To the Editors:
It is known that a number of multidrug-resistant proteins (MRPs) and P-glycoprotein (P-gp/MDR1), all members of the ATP-binding cassette (ABC) superfamily, are able to transport antiretroviral (ARV) drugs1 and that the expression of these transporters, in the presence of HIV infection, could affect the efficacy of antiviral drug therapy by influencing the pharmacokinetics of the ARVs and/or by promoting the development of drug resistance.2-4 It has also been reported that HIV infection itself and/or different ARV treatments can increase the transcription of MRP1, MRP4, and MRP5 in human monocyte-derived macrophages and/or in peripheral blood mononuclear cells (PBMCs),5,6 thus possibly contributing to treatment failure. However, the specific contribution of different ABC proteins to treatment failure has not been defined in detail, and the clinical relevance of these transporters in the treatment of HIV/AIDS still remains questionable, mainly because no prospective studies have been undertaken.
To gain new insights into this phenomenon, we planned to evaluate if mRNA expression of MDR1 and some of the known MRPs was also affected by ARV treatment rather than by HIV infection. Then, 13 HIV-positive treatment-naive patients (11 men and 2 women), who were just starting therapy, were followed for 48 weeks by monitoring the mRNA expression levels of MDR1, MRP1, MRP4, and MRP5. At the baseline, the median CD4 cell count was 99 cells per microliter (range, 10-282 cells/μL) and the median viral load was >109,848 copies per milliliter (range, 6385 to >500,000 copies/mL).
All patients started ARV treatment. Specifically, 5 patients were treated with Kaletra (Abbott Laboratories, Kent, UK) and Truvada (Gilead Sciences, Cambridge, UK), 3 patients were treated with Reyataz (Bristol Myers Squibb, Hounslow, UK) and Truvada, and 5 patients were treated with Sustiva (Bristol Myers Squibb, Hounslow, UK) and Truvada. Once treatment began, blood samples were collected at 12, 24, 36, and 48 weeks. All patients were virologically suppressed during follow-up.
mRNA expression for transporters was evaluated by real-time polymerase chain reaction [TaqMan technology (ABI Prism 7000; Applied Biosystems, Monza, Italy)], using primers and probes as previously described.7 Coamplification of the β-glucuronidase mRNA housekeeping gene (TaqMan endogenous controls; Applied Biosystems) was used to normalize the amount of total RNA present. All samples were amplified in triplicate.
mRNA levels of the above transporters at all the times measured did not significantly differ from the levels detected before treatment began (T0). A high interindividual variability existed both before and after treatment began. To evaluate whether the mRNA transporter expression levels were differently affected by the different therapeutic regimen, samples were divided into 2 groups on the basis of their treatment: protease inhibitor (PI) + nucleoside reverse transcriptase inhibitor (NRTI) (group I) and nonnucleoside reverse transcriptase inhibitor + NRTI (group II). For each patient, the mRNA expression of the transporters was analyzed using the 2−ΔΔCt method,8 which represents the fold change in gene expression, relative to mRNA expression at T0.
As shown in Figure 1, mRNA expression of these transporters appears to be up- or downregulated during treatment, independent of both the type of therapy and the treatment time. For example, in patient 48, treated with PI + NRTI, increased levels of MDR1 mRNA with respect to those detected at T0 with a 7-fold increase at T36. In contrast, in patient 89, decreased expression of MDR1 mRNA was measured at T12, followed by 7-, 2-, and 4-fold increases at T24, 36, and 48, respectively. Interestingly, patient 48, who had exhibited an increase in MDR1 mRNA expression, also had increases in expression levels of MRP1 and MRP4. In most of the patients treated with PI + NRTI, an increase in MRP5 mRNA expression was measured during follow-up. For patients in group II, there was less variability in the expression of MDR1 than in group I. In only 1 patient (patient 54), reductions of 1 log and 0.5 log in mRNA expression for MDR1 were observed at T12 and T48, respectively. In patient 12, there were increases in MRP1 expression at each time point.
In summary, these results showed that no clear interference with the gene expression profiles of ABC transporters during ARV treatment existed at the times studied. These data seem to be in agreement with those from studies that demonstrated that ARVs had no influence on MDR1 or MRP expression in lymphocytes.9 However, other studies have shown a modulation in the expression or activity of ABC transporters by anti-HIV drugs.10,11 Indeed, in our analysis, the mRNA expression of these proteins was differently modulated after treatment began, but this did not apparently depend on the type of therapy. Various anti-HIV drugs have been shown to induce the expression of drug efflux transporters, such as MDR1 and MRP1, in a human intestinal epithelial cell line.10 These data were all obtained from in vitro studies, none of which were conducted on lymphocytes derived from patients undergoing treatment. In our study, the analysis was of PBMCs derived from HIV-infected individuals; hence, it can be speculated that the gene expression of these proteins may be affected not only by ARV therapy but also by host factors that will vary from patient to patient. This hypothesis is supported by other studies, which have suggested that several factors may influence transporter expression within cell membranes, such as cell subsets and their functionality, their activation status, and the development and polymorphism of the coding gene.9,12
The difference in mRNA expression levels detected in PBMCs both before and during ARV therapy do not appear to affect the virological response. Although some patients displayed a greater rise in CD4 cell counts than others, this was not associated with reduced expression of the transporters' mRNA levels. Furthermore, on initiation of ARV treatment, all patients had similar rates of viremia decay despite the different expression levels of mRNA.
