBC samples used in the transport studies were assessed for their relative surface expression of P-gp, MRP1 and MRP2 mRNA. This would provide evidence of the efflux transporters that may have contributed to the reduced intracellular accumulation of lopinavir, and the inhibition of which by the panel of inhibitors demonstrably increased the CAR of lopinavir (Table 1). The relative expression of P-gp is shown in the bottom section of Table 1. P-gp and MRP1 expression in sample 12 were below the level of quantification. Variable expression of MRP1 was observed in the PBMC samples, with sample 9 having the highest MRP1 and sample 8 having the least. Sample 6 had the greatest MRP2 mRNA and sample 1 the least. There were no detectable levels of MRP2 mRNA in samples 3–5.
Ritonavir, amprenavir and atazanavir increase the intracellular concentration of lopinavir
As ritonavir is widely used to increase plasma concentrations of HPIs by reducing first pass metabolism and inhibiting clearance, we investigated whether ritonavir, amprenavir and atazanavir increased the intracellular accumulation of lopinavir in both cultured cells (CEM and CEM variants that overexpress P-gp and MRP1) and PBMCs. We hypothesized that if lopinavir is a substrate of P-gp and MRP and that these HPIs are able to inhibit P-gp and MRP1, overexpressed in CEMVBL and CEME1000 cells, respectively, then co-incubation of lopinavir with the HPIs may increase the intracellular concentration of lopinavir in the P-gp- and MRP1-overexpressing cells. The accumulation of lopinavir in CEM, CEME1000 and CEMVBL cells is shown in Figure 1(b). We observed that overexpression of MRP1 did not reduce the accumulation of lopinavir in the CEME1000 cells, showing identical CARs to the CEM cells. However, the CAR of lopinavir was reduced in CEMVBL (P-gp overexpressing) cells. Ritonavir significantly increased the CAR of lopinavir in CEM, CEMVBL and CEME1000 cells in a concentration-dependent manner (P 
0.01; Figure 1b). Similarly, both amprenavir (at 3–30 µM) and atazanavir (at 10 and 30 µM) significantly increased the CAR of lopinavir in the CEM and its variant cells (Figure 1c and d), but amprenavir was the weakest inhibitor of P-gp in the CEMVBL cells. The concentration-dependent increases in the CAR of lopinavir in the CEM (parental) cells by ritonavir, amprenavir and atazanavir suggest some background levels of P-gp and MRP1 in the cells.
We also observed that amprenavir, atazanavir and ritonavir caused a concentration-dependent increase in the CAR of lopinavir in the PBMCs (Figure 1e). Amprenavir at 3, 10 and 30 µM significantly increased (P 0.01) the CAR of lopinavir by 1.5-, 1.6- and 2-fold, respectively. Atazanavir at 10 and 30 µM also significantly increased the CAR of lopinavir by 1.3- and 1.5-fold, respectively (P < 0.001). Similarly, ritonavir significantly increased the CAR of lopinavir at 10 and 30 µM by 1.5- and 1.8-fold, respectively (Figure 1e). Interestingly, concentrations of amprenavir and atazanavir that significantly increased the CAR of lopinavir in the cultured cells also increased the CAR of lopinavir in PBMCs significantly.
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Clearly, PBMCs support viral replication17 and also express a number of membrane-bound ATP-dependent drug efflux transporters,13–15,24,25 which could actively extrude substrate drugs (e.g. HPIs). Additionally, it has been observed that some antiretrovirals are not only substrates but also inhibitors of drug efflux transporters.9–11,13,26–29 Furthermore, the CAR of some HPIs has been shown to be amenable to manipulation by specific or relatively specific inhibitors of drug efflux transporters.11–13
In order to test the possibility of modulating the intracellular accumulation of lopinavir, we co-incubated the cells with fixed concentrations of specific or relatively specific inhibitors of drug efflux transporters and examined their effects on the transport and accumulation of lopinavir. Our data suggest that the modulation of the intracellular accumulation of lopinavir is, to some extent, dependent on inhibition of P-gp, MRP1 and MRP2. These stem from the observations that tariquidar, MK571, frusemide and dipyridamole all increased the CAR of lopinavir, suggesting that lopinavir, like saquinavir,13 is a substrate of these drug efflux proteins. As probenecid inhibits both MRP and OATP activities (http://www.bigfoot.med.unc.edu/watkinsLab/website/intesinfo.htm), the observed decrease in the CAR of lopinavir by probenecid may be due to inhibition of lopinavir influx. Indeed, we have recently observed that PBMCs express OATP3A1 (OATP-D) and that lopinavir is a substrate of OATP (such as OATP3A1, OATP4A1 and possibly OATP1A2) (O. Janneh, R. C. Hartkoorn, E. Jones, A. Owen, S. A. Ward, D. J. Back and S. H. Khoo, unpublished results). Similarly, OATP isoforms have been shown to be expressed in CEM and its variant cells, with the transport and accumulation of lopinavir found to be amenable to manipulation using estrone-3-sulphate and montelukast, substrates and inhibitors of OATP20,30 (O. Janneh, R. C. Hartkoorn, E. Jones, A. Owen, S. A. Ward, D. J. Back and S. H. Khoo, unpublished results). Although previous data have shown that probenecid increased the intracellular accumulation of the HPIs in cultured cells manipulated to overexpress MRP1,11 it is conceivable that the effects of probenecid on the PBMCs may depend on the relative expression of MRP and OATPs or on the concentration of probenecid used. Other studies have used much higher concentrations12,31 than that used in our studies (at 50 µM). However, our preliminary investigations in PBMCs demonstrated a concentration-dependent decrease in the CAR of lopinavir by probenecid up to 100 µM (data not shown). One criticism that can be levelled against these inhibitors is the lack of specificity. However, although a specific inhibitor allows the definitive characterization of the involvement of a single transporter in the cellular accumulation of a substrate drug, a more ‘promiscuous’ inhibitor can identify the overall impact of multiple transporters. Given that the extrusion (and indeed the intrusion) of most therapeutic drugs, especially the HPIs, is mediated by more than one efflux (and influx) protein, as the target cells express a plethora of efflux (and influx) proteins, an efflux inhibitor (e.g. dipyridamole) with a broad specificity may substantially increase the intracellular concentration of a substrate drug when compared with a relatively specific inhibitor such as tariquidar (Figure 1a). In contrast, a compound that inhibits both influx and efflux transporters may reduce/increase the intracellular concentration of substrate drugs depending on the relative expression of the influx/efflux proteins and also depending on the concentration used of the inhibitor (e.g. effects of MK571, Figure 1a).
