The protease inhibitor ritonavir inhibits the functional activity of the multidrug resistance related-protein 1 (MRP-1)

Abstract

Background 

Efflux pumps situated on the plasma membrane, such as P-glycoprotein (Pgp) and the multidrug resistance related-protein 1 (MRP-1), have been shown to extrude HIV protease inhibitors from the cell. MRP-1 is present on many barrier sites throughout the body, such as the blood–brain and blood–testis interfaces and could reduce the concentration of protease inhibitors in these sanctuary sites for HIV-1 replication. Factors that modulate efflux pump function in vivo are poorly defined.

Objective 

To analyze the inhibitory potential of the anti-retroviral drugs indinavir, amprenavir, ritonavir, lamivudine or zidovudine to modulate MRP-1 function.

Methods 

Effect of anti-HIV drugs on the efflux pump activity of MRP-1 was evaluated in the presence of increasing concentrations of human plasma, using UMCC-1/VP cells which stably over-express MRP-1. MRP-1 activity was abrogated by probenecid. The potential of blocking MRP-1 function for an extended (3 day) time period, was also examined in MRP-1 over-expressing cells cultured with either probenecid or the anti-retroviral drugs and a cytotoxic compound (etoposide) that is transported by MRP-1.

Results 

Ritonavir inhibited the functional activity of MRP-1 similarly to probenecid, as demonstrated by re-sensitization of MRP-1 over-expressing cells to cytotoxic effects of etoposide. Inhibition by ritonavir was inversely related to the concentration of human plasma added to the cells (r²=0.89). Other anti-HIV drugs didn't affect the MRP-1 mediated efflux of etoposide.

Conclusions 

These data may be exploitable to further improve sanctuary site concentrations of anti-HIV or anti-cancer drugs by using ritonavir as a lead compound to develop more potent MRP-1 inhibitors.

Introduction

Highly active antiretroviral therapy has proven successful in treating HIV disease and dramatically delaying the progression of immunodeficiency to AIDS. Nevertheless, many individuals fail drug therapy. In some cases, viruses present during drug failure lack any genotypic viral mutations that induce a drug resistant phenotype. Therefore, the elucidation of possible mechanisms of drug failure in addition to virus mutational changes is necessary. In addition, wild-type virus replication has proven more difficult to inhibit by drugs in many sites throughout the body – such as the testes and central nervous system – than in the blood; low levels of drug are achieved in those tissues [¹]. These sites have been proposed as sanctuaries from drug in which HIV can replicate during treatment [²]. The ability to increase drug concentrations in these tissues – and as such, therapeutic efficacy – could have important clinical implications.

Cellular efflux pumps, situated on the plasma membrane, transport drug out of the cell in an ATP-dependent fashion [³]. They were first identified in studies of cancer chemotherapy and their capability to transport HIV protease inhibitors (PI) led to investigation for effects on antiretroviral drugs. These pumps may contribute to HIV drug delivery, as they are located in high density along the ependymal lining of many interfaces in the body. The multidrug resistance related-protein 1 (MRP-1) is highly expressed and functional at the blood–brain and blood–testes barriers [^4,5]. P-glycoprotein (Pgp) is additionally expressed on ependymal cells and also on CD4, CD8, natural killer and hematopoietic stem cells [^6–8] where the levels of this pump are lower yet clinically relevant, as they are capable of extrusion of PI and thus may limit the intracellular drug concentration.

A number of anti-retroviral drugs, most notably the PI, have been shown to be substrates for both Pgp and MRP-1 [^9,10]. Recent studies have focused intensely on Pgp as a modulator of intracellular PI concentrations [^11,12], while the contribution of MRP-1 to cellular resistance has been less well studied [^13,14]. Presently, no clinical study has shown a cause-and-effect relationship that directly links either Pgp or MRP-1 over-expression and clinical drug evasion, but in vitro results strongly suggest a role for these efflux pumps in the failure of PI therapy. Some investigations reported inhibitors of Pgp or MRP-1, but so far only one investigational MRP-1 inhibitor (MK-571) slightly inhibited efflux of one of the PI [¹³]. While several known modulators of MRP-1 are available for in vitro laboratory experiments, those inhibitors that have clinical potential are still in clinical trials [^3,15].

