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Antimicrobial Agents and Chemotherapy, December 1998, p. 3157-3162, Vol. 42, No. 12
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Human Immunodeficiency Virus Protease Inhibitors Serve as Substrates for Multidrug Transporter Proteins MDR1 and MRP1 but Retain Antiviral Efficacy in Cell Lines Expressing These Transporters

Ranga V. Srinivas,^1,* David Middlemas,² Pat Flynn,¹ and Arnold Fridland¹

Department of Infectious Diseases¹ and Department of Molecular Pharmacology,² St. Jude Children's Research Hospital, Memphis, Tennessee

Received 26 May 1998/Returned for modification 17 August 1998/Accepted 3 October 1998

	ABSTRACT

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The human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs)saquinavir, ritonavir, nelfinavir, and indinavirinteract with the ABC-type multidrug transporter proteins MDR1 and MRP1 in CEM T-lymphocytic cell lines. Calcein fluorescence was significantly enhanced in MDR1⁺ CEM/VBL100 and MRP1⁺ CEM/VM-1-5 cells incubated in the presence of various HIV PIs and calcein acetoxymethyl ester. HIV PIs also enhanced the cytotoxic activity of doxorubicin, a known substrate for MDR1 and MRP1, in both VBL100 and VM-1-5 CEM lines. Saquinavir, ritonavir, and nelfinavir enhanced doxorubicin toxicity in CEM/VBL100 cells by approximately three- to sevenfold. Saquinavir and ritonavir also enhanced doxorubicin toxicity in CEM/VM-1-5 cells. HIV-1 replication was effectively inhibited by the various PIs in all of the cell lines, and the 90% inhibitory concentration for a given compound was comparable between the different cell types. Therefore, overexpression of MDR1 or MRP1 by T lymphocytes is not likely to limit the antiviral efficacy of HIV PI therapy.

	INTRODUCTION

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The human immunodeficiency virus (HIV) protease inhibitors (PIs)saquinavir, ritonavir, nelfinavir, and indinavirwere recently shown to interact with the multidrug resistance protein (MDR1; also known as P-glycoprotein or Pgp) in the human intestinal epithelial cell line CaCO2 (20) and a human carcinoma line, KB-V1 (22). HIV type 1 (HIV-1) primarily replicates in CD4⁺ T lymphocytes and macrophages. Lymphocytes and macrophages also express MDR1, and their expression is regulated in a development- and differentiation-specific manner (21, 33, 34). MDR1 expression is altered in HIV-infected individuals, and HIV-infected individuals show a significant decrease in Pgp⁺ CD4⁺ lymphocyte subsets compared to controls (2, 26). MDR1 is a member of a multigene family of ABC transporter proteins that are characterized by the presence of ATP-binding cassettes (6). A series of related ABC-containing drug transporter genes, designated multiple-drug resistance-associated proteins (MRPs), also determine resistance to various chemotherapeutic agents (6, 7). MRPs are found on the surfaces of lymphocytes and macrophages, but their physiological function is not known (24). Treatment failure due to activation of multidrug transporter proteins has been reported in human cancers including lymphocytic leukemia (12), and T-lymphocytic cell lines expressing various transporters have been established (4).

The recent observations that HIV PIs can interact with MDR1 raise several questions. Long-term treatment with HIV PIs may activate MDR1 expression, and overexpression of MDR1 in T lymphocytes can limit the antiviral efficacy of HIV PIs. Secondly, HIV PIs may interact with other related ABC-type drug transporters. To address these questions, we have investigated the activity of HIV PIs in T-lymphocytic cells that overexpress MDR1 or MRP1.

We show here that HIV PIs interact with and inhibit both MDR1 and MRP1 activity in T cells. Interestingly, HIV PIs were equally effective against HIV in both wild-type and multidrug-resistant T-lymphocytic cell lines, suggesting that cellular resistance to HIV PIs via activation of an ABC-type drug transporter protein may not be a major therapeutic concern.

	MATERIALS AND METHODS

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Cells and viruses. The T-lymphocytic cell line CEM/WTB (CCRF-CEM) and its MDR1⁺ CEM/VLB100 (5) and MRP1⁺ CEM/VM-1-5 (9, 31) variants were obtained from W. T. Beck (University of Chicago, Chicago, Ill.). The neuroblastoma cell line NB1643 and its MDR1⁺ variant NB1643Dox^r are described elsewhere (27). MT-2a human T-cell leukemia virus type 1-transformed T-lymphocytic cell line that is highly sensitive to HIV replicationand the different viruses used in this study were all obtained from the NIH/NIAID AIDS Research and Reference Reagent Program (National Institute of Allergy and Infectious Diseases, National Institutes of Health). The viruses used were HIV-1_IIIB, saquinavir-resistant (Saq^r) HIV-1 (17), and multiple PI-resistant (MPR) HIV-1_{M46I/L63P/V82T/184V} (8).