Ritonavir increases the plasma concentrations of HPIs, via its inhibition of hepatic and/or gastrointestinal cytochrome P450-mediated breakdown32,33 and inhibition of P-gp.34 To explore the possibility that lopinavir/ritonavir, lopinavir/amprenavir and lopinavir/atazanavir interactions at the cellular level involve P-gp and possibly MRP, we investigated whether in vitro and ex vivo incubations of [14C]lopinavir with these HPIs would ‘boost’ the intracellular concentration of [14C]lopinavir in cultured cells with known transporter expression profiles and in isolated PBMCs. Here, we demonstrate that ritonavir, amprenavir and atazanavir significantly increased the intracellular accumulation of lopinavir in both CEM and its variant cells (Figure 1b–d), suggesting the inhibition of P-gp- and MRP1-mediated efflux of lopinavir by these HPIs. Interestingly, we observed that co-incubation of lopinavir in PBMCs with these HPIs, at clinically relevant concentrations,22 also significantly increased the intracellular accumulation of lopinavir (Figure 1e), with ritonavir being the most potent of the HPIs tested. Given that these drugs increased the CAR of lopinavir in cultured cells, we postulate that the observed effects in PBMCs occur via inhibition of P-gp and MRP activities. Previous studies have also reported a reciprocal interaction of lopinavir with ritonavir, that lopinavir in combination with ritonavir, in comparison with other HPIs, increased the intracellular concentrations of ritonavir.35 Here we provide evidence that the augmentation of the intracellular concentration of lopinavir may not be limited to its use with ritonavir; that the intracellular accumulation of lopinavir is also amenable to manipulation by atazanavir and amprenavir.
These data need to be confirmed by in vivo studies especially because discrepancies exist between these in vitro/ex vivo data and an earlier in vivo observation in HIV-infected patients that low-dose ritonavir did not ‘boost’ the intracellular accumulation of saquinavir or indinavir above any plasma ‘boosting’ effect.36 Clearly, the studies were done under markedly different conditions (i.e. in vivo versus ex vivo incubations) with different HPIs. Studies such as the current report are able to tease out mechanisms of intracellular drug accumulation, but their clinical relevance and pharmacodynamic consequences can only be properly evaluated in clinical studies.
We previously demonstrated the presence of MRP2 protein in PBMCs,13 suggesting that the observed increase in CAR with frusemide is due to inhibition of both MRP1 and MRP2. Although we demonstrated variable relative amounts of P-gp, MRP1 and MRP2 mRNA and the inhibition of the activities of these efflux transporters demonstrably modulates the intracellular accumulation of lopinavir, we found no significant correlation between the baseline accumulations of lopinavir to any single transporter. This would suggest that multiple processes control the access and retention of the drug within the cell.
We demonstrate that the accumulation of lopinavir can be manipulated in cultured cells and in primary human lymphocytes using inhibitors that interfere with the activities of P-gp, MRP1 and MRP2. Our in vitro data confirm previous observations that lopinavir is a substrate of these transporters.37,38 Furthermore, we provide direct evidence that ritonavir, amprenavir and atazanavir increased the intracellular concentration of lopinavir, possibly through inhibition of P-gp- and MRP1-mediated efflux of lopinavir. However, we also need to bear in mind that the alleged inhibitors could also modulate lopinavir concentrations by displacement from protein in the incubation, thereby increasing the ‘free’ concentration to be transported in the cell.
Our results provide direct evidence that drug efflux transporters can contribute to reduced intracellular concentrations of HPIs. Thus, if drug efflux contributes to reduced intracellular accumulation and treatment failure, inhibition of all contributing transporters may be a viable strategy that could enhance the clinical efficacy of failing drugs. In summary, our data provide evidence that P-gp, MRP1 and MRP2 are key regulators of the intracellular accumulation of lopinavir and targeted modulation of the activities of these transporters may play a role in enhancing clinical efficacy.
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BSAC funding supported this work. The BSAC Academic Initiative Grant (PC/AI-05) supports O. J. and B. C.
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None to declare.
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