Using MRP-1 over-expressing cell lines and the known MRP-1 inhibitor probenecid, we assessed anti-retroviral drugs as modulators of MRP-1 functional activity. Our results show that ritonavir is able to inhibit MRP-1 function as efficiently as probenecid, and that this effect is blocked at high concentrations of human plasma. Inhibition of MRP-1 function by ritonavir suggests a potential strategy to optimize anti-retroviral drug concentrations at distinct ‘sanctuary’ sites.

Materials and methods

Cell lines

The cell lines used were the UMCC-1 wild-type and the UMCC-1/VP cell lines, the latter of which is derived from the UMCC-1 wild-type line and has been induced to over-express the MRP-1 drug transporter by being continuously grown in the MRP-1 substrate etoposide (VP-16) (Abbott Laboratories, Abbott Park, Illinois, USA) at a concentration of 4 μM. The cells are an adherent cell line and are derived from a non-small cell lung carcinoma [¹⁶]. The parent line does not over-express any known drug transporters, while the VP line exclusively over-expresses MRP-1. Cells were grown in RPMI 1640 (CellGro, Herndon, Virginia, USA) supplemented with 1% L-glutamine (BioWhitaker, Walkersville, Maryland, USA) and 1% penicillin/streptomycin (BioWhitaker) and 10% FCS (BioWhitaker) at 37°C, 5% CO_2, 95% relative humidity. The MRP-1-positive VP line was cultured with 4 μM etoposide, necessary to maintain the over-expression of the MRP-1 protein.

Flow cytometric assay

This assay has been described previously [¹⁷]. Briefly, cells were suspended in complete RPMI and loaded with 0.05 μM of the fluorescent dye calcein AM (Molecular Probes, Eugene, Oregon, USA) for 30 min at room temperature. Cells were then washed twice in cold complete RPMI, split into different tubes, and resuspended in 500 μl of complete RPMI. One tube was placed directly on ice, and served as a zero hour time point. Another tube was placed at 37°C without potential MRP-1 modulators and served as a 2 h control time point. Additional tubes were also placed in the 37°C incubator, supplemented with the known MRP-1 inhibitor probenecid (2 μM; Sigma, St. Louis, Missouri, USA) or another potential modulator: indinavir (crixivan; 0.114 μM; Merck, Whitehouse Station, New Jersey, USA), amprenavir (0.020 μM; Glaxo SmithKline, Research Triangle Park, North Carolina, USA), ritonavir (norvir; 147 μM; Abbott Laboratories), lamivudine (0.55 μM; Glaxo SmithKline) or zidovudine (0.020 μM; Glaxo SmithKline). After 2 h, all tubes were removed from the incubator and placed directly on ice and analyzed within 1 h on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, California, USA). Signals were collected in the FL1 channel using a 525 nM band pass filter. The geometric mean fluorescent intensity was used for all calculations.

For experiments in which increasing amounts of human plasma were added, fresh human plasma was added to the tube containing ritonavir prior to incubation for 2 h at 37°C. The ritonavir concentration was held stable at 147 μM. Samples were analyzed as described above.

Cell culture assay

Cells were grown in either complete RPMI for the wild-type line, or complete RPMI plus 4 μM etoposide for the MRP-1-positive over-expressing line.

The same compounds used for the flow cytometric assay (probenecid, indinavir, amprenavir, ritonavir, lamivudine or zidovudine) were added to the cultures individually at the concentrations given above. After 3 days, cells were trypsinized, washed once in complete RPMI, and assessed for viability using the Trypan blue dye exclusion method.

Calculations

To calculate the amount of calcein dye retained in the cells after 2 h, the following calculation using the mean fluorescent intensity of the dye in the cells (MFI) was used: [(MFI zero hour control – MFI experimental) × 100]/MFI zero hour control, where MFI experimental refers to the assay at the 2 h time point with or without putative modulating agents added.

To determine viability in the cell culture assay, the following calculation was used: (total cell number/total blue cell number) × 100.

