Mitochondrial Toxicity of Tenofovir, Emtricitabine and Abacavir Alone and in Combination with Additional Nucleoside Reverse Transcriptase Inhibitors

Walker 10/10/07 11:53 Page 1075

Antiviral Therapy 12:1075–1085 Mitochondrial toxicity of tenofovir, emtricitabine and abacavir alone and in combination with additional nucleoside reverse transcriptase inhibitors

Nils Venhoff, Bernhard Setzer, Kerstina Melkaoui and Ulrich A Walker*

Medizinische Universitätsklinik, Department of Rheumatology & Clinical Immunology, Freiburg, Germany

*Corresponding author: Tel: +49 761 2703639; Fax: +49 761 2703306; E-mail: [email protected]

Background: Some nucleoside/nucleotide reverse tran- and dose-dependent loss of mtDNA and COXII, decreased scriptase inhibitor (NRTI) combinations cause additive or cell growth and increased levels of lactate and intracel- synergistic interactions in vitro and in vivo. lular lipids. CBV and AZT strongly impaired hepatocyte Methods: We evaluated the mitochondrial toxicity of proliferation and increased lactate and lipid production, tenofovir (TFV), emtricitabine (FTC) and abacavir as but did not induce mtDNA depletion. The dual combina- carbovir (CBV) alone, with each other, and in combination tion of TFV plus 3TC had only minimal toxicity; TFV plus with additional NRTIs. HepG2 human hepatoma cells FTC slightly reduced cell proliferation without affecting were incubated with TFV, FTC, CBV, didanosine (ddI), mitochondrial parameters. All other combinations exhib- stavudine (d4T), lamivudine (3TC) and zidovudine (AZT) ited more pronounced adverse effects on mitochondrial at concentrations equivalent to 1 and 10× clinical endpoints. Toxic effects on mitochondrial parameters

steady-state peak plasma levels (Cmax). NRTIs were also were observed in all combinations with ddI, d4T, AZT or used in double and triple combinations. Cell growth, CBV. TFV and 3TC both attenuated ddI-related cytotoxi- lactate production, intracellular lipids, mtDNA and the city, but worsened the effects of CBV and AZT. mtDNA-encoded respiratory chain subunit II of Conclusions: The data demonstrate unpredicted interac- cytochrome c oxidase (COXII) were monitored for 25 days. tions between NRTIs with respect to toxicological Results: TFV and 3TC had no or minimal toxicity. FTC endpoints and provide an argument against the liberal moderately reduced hepatocyte proliferation indepen- use of NRTI cocktails without first obtaining data from dent of effects on mtDNA. ddI and d4T induced a time- clinical trials.

Introduction

A combination of two nucleoside/nucleotide reverse consist of the dideoxynucleosides zalcitabine (ddC), transcriptase inhibitors (NRTIs) usually forms the didanosine (ddI) and stavudine (d4T), which consis- backbone of antiretroviral regimens. This is mostly tently impair mtDNA replication in hepatocytes and combined with non-nucleoside reverse transcriptase liver tissue [5,6]. These NRTIs also inhibit the expres- inhibitors (NNRTIs) or protease inhibitors. Several sion of mtDNA-encoded respiratory chain subunits, side effects have been associated with prolonged NRTI interfere with cell division, enhance lactate production therapy and their ability to inhibit polymerase-γ, the and increase levels of intracellular lipids. The effects of enzyme that replicates (mtDNA). Reduced levels of the dideoxynucleosides are time- and dose-dependent mtDNA have been observed in several tissues of HIV- [5]. When the dideoxynucleosides are combined with infected patients and can result in decreased levels of each other, their mitochondrial toxicity is increased mtDNA-encoded respiratory chain subunits and [5,6]. The in vitro toxicity of these NRTIs is also repli- impaired oxidative phosphorylation. In the liver, such cated in vivo [6]. The second category of NRTIs is mitochondrial toxicity can manifest as steatosis, represented by substances that do not exhibit mito- steatohepatitis and organ failure and is also associated chondrial toxicity when given without a second NRTI, with hyperlactataemia and lactic acidosis [1–4]. but which might have side effects when administered in NRTI combinations can also cause additive or conjunction with other nucleoside partners. synergistic long-term toxicity [5–7]. It was previously Lamivudine (3TC) belongs in this category, as this suggested that NRTIs can be divided into three cate- compound enhances the cytotoxic effects of zidovu- gories on the basis of their ability to inhibit liver cell dine (AZT), despite the lack of mitochondrial toxi- proliferation with or without simultaneous impairment city when 3TC is given alone [5]. The third category of polymerase-γ [5]. Members of the first category of NRTIs comprises compounds that impair cell