Reagents and chemicals. Calcein acetoxymethyl ester (calcein-AM) was purchased from Molecular Probes (Portland, Oreg.). Reserpine was purchased from Sigma (St. Louis, Mo.). Alamar blue was purchased from Alamar Biosciences (Sacramento, Calif.). Doxorubicin and the various HIV PIs were obtained from the St. Jude pharmacy.

Calcein flux assays. A kinetic fluorimetric assay (11, 27) was used to study MDR1-HIV PI interactions. Briefly, cells were plated in 96-well (Costar) tissue culture plates in medium containing 1 µM reserpine or the various HIV PIs. After a 2-min incubation, calcein-AM was added to a final concentration of 1 µM and the plates were placed in a cytofluorimeter (Millipore, Bedford, Mass.). Fluorescence was measured at regular intervals for 30 to 60 min with 485-nm (bandwidth, 20 nm) excitation and 530-nm (bandwidth, 25 nm) emission filters. The rate of calcein accumulation in the absence or presence of the modulators was calculated by linear regression analysis of the data with Prism II software (GraphPad). The optimal treatment regimens of different HIV PIs readily achieve a concentration in plasma of ~5 µM. For example, the concentrations in plasma of HIV PIs range from 4 to 11 µM for ritonavir and from 7 to 16 µM for indinavir (3). Therefore, the concentrations of the HIV PIs used in this study are clinically relevant.

Flow cytometry. The interaction of HIV PIs with MRP1 (and MDR1) was investigated by monitoring calcein accumulation by flow cytometry, as described before (11). Briefly, cells (2 × 10⁵/ml) were incubated for 1 h at 37°C with 1 µM calcein-AM in the absence or presence of HIV PI (5 or 50 µM) or other modulators (1 µM reserpine or verapamil). They were centrifuged and resuspended in phosphate-buffered saline and analyzed in a FACScan flow cytometer (Becton Dickinson Medical Systems, Sharon, Mass.) equipped with an argon laser. The fluorescence of 30,000 events was measured at an extinction wavelength of 488 nm. The logarithmic signals were converted to values on a linear scale and expressed as relative fluorescence units to calculate mean fluorescence.

Cytotoxicity assays. The cytotoxicity of doxorubicin to different CEM cell lines was determined by a chromogenic dye conversion assay (30). Briefly, 10⁵ cells (in a total volume of 200 µl) were seeded in 96-well tissue culture plates in the presence or absence of the test compounds. Serial fivefold dilutions of doxorubicin, at concentrations ranging from 1 µM to 0.3 nM, were tested, and the HIV PIs were tested at a concentration of 5 µM. After 48 h, 25 µl of Alamar blue reagent was added to each well and incubated for 2 to 4 h, and the absorption at 570 nm was determined as a measure of viable cell numbers.

Antiviral assays. Virus yield reduction assays were used to monitor the antiviral activity of the various compounds (30). Briefly, the various cells were infected with HIV-1 at a multiplicity of infection of 0.01, and the virus-infected cells were seeded at a concentration of 0.2 × 10⁶ cells/ml in media containing varying concentrations of the drugs. Serial fivefold dilutions of HIV PIs, at concentrations ranging from 1 µM to 0.3 nM, were tested. After 5 days of incubation, the p24 antigen levels in the culture supernatants were determined with an in-house antigen capture assay kit (29). The antiviral effects of PIs on CEM/WTB, CEM/VBL100, and CEM/VM-1-5 cells were also confirmed by infectious virus yield reduction, with MT-2 cells as targets for virus titration.

	RESULTS

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Effects of HIV PIs on calcein-AM flux in MDR1⁺ cells. Calcein-AM, a nonfluorescent acetoxymethyl ester of calcein, is a substrate for MDR1 (13-16, 18). It is readily internalized by viable cells and hydrolyzed by cellular esterases. The product calcein is a charged membrane-impermeable fluorescent molecule. Calcein is not a substrate for MDR1. Calcein fluorescence therefore corresponds to the intracellular levels of calcein-AM, and the factors affecting calcein fluorescence may be summarized by the equation K_observed = K_{calcein-AM influx}  K_{calcein-AM efflux}. The influx and efflux of membrane permeable calcein-AM by passive diffusion occurs at the same rate in both wild-type and MDR1⁺ cells. However, cells expressing MDR1 show an additional Pgp-mediated efflux of calcein-AM. The net increase in calcein-AM efflux by MDR1⁺, compared to wild-type cells, leads to a reduction in calcein fluorescence. MDR1-inhibitors inhibit Pgp-mediated efflux of calcein-AM and enhance calcein fluorescence in MDR1⁺ cells but have no effect on wild-type cells. An increase in calcein fluorescence in the presence of known MDR1-specific inhibitors has been used to identify cells showing MDR1 activity. Similarly, enhancement of calcein fluorescence in an MDR1⁺ cell, but not in the wild-type parent, identifies compounds that can interact with MDR1 (13-16, 18).

