Raltegravir does not revert efflux activity of MDR1-P-glycoprotein in human MDR cells
- 3k Downloads
Raltegravir (Isentress®)(RALT) has demonstrated excellent efficacy in both treatment-experienced and naïve patients with HIV-1 infection, and is the first strand transfer integrase inhibitor to be approved for use in HIV infected adults worldwide. Since the in vivo efficacy of this class of antiviral drugs depends on their access to intracellular sites where HIV-1 replicates, we analyzed the biological effects induced by RALT on human MDR cell systems expressing multidrug transporter MDR1-P-glycoprotein (MDR1-Pgp).
Our study about RALT was performed by using a set of consolidated methodologies suitable for evaluating the MDR1-Pgp substrate nature of chemical and biological agents, namely: i) assay of drug efflux function; ii) analysis of MDR reversing capability by using cell proliferation assays; iii) monoclonal antibody UIC2 (mAb) shift test, as a sensitive assay to analyze conformational transition associated with MDR1-Pgp function; and iv) induction of MDR1-Pgp expression in MDR cell variant subjected to RALT exposure.
Functional assays demonstrated that the presence of RALT does not remarkably interfere with the efflux mechanism of CEM-VBL100 and HL60 MDR cells. Accordingly, cell proliferation assays clearly indicated that RALT does not revert MDR phenotype in human MDR1-Pgp expressing cells. Furthermore, exposure of CEM-VBL10 cells to RALT does not induce MDR1-Pgp functional conformation intercepted by monoclonal antibody (mAb) UIC2 binding; nor does exposure to RALT increase the expression of this drug transporter in MDR1-Pgp expressing cells.
No evidence of RALT interaction with human MDR1-Pgp was observed in the in vitro MDR cell systems used in the present investigation, this incorporating all sets of studies recommended by the FDA guidelines. Taken in aggregate, these data suggest that RALT may express its curative potential in all sites were HIV-1 penetrates, including the MDR1-Pgp protected blood/tissue barrier. Moreover RALT, evading MDR1-Pgp drug efflux function, would not interfere with pharmacokinetic profiles of co-administered MDR1-Pgp substrate antiretroviral drugs.
KeywordsRaltegravir MDR1-Pgp Drug substrate MDR1-Pgp induction Antiretroviral treatment
- MDR1-Pgp and ABCB1
Quinolonyl diketoacid derivatives
The suboptimal penetration of antiretroviral agents into sanctuary sites such as the central nervous system or into target CD4 cells may contribute to viral persistency. Drug transporters are viewed as one of the major mechanisms which account for suboptimal tissue concentrations of antiretroviral agents. MDR1-P-glycoprotein (MDR1-Pgp, ABCB1), as well as other ABC family members of structurally and functionally related proteins, is a plasma membrane transporter which participates in the transport of a wide variety of drugs, including anti-cancer chemotherapeutics  and antiretroviral compounds . The antiviral agent Raltegravir (Isentress®)(RALT) is the first integrase inhibitor (IIN) to be approved for treatment of HIV infection in adults [3, 4]. However, the involvement of human drug transporters in RALT absorption, disposition, metabolism and excretion (ADME) has not been fully investigated. RALT has been described as being an MDR1-Pgp substrate [5, 6], but there are still few data in the public domain, which are not even definitive.
As for all known anti-retrovirals, the emergence of viral mutations conferring resistance to antiretroviral agents has been documented for this compound . However, drug resistance may also be caused by the biological activity of MDR1-Pgp and/or other members of the ABC transporter family which, through intercepting drugs by means of the binding transport sites within the MDR1-Pgp binding pocket, delivers them out of the cells via an ATP dependent mechanism [8, 9]. MDR1-Pgp was initially studied in the setting of anticancer treatment; it was identified as the biological entity conferring the multidrug resistance (MDR) in tumor cells, this by reducing the level of cytotoxic drug under sub-lethal concentration . In vitro and in vivo studies have shown that all protease inhibitors display a high affinity for MDR1-Pgp [11, 12, 13], as well the CCR5 inhibitor maraviroc [6, 14] and quinolonyl diketoacid derivatives (DKA) with anti-integrase activity . These latter compounds, although different in chemical structure from RALT, exert a similar inhibition on strand transfer activity of HIV-1 integrase. Since in vivo efficacy of this class of drugs depends on their access to intracellular sites where HIV-1 replicates, and given that limited information exists on RALT interaction with human MDR1-Pgp expressing cells, we performed a set of well-established in vitro studies on the human CD4 positive lymphoblastoid CCRF-CEM cell line and its derivative MDR variants, in line with FDA concept paper on drug interactions . In order to strengthen the data about the interaction between RALT and human MDR1-Pgp, we incorporated an additional human MDR cell system in this investigation. In line with FDA recommendations, we evaluated RALT as substrate, inhibitor and inducer of MDR1-Pgp by performing the following studies: i) inhibition of drug transport function by using the classical efflux assay ; ii) down-modulation of multidrug resistance (MDR) phenotype in cell proliferation assay ; iii) up-modulation of the monoclonal antibody (mAb) UIC2 epitope in MDR cells during MDR1-Pgp-mediated drug transport ; and iv) induction of MDR1-Pgp expression by exposing MDR CEM-VBL10 cells to MDR1-Pgp substrates .
