JAIDS Journal of Acquired Immune Deficiency Syndromes:
Letters to the Editor
Atazanavir Inhibits P-Glycoprotein and Multidrug Resistance-Associated Protein Efflux Activity
Lucia, Mothanje Barbara MD, PhD*; Golotta, Caterina MD†; Rutella, Sergio MD, PhD*; Rastrelli, Elena MD*; Savarino, Andrea MD*; Cauda, Roberto MD, PhD*
*Department of Infectious Diseases, Catholic University, Rome, Italy, †Department of Hematology, Catholic University, Rome, Italy
To the Editor:
P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP) are the best studied energy-dependent adenosine triphosphate (ATP)-binding cassette (ABC) drug efflux pumps.1,2 P-gp and MRP function as major transporters for the HIV-protease inhibitors (PIs),3,4 and for this reason, their expression and function are determinants of intracellular concentrations of these agents.5 A variety of cell types directly involved in HIV pathobiology express both P-gp and MRP (eg, peripheral blood lymphocytes [PBLs], bone marrow stem cells).6,7 Among the PIs, ritonavir (RTV) is the most potent inhibitor of P-gp and MRP efflux, although all the PIs are substrates for P-gp and MRP-1 and, to some degree, also inhibitors,3,8,9 whereas they do not significantly induce P-gp/MRP expression in normal PBLs.10,11 Atazanavir (ATV) is a potent novel azapeptide inhibitor of the HIV-1 protease with a pharmacokinetic profile permitting once-daily administration and a lower degree of dyslipidemia.12 The objective of this study was to analyze the effect of ATV on P-gp and MRP function in normal PBLs as well as in hemopoietic stem cells (HSCs) from human umbilical cord blood cells (UBCs), which provide a powerful in vitro model of normal human hematopoiesis.13 Cells were coincubated with the following HIV PIs: ATV (Bristol-Myers Squibb), RTV (Abbott Laboratories), saquinavir (SQV; Roche Products), and indinavir (IDV; Merck Sharp & Dohme). PBLs were collected by 5 healthy volunteers. UBCs were obtained from 3 informed mothers undergoing cesarean delivery of full-term infants in accordance with hospital ethical regulations. Mononuclear cells were separated by Ficoll-Hypaque density centrifugation, washed, and subsequently incubated with Miltenyi MultiSort anti-CD34 MicroBeads (Miltenyi Biotec; 100 μL/108 cells); after extensive washing, they were processed using a VarioMACS magnetic device to remove non-CD34+ cells, as previously described.14 CD34+ cells were eluted after removal of unwanted unbound cells by discontinuation of the magnetic fields in the VarioMACS device. All CD34+ immunoselected samples had CD34 cell purity greater than 95%. P-gp and MRP activity was evaluated using functional assays for rhodamine 123 (Rh123; Sigma Chemicals)15 and carboxyfluorescein diacetate (CF; Molecular Probes, Eugene, OR),16 respectively, with or without ATV (0.2-10 μM), RTV (3 μM), SQV (3 μM), and IDV (3 μM), as previously reported.8,17 The anthracycline compound doxorubicin (DOX; Adria Laboratories, Columbus, OH), a common P-gp/MRP naturally fluorescent substrate,18 was used for HSC functional analysis. A human acute lymphoblastic leukemia cell line (CEM) and its vinblastine-resistant subline (CEM/VBL100), kindly provided by Dr. M. Cianfriglia (Istituto Superiore di Sanità, Rome, Italy), were used as negative and positive controls, respectively, for P-gp expression and function. Results were expressed as percentages of dim and bright cells in the case of bimodality of the fluorescence during dye efflux or as mean fluorescence intensity (MFI) when fluorescence distribution in a cell population was Gaussian.8,17 P-gp phenotype was detected by using the specific monoclonal antibody MRK-16 (10 μg/mL; Kamiya Biomedicals, Seattle, WA), as previously reported.8
We investigated whether ATV alone or in combination with other PIs (ie, RTV, SQV) inhibits P-gp and MRP function in normal PBLs. Rh123 retention was studied in the absence and presence of increasing ATV concentrations corresponding to its steady-state pharmacokinetic profile.19 ATV increased Rh123 retention in a concentration-dependent manner, effectively blocking efflux at 10 μM (Fig. 1). We also observed an augmented inhibitory effect between ATV and RTV or SQV (see Fig. 1). ATV also significantly inhibited MRP efflux function (data not shown) as well as DOX efflux by HSCs (see Fig. 1). P-gp expression of drug-selected CEM/VBL100 was higher than that of the corresponding parental cells (3% CEM/P-gp+, 100% CEM/VBL100/P-gp+), and higher doses of ATV were required to inhibit Rh123 efflux (CEM-MFI: efflux 169, ATV-10 μM: efflux 207, ATV-50 μM: efflux 389; CEM/VBL100-MFI: efflux 131, ATV-10 μM: efflux 190, ATV-50 μM: efflux 295, ATV-100 μM: efflux 411), suggesting that ATV may be a possible P-gp/MRP substrate and competitively inhibit P-gp/MRP function as a drug efflux pump.