We used two sets of parental and MDR1⁺ cell linesCEM/WTB and CEM/VBL100 along with NB1643 and NB1643Doxto study MDR1 and HIV PI interactions. Figure 1 shows calcein-AM flux in CEM cells. CEM/VBL100 cells accumulated calcein at a significantly lower rate than did the wild-type CEM/WTB cells (P < 0.05) (Fig. 1A). Reserpine, an MDR1 antagonist, significantly enhanced calcein fluorescence in CEM/VBL100 cells (Fig. 1B) but had very little effect on calcein accumulation in CEM/WTB cells (Fig. 1C), thus suggesting a functional MDR1 activity in CEM/VBL100 cells. Figure 2 summarizes the effects of various HIV PIs on calcein accumulation in CEM/VBL100 cells. At a concentration of 5 µM, ritonavir, nelfinavir, and indinavir showed significant increases in calcein fluorescence in CEM/VBL100 cells. Maximal increase was observed with ritonavir, followed by nelfinavir and indinavir. However, at a higher concentration (50 µM), saquinavir also enhanced calcein fluorescence in CEM/VBL100 cells (see Fig. 4). Thus, all four HIV PIs interact with MDR1, with affinities in the order ritonavir > nelfinavir > indinavir > saquinavir, by calcein flux assays. Similar results were obtained with the MDR1⁺ neuroblastoma cell line NB1643Dox (Fig. 3). The PIs did not affect calcein accumulation in CEM/WTB cells (data not shown).

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Calcein-AM flux in multidrug-resistant CEM cells. Calcein flux was measured as described in Materials and Methods. Relative fluorescence units (F) (mean ± standard deviation) are shown. Panel A shows the rate of accumulation of calcein in MDR1+ CEM/VBL100 (broken line) and wild-type CEM/WTB (solid line) cells. The effects of reserpine on the calcein flux in CEM/VBL100 and CEM/WTB cells are shown in panels B and C, respectively. Calcein fluorescence of CEM/VBL100 cells was significantly enhanced when incubated with the MDR1 antagonist reserpine (panel B, solid line) compared to CEM/VBL100 cells incubated with saline (panel B, broken line). By contrast, the calcein fluorescence of wild-type CEM/WTB cells was not different when incubated in the presence of either reserpine (hatched line) or saline (solid line). The P values indicate the statistical differences between the slopes of calcein fluorescence in CEM/WTB and in CEM/VBL100 cells (A) or between cultures incubated with reserpine and controls (B and C).

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Effects of various HIV PIs on calcein accumulation in MDR1+ CEM/VBL100 cells. The rates of accumulation of calcein in CEM/VBL100 cells incubated with 5 µM (each) saquinavir (A), ritonavir (B), indinavir (C), or nelfinavir (D) are shown. The broken lines show results of control cultures incubated with saline, while the solid lines show results of cultures incubated with HIV PI. The P values indicate the statistical differences between the slopes of calcein fluorescence in cultures incubated with HIV PIs and in controls. A statistically significant increase in calcein fluorescence was seen in the presence of ritonavir, nelfinavir, and indinavir, suggesting that they all inhibit MDR1. Significant differences were also observed with saquinavir, albeit at higher (50 µM) concentrations (data not shown).

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Effects of various HIV PIs on calcein accumulation in MDR1+ NB1643Doxr cells. The rates of accumulation of calcein in NB1643Doxr cells incubated with 5 µM (each) ritonavir, nelfinavir, indinavir, or saquinavir are shown. The broken lines show results of control cultures incubated with saline, while the solid lines show results of cultures incubated with HIV PI. The P values indicate the statistical differences between the slopes of calcein fluorescence (F) in cultures incubated with HIV PIs and in controls.