Results and discussion
Assessment of MDR1-Pgp expression level in human MDR cell lines
UIC2 shift assay
Induction of MDR1-Pgp expression in MDR cells
Several clinical trials have shown a sustained antiretroviral effect and a good tolerability of RALT in naive and treatment-experienced HIV-1 infected patients . Previous investigations have already reported that RALT has a low propensity for involvement in drug–drug interactions . Update studies on pharmacology profile of RALT are described in the recently published review article by Brainard et al. , which reports that RALT is not an inhibitor of the major CYP isozymes, including CYP3A4, UGTs, and MDR1-Pgp. Additionally, it has been reported that RALT is not an inducer of CYP3A4 RNA expression or CYP3A4-dependent testosterone 6 beta-hydroxylase activity . In previous studies conducted by our group, a series of diketoacid-containing derivatives (DKA) functioning as inhibitors of HIV-1 integrase have been described as being MDR1-Pgp ABCB1 substrates  with strong MDR1-Pgp inhibitory activity. Elvitegravir [4, 6], which has a biochemical formulation similar to DKA, shows marked drug interaction with MDR1-Pgp multi-drug transporter and acts as a strong MDR reversing agent . Our study performed with human MDR cell lines clearly shows that the RALT compound does not inhibit MDR1-Pgp mediated drug transport function. The different level of cytotoxic effect exerted by RALT on drug sensitive/resistant cell pairs (Figure 4) and the low shift of a small fraction of MDR cell population incubated in presence of Calcein-AM may be cell type and dye-substrate related, and not sufficient to establish the existence of an authentic interaction with MDR1-Pgp. In this context, Zambruski et al. , include elvitegravir, vicriviroc and to a lesser extent RALT in the list of MDR1-Pgp substrates. However, our own findings concerning RALT seem to suggest otherwise, a possible explanation being in the different cell system used and in the interpretation of data. Again in this regard, Moss et al. showed that RALT has minimal interactions with known drug transporters, and that the rate of MDR1-Pgp-mediated transport in vitro is so low that the potential for interactions of this entity is expected to be small . The very low rate of RALT transport by MDR1-Pgp expressing cells may explain the absence of major drug interactions with known potent MDR1-Pgp inhibitors. Furthermore, very recently, Tempestilli et al.,  showed that darunavir, unlike RALT, may modify the expression and functionality of MDR1-Pgp on human lymphocytes. Taken in aggregate, the above mentioned studies are consistent with a previous report where the co-administration of low-dose ritonavir had no major effect on RALT pharmacokinetics, and no dose adjustment was required for patients .
Overall, these findings suggest that, in addition to its well known efficacy and safety, RALT may present an advantage in respect to other anti-retrovirals that are MDR1-Pgp substrate. Indeed, RALT’s biological properties may endow it with a higher therapeutic potential against HIV-1 residing in sanctuaries sites pharmacologically protected by MDR1-Pgp expressed on blood tissue barriers. However, in this context, it is important to remember that, despite MDR1-Pgp is the first discovered and probably the most widely studied ABC transporter protein, there are other ABC transporters involved in clinical MDR and in drug absorption and distribution; these include multidrug resistance proteins (MRPs, ABCCs) and breast cancer resistant protein (BCRP, ABCG2) [32, 33]. In particular, MRP1, MRP2, MRP4 and BCRP/ABCG2, together with MDR1-Pgp, are present on many barrier sites such as the blood-brain barrier and on many circulating cells such as lymphocytes, and consequently they could contribute to reduce antiretroviral agents in sanctuary or HIV-1 target sites .