Although PIs represent a clear advance in the management of HIV disease, drug resistance, side effects, and drug interactions are common. To understand and predict pharmacokinetics and drug interactions, it is essential to determine the interaction of these agents with drug transporters of the ABC family that have been shown to control the intracellular concentrations of PIs.5 ATV is a new once-daily administered PI with excellent anti-HIV activity that is metabolized by cytochrome CYP3A substrate.12,19 It is not known, however, whether ATV interacts with drug transporter proteins because it differs from the peptidomimetic PIs (eg, ritonavir, nelfinavir) by its C-2 symmetric chemical structure.19 Our results indicate that ATV is a possible substrate for P-gp and MRP in HIV-target cells such as normal PBLs and, like other PIs, competitively inhibits their efflux activity in a dose-dependent manner. The concentrations of ATV used here are comparable to those achieved clinically.19 We also observed an increased inhibitory effect of ATV in combination with RTV and SQV that follows the hierarchy ATV+RTV > ATV+SQV, which is most likely attributable to the higher inhibitory potential shown by ATV compared with the older PIs. These data support a recent observation that suggests a predominant effect of RTV on CYP3A4 metabolism and of ATV on ABC transporter activity when ATV, RTV, and SQV are coadministered.20 In addition, we observed a significant ATV-mediated inhibition of the efflux of the anthracycline compound DOX by HSCs from human UBCs. Because P-gp and MRP transport a broad range of anticancer agents (ie, anthracyclines, Vinca alkaloids, taxanes),21 our results help to explain the higher hematologic toxicity induced by the combination of PI-containing highly active antiretroviral therapy (HAART) plus chemotherapy in patients with AIDS-related neoplasms receiving MDR-related cytotoxic agents.22,23 The modulator effect exerted by PIs when used in conjunction with anticancer drugs may enhance the cytotoxicity of chemotherapy drugs as a result of reduced drug efflux and increased intracellular concentration.
Mothanje Barbara Lucia, MD, PhD*
Caterina Golotta, MD†
Sergio Rutella, MD, PhD*
Elena Rastrelli, MD*
Andrea Savarino, MD*
Roberto Cauda, MD, PhD*
*Department of Infectious Diseases Catholic University Rome, Italy †Department of Hematology Catholic University Rome, Italy
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8. Lucia MB, Rutella S, Leone G, et al. HIV-protease inhibitors contribute to P-glycoprotein efflux function defect in peripheral blood lymphocytes from HIV-positive patients receiving HAART. J Acquir Immune Defic Syndr. 2001;27:321-330.
9. Srinivas R, Middlemas D, Flynn P, et al. Human immunodeficiency virus protease serve as substrates for multidrug transporter MDR1 and MRP1 but retain antiviral efficacy in cell lines expressing these transporters. Antimicrob Agents Chemother. 1998;42:3157-3162.
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11. Lucia MB, Savarino A, Straface E, et al. Role of lymphocyte multidrug resistance associated protein 1 (MRP1) in HIV infection: expression, function, and consequences of inhibition. J Acquir Immune Defic Syndr. (In press).
12. Havlir DV, O'Marro SD. Atazanavir: new option for treatment of HIV infection. Clin Infect Dis. 2004;38:1599-1604.
13. Leglise MC, Darodes de Tailly P, Vignot JL, et al. A cellular model for drug interactions on hematopoiesis: the use of human umbilical cord blood progenitors as a model for the study of drug-related myelosuppression of normal hematopoiesis. Cell Biol Toxicol. 1996;12:39-53.
14. Rutella S, Bonanno G, Marone M, et al. Identification of a novel subpopulation of human cord blood CD34-CD133-CD7-CD45+ lineage-cells capable of lymphoid/cell differentiation after in vitro exposure to IL-15. J Immunol. 2003;15:2977-2988.
15. Lee JS, Paull K, Alvarez M, et al. Rhodamine efflux patterns predict P-glycoprotein substrates in the National Cancer Institute drug screen. Mol Pharmacol. 1994;46:627-638.
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17. Savarino A, Lucia MB, Rastrelli E, et al. Anti-HIV effects of chloroquine. Inhibition of viral particle glycosylation and synergism with protease inhibitors. J Acquir Immune Defic Syndr. 2004;35:223-232.
18. Harbottle A, Daly AK, Campbell C. Role of glutathione s-transferase P1, P-glycoprotein and multidrug resistance-associated protein 1 in acquired doxorubicin resistance. Int J Cancer. 2001;92:777-783.
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20. Boffito M, Kurowski M, Kruse G, et al. Atazanavir enhances saquinavir hard-gel concentrations in a ritonavir-boosted once-daily regimen. AIDS. 2004;18:1291-1297.
21. Fischer G, Lum B, Sikic BJ. Clinical studies with modulators of multidrug resistance. In: Fischer G, Sikic B, eds. Drug Resistance Clinical Oncology and Hematology. Philadelphia: WB Saunders; 1995:363-382.
22. Spina M, Gabarre J, Rossi G, et al. Stanford V regimen and concomitant HAART in 59 patients with Hodgkin disease and HIV infection. Blood. 2002;100:1984-1988.
23. Bower M, McCall-Peat N, Ryan N, et al. Protease inhibitors potentiate chemotherapy induced neutropenia. Blood. 2004;104:2943-2946.
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