Lack of a Metabolic and Antiviral Drug Interaction Between Tenofovir
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Antiviral Therapy 10:451–457 Lack of a metabolic and antiviral drug interaction between tenofovir, abacavir and lamivudine Adrian S Ray1*, Florence Myrick2, Jennifer E Vela1, Loren Y Olson1, Eugene J Eisenberg1, Katyna Borroto- Esoda2, Michael D Miller1 and Arnold Fridland1 1Gilead Sciences, Inc, Foster City, CA, USA 2Gilead Sciences, Inc, Durham, NC, USA *Corresponding author: Tel: +1 650 571 7697; Fax: +1 650 522 5536; E-mail: [email protected] Objective: An anti-HIV regimen composed of the nucleo- presence or absence of 3TC was studied in stimulated side/nucleotide reverse transcriptase inhibitors (NRTIs) PBMCs infected with the HXB2 strain of HIV-1. tenofovir (TFV) disoproxil fumarate (TDF), abacavir (ABC) Results: Levels of the active metabolites produced from and lamivudine (3TC) has performed poorly in patients. TFV and ABC after incubation with CEM or PBMCs This study evaluated the combination of TFV, ABC and showed no significant change upon introduction of the 3TC for metabolic or antiviral antagonism in vitro. other NRTI. Moreover, the pool sizes for the natural Design: Procedures were developed to evaluate the in substrates of 2′-deoxyadenosine triphosphate and 2′- vitro metabolism and antiviral activity of drug combina- deoxyguanosine triphosphate were also unchanged. In tions of TFV, ABC and 3TC in cell types relevant for HIV anti-HIV assays in PBMCs, the combination of TFV and infection. ABC was found to be additive with respect to inhibition Methods: Anabolism of combinations of TFV and ABC of HIV replication. Addition of 3TC to the combination did were studied over a 24 h period in the human T not result in synergistic or antagonistic effects. leukaemic CEM lymphoblast cell line and human primary Conclusions: The poor efficacy of the triple NRTI regimen peripheral blood mononuclear cells (PBMCs) stimulated of TDF, ABC and 3TC is probably not due to a metabolic with human interleukin-2 and phytohaemagglutinin. The drug interaction resulting in antagonism of antiviral anti-HIV activity of combinations of TFV and ABC in the activity. Introduction High rates of virological non-response and virological the combination and (iii) a pharmacological drug inter- failure have been observed in several clinical trials action between TDF and ABC. The lack of a systemic assessing the ability of triple nucleoside reverse tran- pharmacokinetic drug interaction between TDF and scriptase inhibitor (NRTI) therapies to suppress HIV ABC in plasma has already been established [4]. infection. Study ESS30009 was conducted in 194 treat- The oral prodrug TDF is converted to TFV upon ment-naive patients and compared the efficacy of teno- absorption. Within cells, both ABC and TFV require fovir (TFV) disoproxil fumarate (TDF)/abacavir intracellular phosphorylation to their nucleoside (ABC)/lamivudine (3TC) to efavirenz/ABC/3TC, all in triphosphate analogue forms in order to have antiviral once-daily regimens. In an unplanned interim analysis, activity. The intracellular anabolism of ABC to its active a 49% virological failure rate was observed at 12 2′-deoxyguanosine triphosphate (dGTP) analogue, weeks in the triple NRTI arm versus 5% in the carbovir triphosphate (CBV-TP) is complex. ABC is efavirenz-containing arm [1]. Other studies with the first phosphorylated by adenosine monophosphate same combination of TDF/ABC/3TC also found poor phosphotransferase to ABC monophosphate, which is response and a high incidence of viral resistance muta- then deaminated to carbovir monophosphate (CBV- tions [2,3]. Since TDF and ABC have each been used MP) and then phosphorylated to CBV-TP via two successfully in regimens with 3TC, a problem between cellular kinases (Figure 1A) [5]. TFV follows a different TDF and ABC has been surmised. These results have led anabolic pathway to the active 2′-deoxyadenosine to the proposal of different mechanisms for the poor triphosphate (dATP) analogue TFV diphosphate (TFV- efficacy of triple NRTI regimens containing TDF and DP). The activation pathway involves the formation of ABC, including (i) intrinsic poor potency of all triple a single key intermediate, TFV-MP, by the activity of NRTI regimens, (ii) a low barrier for viral resistance to adenylate kinase 2 followed by further phosphorylation © 2005 International Medical Press 1359-6535 451 AS Ray et al. Figure 1. Activation pathways for ABC and TFV A O HN HN N N N H2N N N N H2N N AMP O HO –O PO Carbovir-TP O– AMP Cellular Cellular ABC phosphotransferase deaminase Carbovir-MP kinases B NH2 N N N O N –O PO Tenofovir-DP O– Adenylate Cellular kinase 2 kinases TFV (A) Anabolism of ABC includes deamination as well as phosphorylation [5]. (B) Phosphorylation pathway for TFV based on that determined for the acyclic nucleotide analogue adefovir [21]. Carbovir-MP, carbovir monophosphate; carbovir-TP, carbovir triphosphate; tenofovir-DP; tenofovir diphosphate. ABC, abacavir; TFV, tenofovir. to the active metabolite TFV-DP (Figure 1B) [6]. TFV- by GlaxoSmithKline (Research Triangle Park, NC, DP and CBV-TP compete with the intracellular dATP USA). All other chemicals used were the highest grade and dGTP pools, respectively, for incorporation by the available from Sigma-Aldrich, Inc (St Louis, MO, USA). viral RT. Due to their lack of 3′-hydroxyl groups, incorporation of these nucleotide analogues causes Cell culture chain termination of viral reverse transcripts and inhi- Human T leukaemic CCRF-CEM cells were obtained bition of viral replication. from the American Type Culture Collection (Manassas, In this article, we further examine the potential for a VA, USA) and were maintained in RPMI 1640 supple- drug interaction between TFV and ABC by studying mented with 10% heat-inactivated fetal bovine serum the in vitro intracellular pharmacology and anti-HIV (FBS), 100 units/ml penicillin G and 100 µg/ml strepto- activity of the two agents incubated either alone or in mycin sulphate. Stanford Blood Center (Palo Alto, CA, combination in cell types relevant for HIV infection. USA) and the American Red Cross (Durham, NC, USA) supplied buffy coats prepared from the blood of Materials and methods healthy donors. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by density Chemicals gradient centrifugation on Ficoll-Paque Plus using stan- Cell culture supplies were purchased from Irvine dard techniques as recommended by the manufacturer Scientific (Santa Ana, CA, USA). TFV and TFV-DP as (Amersham Pharmacia Biotech, Inc, Piscataway, NJ, well as adefovir-DP (used as an internal standard) were USA). Two additional washes in RBC lysis buffer synthesized by Gilead Sciences, Inc (Foster City, CA, (eBioscience, San Diego, CA, USA) were performed for USA). Stable isotope-labelled 13C15N dATP and dGTP PBMC preparations used in assays for triphosphate were purchased from Spectra Stable Isotopes evaluations. PBMC pellets were resuspended in 1640 (Columbia, MD, USA). TFV-MP was synthesized by Dr RPMI medium containing 10% heat-inactivated FBS, Ivan Rosenberg of the Institute of Organic Chemistry 2 mM L-glutamine and penicillin/streptomycin. Cells and Biochemistry, Czech Academy of Sciences, Prague, were stimulated with 20 U/ml of human interleukin-2 Czech Republic. ABC, 3TC and CBV-TP were provided (IL-2; Roche Diagnostics Corp, Indianapolis, IN, USA) 452 © 2005 International Medical Press Combination of tenofovir, abacavir and lamivudine and 1–5 µg/ml of phytohaemagglutinin from Phaseolus Antiviral synergy vulgaris (PHA-P; Sigma-Aldrich, Inc) for 48–72 h at TFV and ABC were tested in combination for antiviral 37°C and subsequently cultured in the same medium activity against the HXB2 laboratory strain of HIV-1 in without PHA-P. PBMCs. Stimulated PBMCs were infected with virus at a multiplicity of infection of 0.001 for 4 h, washed Intracellular metabolism once with complete media and plated in 96-well plates TFV or ABC either alone or in combination at concen- containing various concentrations of test compound. trations of 10–100 µM were added to CEM cells The final concentration of cells was 1 million PBMC seeded at 0.2 million/ml or PBMCs seeded at per well. Compounds were plated in a checkerboard 0.5 million/ml. At 2 and 24 h, aliquots of cells taken fashion with the starting concentration for each for analyses were separated from the media and extra- compound fixed at three- to fourfold above the EC50 cellular drug by spinning through oil [7]. Isolated cells value for the specific compound. Subsequent twofold were resuspended in 70% methanol in order to extract serial dilutions were then performed across the plate. intracellular metabolites. After drying by vacuum, Five plates for each drug combination were tested. The ° samples were resuspended in an injection buffer plates were incubated for 4 days at 37 C in 5% CO2 containing 10 mM tetrabutyl ammonium hydroxide and the level of virus replication monitored by evalua- (TBAH) and 10 mM ammonium phosphate pH 7.0 at tion of HIV-1 p24 antigen in the cell culture super- concentrations of 1–5 million cells per 20 µl, and used natant of each well (Coulter HIV-1 p24 antigen assay). for analysis of intracellular anabolites. Combinations of 3TC and 3TC were included as Transient ion-pairing high performance liquid chro- controls for additivity. Combinations of TFV with 3TC matography (HPLC) coupled to positive ion electro- and ABC with 3TC were also evaluated as positive spray tandem mass spectrometry (LC/MS/MS) was controls as these compounds have demonstrated good used to analyse TFV and ABC in their phosphorylated clinical activity when used in combination. forms for uptake and egress studies. Earlier methods Results from the antiviral assays were analysed using for nucleoside triphosphate analysis using TBAH as an the isobologram method [9] and the MacSynergy ion pair [8] were modified to include an acetonitrile program [10] to determine potential synergistic and/or step gradient in order to attain sufficient retention of antagonistic activities. The isobologram evaluation is TFV and TFV-MP on an Xterra MS, C18, 3.5 µm, based on the Lowe additivity model.