Our investigations demonstrate that RALT is ineffective in inhibiting drug efflux and in down-modulating the MDR phenotype of human CEM and HL60 MDR cells to an extent considered relevant in vivo by FDA guidelines . In addition, exposure of CEM-VBL10 to RALT does not induce the functional conformation of MDR1-Pgp intercepted by the shift of UIC2 mAb binding. Furthermore, in contrast to other licensed anti-HIV-1 drugs such as the protease inhibitors, RALT has proved to be ineffective in inducing an increase of MDR1-Pgp expression level in MDR cells in culture conditions. The absence of remarkable RALT/MDR1-Pgp interaction may represent a medically relevant property, although at present its impact in the clinical setting is not totally clear. Further studies are warranted to better define i) the mechanisms underlying the profound functional differences of RALT in comparison with other IINs which behave as MDR-Pgp substrates and MDR reversing agents and, ii) the potential involvement of other ABC drug transporters in RALT absorption and disposition.
The RALT was a kind gift of the Merck company (Pomezia, Rome, Italy); Verapamil (Isoptin) was purchased by Abbott (Latina, Italy); Vinblastine (Velbe) by Eli Lilly (Paris, France); Rhodamine-123 was purchased from Sigma (St. Louis, MO). Vinblastine-bodipy (VBL-bodipy) and Calcein acetoxymethlyl ester (Calcein-AM) were purchased from Molecular Probes (Eugene, OH).
The multidrug resistant (MDR) variants CEM-VBL10 and CEM-VBL100 cells were isolated by stepwise selection of the parental drug sensitive CCRF-CEM (CEM) in the presence of increasing concentrations of VBL [up to the final concentration of 10 and 100 ng/mL, respectively]. Cells were grown under standard conditions for mammalian cells cultured in suspension. The basic medium (BM) for cell culturing consisted of RPMI-1640 supplemented with 10% foetal calf serum (FCS), L-glutamine (2 mM) penicillin (100 U/mL) and streptomycin (100 U/mL). All these components were purchased from Hyclone (Logan, Utah, USA). Identical culture conditions were adopted for the multidrug resistant (MDR) variants HL60-DNR, kindly provided by Dr. Ruoping Tang (Hopitaux de Paris, Paris, France).
MDR efflux assay
CEM-VBL100 and HL60-DNR cell lines (1 × 106) were loaded with Rh123 (5 μg/mL)(or with VBL-bodipy, 50 ng/mL, or Calcein-AM, 50 ng/mL) in 1 mL of BM in the presence of RALT (concentrations: 50 and 25 μg/mL) or Vrp (2.5 μg/mL) for 1 h at 37°C. The cells were incubated with Rh123 at the indicated concentrations or with drug diluents (DMSO: 0.5%; H2O). At the end of incubation, the cells were washed in serum-free medium and re-suspended in BM in the presence of RALT or Vrp (drug diluents was added in control samples) for a further 1 h at 37°C. Finally, cells were washed twice with ice cold phosphate-buffered saline (PBS)/FACS, and analyzed in a flow cytometer (FACScan, Becton Dickinson, San Josè, CA).
Cell proliferation assay
The parental drug sensitive and their MDR derivative cell lines in exponential phase of growth were collected, extensively washed with warm RPMI-1640 and seeded (in triplicate) in 96-well microtiter Costar plates (Costar, Rochester, NY) at a density of 5×103 cells/mL.
For MDR chemosensitization studies, the cells were cultured in BM containing increasing concentrations of VBL ranging from 0 to 10 μg/mL; in parallel, MDR cell cultures containing the different concentrations of VBL were grown in the presence of RALT (12.5 μg/mL) dissolved in water. As a control the MDR reversing agent Vrp was used at the concentration of 2.5 μg/mL in an additionally parallel culture. In growth inhibition assays RALT was tested alone at 4 concentrations spread over a range between 0 and 100 μg/mL. For all above described experiments cell survival was determined by WST-1 assay (PreMix WST-1 cell proliferation kit, Vinci Biochem, Firenze, Italy) after 72 h treatment at 37˚C in 5% CO2. The values describing the concentration-response profiles are calculated as % of appropriated control and represent the mean of three independent experiments, each done in triplicate. The GraphPad Prism statistical analysis program was used.