Results

Flow cytometric assay

As expected, the UMCC-1 wild-type cell line failed to efflux appreciable amounts of calcein after the 2 h incubation (average retention, 88% ± 7%; n = 5). The UMCC-1 VP cell line effluxed a substantial amount of dye after 2 h, with average retention of the dye being only 34% in the absence of the known inhibitor probenecid. When probenecid was added to the culture, the retention increased dramatically, with an average retention of 83%, showing a direct and specific inhibition of the MRP1 efflux pump by this compound. When anti-retroviral agents were added, only ritonavir inhibited the functional activity of MRP1, with a markedly increased retention of calcein (average retention, 83% ± 8%; n = 4). All of the other drugs failed to inhibit functional activity, with retentions being comparable to the efflux in the absence of an inhibitor: indinavir, 35%; lamivudine, 35%; zidovudine, 29%; amprenavir, 32% (numbers represent mean of four experiments) (Fig. 1). None of these compounds, including ritonavir, effected the retention of dye levels in the MRP-1 negative, UMCC-1 wild-type cell line (data not shown).

Fig. 1.: Inhibition of MRP-1 functional activity: percent of dye retained in the presence of a modulator. MRP-1-positive UMCC-1/VP cells were loaded with calcein and allowed to efflux the dye. Zero hour is the 100% zero hour control value; the 2 h assay shows a significant efflux of dye with only 34% retention. This efflux is reversed by the known MRP-1 modulator probenecid (retention, 83%). Ritonavir also inhibited functional activity, with retention being 83% in the presence of this PI. No other antiretroviral agent inhibited efflux, with all retentions being similar to the 2 h control (retentions < 40% in all cases). Assay conditions are represented on the x-axis, dye retained is shown on the y-axis. Values represent the mean of at least three independent experiments.

When the ritonavir concentration was held stable and the human plasma concentration was increased, the inhibition by ritonavir decreased in a linear fashion (Pearson correlation coefficient = 0.89). Fig. 2 demonstrates that at lower plasma levels (5–25%) ritonavir still inhibited MRP-1 functional activity. At protein levels similar to those in blood (85% plasma) the inhibitory potential of ritonavir was totally abrogated. The inhibition by ritonavir was evident at lower levels of plasma expected to mimic plasma protein concentration on the central nervous system (CNS) side of the blood–CNS barrier. At levels (45%) that mimic the maximum concentration of plasma proteins expected in normal cerebrospinal fluid (CSF) ritonavir showed inhibition but at a decreased level. The inhibitory effect of probenecid was not abrogated by increasing plasma concentration (data not shown).

Fig. 2.: MRP-1 inhibition by ritonavir: Effects of increasing human plasma concentration.MRP-1-positive UMCC-1/VP cells were loaded with dye, and different concentrations of fresh human plasma were added to the tube together with ritonavir (147 μM). As is evident, the ability of ritonavir to inhibit MRP-1 decreases inversely with the amount of plasma present (r ²=0.89). The assay conducted with no inhibitor and in the presence of probenecid (10% plasma) in the last two columns, respectively, are given as a reference point. The x-axis depicts the increasing plasma concentration, and the y-axis shows percentage of dye retained within the cell. Values represent the mean of four independent experiments.

Etoposide cytotoxicity assay

MRP-1 function can protect MRP-1 over-expressing cells from etoposide cytotoxicity. After 3 days of etoposide exposure in either normal growth medium or medium supplemented with probenecid or an antiretroviral agent, MRP-1 over-expressing cell viability was assessed. Fig. 3 shows that the known inhibitor probenecid effectively blocked MRP-1 functional activity, with an effect of increased cytotoxicity [cell viability was only 5 ± 5% (n = 3) with probenecid and always > 90% in the absence of an MRP-1 inhibitor]. Ritonavir also effectively inhibited MRP-1 activity, with cell viability being only 7 ± 4% (n = 3) when this PI was added to the culture. No other anti-retroviral agent showed any inhibition of MRP-1 mediated etoposide efflux, as cell viability was > 85% for all other cell cultures tested (n = 3). There was no significant difference in cytotoxicity of any of the wild-type cell lines not expressing MRP-1 regardless of what compound was added (data not shown). These data indicate that the anti-retroviral drug itself was not directly cytotoxic, but rather that those compounds inhibiting MRP-1 function permitted the intracellular accumulation of the cytotoxic topoisomerase inhibitor, etoposide.