These data apparently suggest that these transporters do not affect responses to ARV treatment. However, before any final conclusions can be drawn on the role of these efflux pumps in HIV therapy, it should be noted that real-time polymerase chain reaction, used here to evaluate the mRNA levels, is an indirect quantification method that may not reflect the levels of protein expression. Although several studies have reported good correlation between gene and protein expression for some transporters,13 we consider that to exclude the possibility that ABC proteins may contribute to the failure of ARV treatment, the amounts of proteins produced should be measured and their functionality evaluated. Furthermore, studies of large numbers of patients are needed to clarify this issue. In this study, because of the small sample size and the high interindividual variability, there was inadequate statistical power to detect modest differences at the different times analyzed.
Nevertheless, this prospective study suggests strongly that the mRNA levels of these membrane proteins are affected by several factors, which are reflected in the high interindividual variability observed before and during follow-up. At the end of the follow-up period, all patients had undetectable viremia. A longer follow-up period is needed to evaluate whether high expression levels of these transporters were associated with the early appearance of drug-resistant viral variants.
*Department of Molecular Medicine, “Sapienza” University of Rome, Rome, Italy
†Department of Hygiene, Public Health and Infectious Diseases, “Sapienza” University of Rome, Rome, Italy
1. Weiss J, Haefeli WH. Impact of ATP-binding cassette transporters on human immunodeficiency virus therapy. Int Rev Cell Mol Biol
2. Köck K, Grube M, Jedlitschky G, et al. Expression of adenosine triphosphate-binding cassette (ABC) drug transporters in peripheral blood cells: relevance for physiology and pharmacotherapy. Clin Pharmacokinet
3. Jones K, Bray PG, Khoo SH, et al. P-glycoprotein and transporter MRP1 reduce HIV protease inhibitor uptake in CD4 cells: potential for accelerated viral drug resistance? AIDS
4. Turriziani O, Di Marco P, Antonelli G, et al. May the drug transporter P glycoprotein affect the antiviral activity of human immunodeficiency virus type 1 proteinase inhibitors? Antimicrob Agents Chemother
5. Jorajuria S, Dereuddre-Bosquet N, NaissantStorck K, et al. Differential expression levels of MRP1, MRP4, and MRP5 in response to human immunodeficiency virus infection in human macrophages. Antimicrob Agents Chemother
6. Turriziani O, Gianotti N, Falasca F, et al. Expression levels of MDR1, MRP1, MRP4, and MRP5 in peripheral blood mononuclear cells from HIV infected patients failing antiretroviral therapy. J Med Virol
7. Taipalensuu J, Törnblom H, Lindberg G, et al. Correlation of gene expression of ten drug efflux proteins of the ATP-binding cassette transporter family in normal human jejunum and in human intestinal epithelial Caco-2 cell monolayers. J Pharmacol Exp Ther
8. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 [-Delta Delta C(T)] method. Methods
9. Giraud C, Manceau S, Declèves X, et al. Influence of development, HIV infection, and antiretroviral therapies on the gene expression profiles of ABC transporters in human lymphocytes. J Clin Pharmacol
10. Perloff MD, Von Moltke LL, Marchand JE, et al. Ritonavir induces P-glycoprotein expression, multidrug resistance-associated protein (MRP1) expression, and drug transporter-mediated activity in a human intestinal cell line. J Pharm Sci
11. Weiss J, Theile D, Ketabi-Kiyanvash N, et al. Inhibition of MRP1/ABCC1, MRP2/ABCC2, and MRP3/ABCC3 by nucleoside, nucleotide, and non-nucleoside reverse transcriptase inhibitors. Drug Metab Dispos
12. Fellay J, Marzolini C, Meaden ER, et al. Response to antiretroviral treatment in HIV-1-infected individuals with allelic variants of the multidrug resistance transporter 1: a pharmacogenetics study. Lancet
13. Young LC, Campling BG, Cole SP, et al. Multidrug resistance proteins MRP3, MRP1, and MRP2 in lung cancer: correlation of protein levels with drug response and messenger RNA levels. Clin Cancer Res