Monoclonal antibodies and UIC-2 Shift assay
The mAb UIC2  was kindly provided by Dr. E. Mechetner (Chemicon Inc, Temecula, CA). For determination of MDR1-Pgp expression, the mAb MM4.17 recognizing an extracellular MDR1-P-gp epitope on intact/living human MDR cells  was used (data not shown). Both UIC2 and MM4.17 mAbs were used in a highly purified form. The UIC2 shift assay was performed under physiological conditions as previously described . CEM-VBL10 cells (1×106) were resuspended in 1 mL of PBS containing 2% FCS and allowed to equilibrate at 37°C in a water bath for 10 min. The RALT was added to samples (final concentration 25 and 50 μg/mL) and incubated for additional 15 min at 37°C with purified UIC2 mAb (final concentration 12.5 μg/mL). VBL (10 μg/mL), a well known UIC2 shifting agent, was used as positive control do detect the conformation of MDR1-Pgp during drug efflux function. Cells were then washed twice in ice-cold PBS containing 2% FCS with 0.01% sodium azide (Shift Stop Buffer, SSB), stained on ice in SSB for additional 15 min with 5 μg/mL of fluorescein-conjugated goat-antimouse antibody (FITC-GAM, Cappel, West Chester, Pa, USA), washed twice with ice cold PBS/FACS and maintained in ice until flow cytometry analysis.
Induction of MDR1-Pgp expression in MDR cells
For the evaluation of the induction of MDR phenotype, CEM-VBL10 cells in exponential phase of growth were collected, extensively washed with warm RPMI-1640 and resuspended at the concentration of 5 × 104 cells/mL in BM alone, or in the presence of different VBL concentrations (from 100 ng/mL to 12.5 ng /mL) or RALT (from 100 μg/mL to 12.5 μg/mL) and were seeded in 24-wells Costar plates (Costar, Rochester, NY) for 96 h. At the end of the incubation, the cells were harvested, washed with BM alone, and incubated with 12.5 μg/mL of mAb MM4.17. After 30 min of incubation at 4°C, the cells were washed, pelleted, resuspended and incubated for an additional 30 min at 4°C in the presence of fluorescein-conjugated goat antimouse antibody (FITC-GAM, Cappel). After incubation, the cells were washed, resuspended in PBS and processed for flow cytometry analysis.
All the experiments were repeated at least thrice. The significance was assessed by Student's t-test and the criterion for statistical significance was set at P < 0.05.
This work has been possible thanks to funding received from EC in the Project: The European AIDS Treatment Network, (NEAT) with contract number LSHP-CT-2006-037570, which is a FP 6 Network of Excellence within the “Integrating and Strengthening the European Research Area”. This work was supported by AIDS grants of Istituto Superiore di Sanità and Italian Ministry of Health, and partly by an ISS-NIH research grant.
It was also supported by a research grant from the Investigator Initiated Studies Program of Merck Sharp & Dohme Corp. The opinions expressed in this paper are those of the authors and do not necessarily represent those of Merck Sharp & Dohme Corp. We thank Mrs Stefania Donnini for secretarial support and Martin Bennett for his help in revising the manuscript for grammar and style.
- 3.Summa V, Petrocchi A, Bonelli F, Crescenzi B, Donghi M, Ferrara M, Fiore F, Gardelli C, Gonzalez Paz O, Hazuda DJ, Jones P, Kinzel O, Laufer R, Monteagudo E, Muraglia E, Nizi E, Orvieto F, Pace P, Pescatore G, Scarpelli R, Stillmock K, Witmer MV, Rowley M: Discovery of raltegravir, a potent, selective orally bioavailable HIV-integrase inhibitor for the treatment of HIV-AIDS infection. J Med Chem. 2008, 51: 584-237.CrossRefGoogle Scholar
- 4.Pandey KK: Raltegravir in HIV-1 infection: safety and efficacy in treatment-naïve patients. Clin Med Rev Ther. 2012, 2011: 13-30.Google Scholar
- 7.Malet I, Delelis O, Valantin MA, Montes B, Soulie C, Wirden M, Tchertanov L, Peytavin G, Reynes J, Mouscadet JF, Katlama C, Calvez V, Marcelin AG: Mutations associated with failure of raltegravir treatment affect integrase sensitivity to the inhibitor in vitro. Antimicrob Agents Chemother. 2008, 52: 1351-1358. 10.1128/AAC.01228-07.CrossRefPubMedPubMedCentralGoogle Scholar