Fig. 3.: Percent viability 3 days after addition of anti-retroviral agents. MRP-1-positive UMCC-1/VP cells were grown in medium containing etoposide, and MRP-1 modulators were added to the culture. The x-axis represents the compound added, either none, probenecid, or an anti-retroviral agent, and the y-axis represents the percent of cells that were viable. The known inhibitor – probenecid – effectively blocked MRP-1-mediated etoposide efflux and induced almost 100% cellular necrosis; ritonavir produced similar results. No other antiretroviral agent had any effect on cytotoxicity (> 85% viability in all other cultures). Data represent the mean of three separate experiments.

Discussion

As has been well documented in a variety of neoplastic conditions, the existence of cellular efflux pumps is a key factor in drug delivery and potential ultimate resistance to these drugs [³]. Recent reports have focused on the role of Pgp in the cellular efflux of antiretroviral drugs. Reports have shown that PI are substrates for the MRP-1 efflux pump as well as for Pgp. Ritonavir has previously been shown to abrogate Pgp function [^12,18]. MRP-1 shares many similarities to Pgp in both substrate specificity and location, and is situated on many sites throughout the body, including all peripheral blood and bone marrow cells [¹⁹], tissues of renal, duodenal, pancreatic, placental and hepatic origin, and others [²⁰]. Additionally, MRP-1 is located in body sites of great clinical concern for HIV drug delivery such as the blood–brain barrier and the testes, and as such, the ability to alter MRP-1 function in addition to Pgp function has attractive clinical potential.

Our data show that ritonavir is capable of inhibiting MRP-1 function in a short time frame of 2 h. Additionally, when the concentration of plasma is increased, the inhibitory potential of ritonavir is eventually ablated, consistent with known binding of ritonavir by plasma proteins such as α-1 acid glycoprotein [²¹]. Ritonavir retained inhibitory potential up to 55% plasma concentration in our experiments. The physiologic concentration of plasma proteins in both the brain and the testes may well be in this lower range (e.g., < 55%) suggesting the possibility of some inhibition of MRP-1 function in vivo on the ependymal lining at these sites [^4,22,23].

In cell culture experiments, ritonavir was able to inhibit MRP-1 functional activity for a longer duration than the 2 h needed for the flow cytometric assay. Intracellular accumulation of an MRP-1 substrate – in this case, etoposide – caused death of MRP-1 over- expressing cells only when ritonavir or probenecid was present to inhibit MRP-1 mediated efflux. Other antiretroviral drugs tested did not show evidence of MRP-1 inhibition in this etoposide cytotoxicity assay, nor in the shorter-term flow cytometry assay. Effects of drug combinations of ritonavir plus other anti-retroviral agents on MRP-1 mediated transport were not tested. The antiretroviral efficacy of the PI was not evaluated in this study, as the UMCC-1 cell line does not support HIV replication. While the UMCC-1/VP cells used here exhibit very high levels of MRP-1, the expression level of MRP-1 in vivo is much less than on laboratory cultured cell lines. This latter fact may indeed represent a potential limitation of this assay system in evaluating clinically relevant MRP-1 inhibitors, yet it may also underscore the fact that high levels of ritonavir were used to achieve inhibition in this study and that lower levels may achieve at least partial inhibition in vivo.

Further research is needed to identify the complexity of the tightly regulated processes of drug entry in cells, intracellular accumulation and efflux. However, these data suggest an additional possible explanation for the observed beneficial effect of ritonavir-boosted PI regimens. While it is of interest to study whether MRP-1 is inhibited to some extent in tissues of subjects receiving ritonavir, the lower doses of ritonavir now common in ritonavir-boosted PI regimens may not achieve concentrations in vivo analogous to those studied here in vitro. However the identification here that ritonavir can modulate MRP-1 function does suggest that it could be a useful lead compound for development of a related dual Pgp/MRP-1 inhibitor. Greater inhibitory potency and reduced plasma protein binding might be developed by further medicinal chemistry manipulation of the lead ritonavir structure.