Effects of HIV PIs on calcein accumulation in MRP⁺ cell lines. Calcein, the membrane-impermeable intracellular cleavage product of calcein-AM, is a substrate for MRP1. Calcein-AM-labeled MRP1⁺ cells extrude calcein and show diminished fluorescence, and inhibitors of MRP1 restore calcein fluorescence. We therefore used the parental CEM/WTB and MRP1⁺ CEM/VM-1-5 cell pairs to study HIV PI-MRP1 interactions. MDR1⁺ CEM/VBL100 was also included, as a control. As shown in Fig. 4, calcein fluorescence was greatly reduced in CEM/VBL100 and CEM/VM-1-5 cells compared with CEM/WTB cells. Saquinavir, ritonavir, nelfinavir, and indinavir all enhanced calcein fluorescence in both VBL100 and VM-1-5 cells (Fig. 4), suggesting that the HIV PIs interact with and inhibit both MDR1 and MRP1 activities.

View larger version (30K): [in this window] [in a new window]   FIG. 4.   Flow cytometry of calcein-AM-labeled CEM/VM-1-5 cells. MRP1+ CEM/VM-1-5 cells were incubated with calcein-AM in the absence or presence of 50 µM saquinavir, ritonavir, indinavir, or nelfinavir and analyzed by flow cytometry, as described in Materials and Methods. Means (± standard deviations) of three independent experiments are shown. The P values indicate the statistical differences between mean calcein fluorescence (F) in cultures incubated with HIV PIs and in controls.

Chemosensitization of multidrug-resistant CEM cells by HIV PIs. We also investigated whether HIV PIs function as competitive inhibitor of MDR1 or MRP1 and reverse the drug resistance phenotype in cells expressing these transporters. We used doxorubicin for these studies, since it is a substrate for both MDR1 and MRP1. The cytotoxic activities of doxorubicin against CEM/WTB, CEM/VBL100, and CEM/VM-1-5 cells were determined by dye conversion assays, and the results are shown in Table 1. The cytotoxicity of doxorubicin against CEM/VBL100 cells was enhanced three- to sevenfold in the presence of 5 µM saquinavir, ritonavir, or nelfinavir, but not indinavir. Saquinavir and ritonavir, but not indinavir and nelfinavir, also caused a threefold increase in the cytotoxic activity of doxorubicin against MRP1⁺ CEM/VM-1-5 cells. Chemosensitization by HIV PIs was only partial and did not restore doxorubicin susceptibility to wild-type levels. Moreover, the degree of chemosensitization did not correlate with the degree of MDR1 or MRP1 inhibition observed by calcein flux assay. The reasons for these discrepant findings are not clear, and they underscore the complexities in reversing multidrug resistance. Pgp contains two distinct sites for drug binding and transport that interact in a complex manner (10, 28). Rhodamine 123 and Hoescht 33342 bind to distinct sites but stimulate the Pgp-mediated transport of each other. Colchicine and quercetin stimulate rhodamine transport and inhibit Hoescht 33342 transport, while doxorubicin and daunorubicin exert the opposite effects. Some compounds (e.g., vinblastine) inhibit the transport of both dyes. It is possible that doxorubicin, calcein-AM, and the HIV PIs interact with these two substrate-binding sites of Pgp with different affinities, thus explaining some of the discrepancies between the calcein flux and doxorubicin sensitization assays.

View this table: [in this window] [in a new window]   TABLE 1.   Cytotoxicities of doxorubicin to different CEM cells

Antiviral efficacy of HIV PIs in multidrug-resistant cells. We investigated the antiviral efficacy of HIV PIs by yield reduction assays. We first compared the activity of different PIs against HIV-1_IIIB, a saquinavir-resistant HIV-1, and an MPR HIV in MT-2 cells (Table 2). All of the compounds effectively inhibited HIV-1_IIIB, and the 90% inhibitory concentrations (IC₉₀s) ranged from 4 to 62.5 nM. These numbers are comparable to the antiviral activities reported in the product literature inserts supplied with the different HIV PI formulations: nelfinavir (95% effective concentration [EC₉₅], 7 to 196 nM), indinavir (EC₉₅, 25 to 100 nM), saquinavir (EC₅₀, 1 to 30 nM), and ritonavir (EC₅₀, 3.8 to 153 nM). However, it is important to note that all HIV PIs bind to plasma protein, albeit to varying degrees (3). Therefore, the actual concentrations in plasma required to inhibit HIV in vivo may vary for the different compounds. Saquinavir-resistant HIV-1 was ~100-fold less sensitive (90% effective dose [ED₉₀], ~100 nM) to saquinavir but was inhibited by other PIs at concentrations that were effective against HIV-1_IIIB. By contrast, HIV-1_{M46I/L63P/V82T/184V} was resistant to saquinavir, indinavir, nelfinavir, and indinavir.