- 14.Walker DK, Bowers SJ, Mitchell RJ, Potchoiba MJ, Schroeder CM, Small HF: Preclinical assessment of the distribution of maraviroc to potential human immunodeficiency virus (HIV) sanctuary sites in the central nervous system (CNS) and gut-associated lymphoid tissue (GALT). Xenobiotica. 2008, 38: 1330-1339. 10.1080/00498250802447409.CrossRefPubMedGoogle Scholar
- 15.Di Santo R, Costi R, Roux A, Artico M, Lavecchia A, Marinelli L, Novellino E, Palmisano L, Andreotti M, Amici R, Galluzzo CM, Nencioni L, Palamara AT, Pommier Y, Marchand C: Novel bifunctional quinolonyl diketo acid derivatives as HIV-1 integrase inhibitors: design, synthesis, biological activities, and mechanism of action. J Med Chem. 2006, 49: 1939-1945. 10.1021/jm0511583.CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Huang SM, Strong JM, Zhang L, Reynolds KS, Nallani S, Temple R, Abraham S, Habet SA, Baweja RK, Burckart GJ, Chung S, Colangelo P, Frucht D, Green MD, Hepp P, Karnaukhova E, Ko HS, Lee JI, Marroum PJ, Norden JM, Qiu W, Rahman A, Sobel S, Stifano T, Thummel K, Wei XX, Yasuda S, Zheng JH, Zhao H, Lesko LJ: New era in drug interaction evaluation: US Food and Drug Administration update on CYP enzymes, transporters, and the guidance process. J Clin Pharmacol. 2008, 48: 662-670. 10.1177/0091270007312153.CrossRefPubMedGoogle Scholar
- 18.Cianfriglia M, Dupuis ML, Molinari A, Verdoliva A, Costi R, Galluzzo CM, Andreotti M, Cara A, Di Santo R, Palmisano L: HIV-1 integrase inhibitors are substrates for the multidrug transporter MDR1-P-glycoprotein. Retrovirology. 2007, 7: 4-17.Google Scholar
- 19.Mechetner EB, Schott B, Morse BS, Stein WD, Druley T, Davis KA, Tsuruo T, Roninson IB: P-Glycoprotein function involves conformational transitions detectable by differential immunoreactivity. Proc Natl Acad Sci USA. 1997, 94: 12908-12913. 10.1073/pnas.94.24.12908.CrossRefPubMedPubMedCentralGoogle Scholar
- 21.Cianfriglia M, Willingham MC, Tombesi M, Scagliotti GV, Frasca G, Chersi A: P-glycoprotein epitope mapping. I: Identification of a linear human-specific epitope in the fourth loop of the Pglycoprotein extracellular domain by MM4.17 murine monoclonal antibody to human multi-drug-resistant cells. Int J Cancer. 1994, 56: 153-160.CrossRefPubMedGoogle Scholar
- 24.Nagy H, Goda K, Fenyvesi F, Bacsó Z, Szilasi M, Kappelmayer J, Lustyik G, Cianfriglia M, Szabó G: Distinct groups of multidrug resistance modulating agents are distinguished by competition of P-glycoprotein-specific antibodies. Biochem Biophys Res Commun. 2004, 315: 942-949. 10.1016/j.bbrc.2004.01.156.CrossRefPubMedGoogle Scholar
- 28.Markowitz M, Morales-Ramirez JO, Nguyen B-Y, Kovacs CM, Steigbigel RT, Cooper DA, Liporace R, Schwartz R, isaacs R, Gilde LR, Wenning L, Zhao J, Teppler H: Antiretroviral activity, pharmacokinetics, and tolerability of MK-0518 a novel inhibitor of HIV-1 integrase, dosed as monotherapy for 10 days in treatment- naive HIV-1-infected individuals. J Acquir Immune Defic Syndr. 2006, 43: 509-515. 10.1097/QAI.0b013e31802b4956.CrossRefPubMedGoogle Scholar
- 30.Tempestilli M, Gentilotti E, Tommasi C, Nicastri E, Martini F, De Nardo P, Narciso P, Pucillo LP: Determination of P-glycoprotein surface expression and functional ability after in vitro treatment with darunavir or raltegravir in lymphocytes of healthy donors. Int Immunopharmacol. 2013, 16: 492-497. 10.1016/j.intimp.2013.05.003.CrossRefPubMedGoogle Scholar
- 31.Iwamoto M, Wenning LA, Petry AS, Laethem M, De Smet M, Kost JT, Breidinger SA, Mangin EC, Azrolan N, Greenberg HE, Haazen W, Stone JA, Gottesdiener KM, Wagner JA: Minimal effects of ritonavir and efavirenz on the pharmacokinetics of raltegravir. Antimicrob Agents Chemother. 2008, 52: 4338-4343. 10.1128/AAC.01543-07.CrossRefPubMedPubMedCentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/2050-6511/14/47/prepub
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.