View this table: [in this window] [in a new window]   TABLE 2.   Antiviral effects of different HIV PIs in MT-2 cells

Table 3 summarizes the antiviral potencies of various HIV PIs in different CEM cell lines. The ED₉₀s of the various PIs against HIV-1_IIIB were only marginally increased (less than twofold) in VBL100 and VM-1-5 cells compared to CEM/WTB cells. This observation may be explained by the fact that HIV PIs inhibit MDR1 and MRP1 only at concentrations ~1,000-fold greater than the concentrations required to inhibit HIV replication. Therefore, intracellular drug concentrations in MDR1⁺ or MRP1⁺ cells are likely to differ only at high concentrations in plasma. The differences are not likely to be significant at concentrations corresponding to 90% infective doses of these drugs. We are currently trying to determine the intracellular steady-state concentrations of HIV PIs in the different cell types. In preliminary studies, duplicate sets of wild-type and mutant CEM cell lines were incubated in the presence of 50 µM ritonavir for 4 h. The cells were separated from drug-containing medium by centrifugation over a nyosil oil cushion. The cell pellets were washed in phosphate-buffered saline and extracted with 80% methanol. The methanol extracts were evaporated, reconstituted in assay buffer, and analyzed for ritonavir levels by high-performance liquid chromatography (HPLC). The areas of the peak corresponding to ritonavir were only slightly smaller in CEM/VBL100 (0.275 ± 0.034 cm²) and CEM/VM-1-5 (0.269 ± 0.010 cm²) cells than in CEM/WTB cells (0.484 ± 0.034 cm²).

View this table: [in this window] [in a new window]   TABLE 3.   Antiviral effects of different HIV PIs in CEM cells

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The present study confirms earlier observations that all of the currently licensed HIV PIs interact with MDR-1 (1, 20, 23) and extends those observations to show that they interact with MRP1, another ABC-type drug transporter protein, as well. MDR1-HIV PI interactions affect intestinal absorption and disposition of HIV PIs. The concentrations of HIV PIs after oral administration are 2- to 5-fold higher in plasma and >10-fold higher in cerebrospinal fluid of mdr1a^/ knockout mice (20) than in normal mice. Unlike MDR1, MRP1 is poorly expressed in the liver and intestine, but it is expressed in all other major organs, including blood (35). The implications of MRP1-PI interactions are presently unclear. MRP1 knockout mice show increased levels of glutathione and increased sensitivity to anticancer agents (25, 32), suggesting possible alterations in drug disposition. Pharmacokinetic studies of HIV PIs in MRP1 knockout mice may provide clues to the possible consequences of MRP1-HIV PI interactions. Therapeutic use of MDR1 (or MRP1) inhibitors to increase levels of HIV PIs in plasma and the central nervous system remains a possibility.

HIV PI recognition by MRP1 may also have other physiological consequences. For example, MRP1 facilitates the secretion of leukotrenes, the major mediators of inflammation. Recent studies show continued immunological improvement despite virological failure among patients treated with HIV PIs (19). These observations raise the possibility that some of the therapeutic benefits of HIV PIs may be due to anti-inflammatory activity exerted by its interactions with MRP1 as well.

Finally, HIV PIs are nontoxic to cells and are therefore unlikely to induce overexpression of MDR1 or MRP1 in T cells and other tissues. Nevertheless, further studies are required to validate this point. Viral resistance to HIV PIs is responsible for treatment failure in one-half to one-third of the patients failing therapy. While noncompliance has largely been held responsible for the remainder of treatment failures, other physiological factors may also play a role. We are currently investigating whether activation of multidrug transporters plays any role in failure of PI therapy.

	ACKNOWLEDGMENTS

This work was supported in part by Public Health Service grants RO1 AI27652 and UO1-AI32908, by Cancer Center (CORE) grant P30 CA21765 from the NIH, and by the American Lebanese Syrian Associated Charities.

We thank Chris Guglielmo and Frank Pinkerton for assistance with flow cytometry and HPLC, respectively. We are grateful to W. T. Beck for providing the various CEM cell lines used in this study. We thank R. Gallo for HIV-1_IIIB, N. Roberts and P. Tomilson for saquinavir-resistant HIV-1, E. Eminii and W. Schleif for HIV-1_{M46I/L63P/V82T/184V}, and D. Richman for MT-2 cells, all through the NIH/NIAID AIDS Research and Reference Reagent Program.

	FOOTNOTES

^* Corresponding author. Mailing address: Department of Infectious Diseases, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. Phone: (901) 495-2359. Fax: (901) 495-3099. E-mail: ranga.srinivas{at}stjude.org.

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Antimicrobial Agents and Chemotherapy, December 1998, p. 3157-3162, Vol. 42, No. 12
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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