N Venhoff et al.

growth, but which maintain mtDNA levels and 3TC, was from Moravek Biochemicals (Brea, CA, USA). mtDNA-encoded respiratory chain subunits. AZT Pepstatin A was from Boehringer Mannheim belongs to this category [5]. (Mannheim, Germany). Anti-mouse monoclonal anti- Nowadays, NRTIs form the backbone of combination bodies raised against subunits II and IV of cytochrome c antiretroviral therapies, containing one or even two of the oxidase (anti-COXII and anti-COXIV, respectively) were relatively new nucleoside analogues, namely emtric- obtained from Molecular Probes (Leipzig, Germany) and itabine (FTC), abacavir (ABC) and TDF — the disoproxil Gibco (Karlsruhe, Germany). fumarate prodrug of tenofovir (TFV). The toxicological interaction of these substances with many NRTI partners Cell culture

has not been systematically investigated, although there HepG2 cells were propagated at 37°C and 5% CO2 in are some indications for adverse interactions. Dulbecco’s modified Eagle’s medium (DMEM), TFV is the only nucleotide reverse transcriptase containing 4.5 g/l glucose and 110 mg/l pyruvate, inhibitor used in HIV therapy and is a weak inhibitor supplemented with 10% fetal bovine serum, 50 U/ml of polymerase-γ in vitro [8]. Although clinical trials streptomycin, 50 U/l penicillin and 0.25 amphotericin indicate a good antiretroviral potency and tolerability B. A total of 2.7×106 HepG2 cells were seeded during of TDF, there have been sporadic reports of pancre- logarithmic growth at day 1. At days 5, 10, 15, 20 and atitis, lactic acidosis and death after TDF addition to 25, the cells were harvested and counted in an auto- NRTIs [9,10]. A role in the intracellular deactivation of mated counter (CASY, Schärfe, Reutlingen, Germany), the active ddI metabolite has also been reported and 2.7×106 cells were replated in new flasks. [11,12]. In the kidney, TFV is thought to interfere with Medium was renewed on the days of trypsinization glomerular and tubular functions [13,14]. and on every third day after replating. NRTIs were added FTC is a 5-fluorinated derivative of 3TC. In vitro, in concentrations corresponding to the steady-state peak × FTC is 4–10 more potent against HIV than 3TC plasma levels (Cmax) in humans during HIV therapy, for [15,16]. Toxicity studies in a variety of cell lines have example, 1.2 μM of TFV [24], 7.3 μM of FTC [24], 11.7 confirmed that 3TC and FTC monoexposure is essen- μM of CBV [25], 7.1 μM of AZT [26], 4 μM of d4T [27], tially not toxic [17]. 8.3 μM of 3TC [26] and 11 μM of ddI [12]. Additionally, × Carbovir (CBV), the active form of ABC, is a concentrations corresponding to 10 Cmax were tested. guanosine nucleoside analogue and is often combined All compounds were dissolved in medium except AZT, with 3TC. The main side effect associated with this which was first dissolved in 2.5% of dimethyl sulphoxide drug is a hypersensitivity reaction. To date, there is no (DMSO). The final DMSO concentration for the HepG2 evidence of mitochondrial toxicity [18]. cells exposed to AZT was 0.0025% to 0.00025%. Each New evidence has suggested that mtDNA depletion experiment was performed in independent triplicates; could also result from mechanisms unrelated to direct two separate cultures were started on the same day, a polymerase-γ inhibition. For example, the inhibition of third culture was started on a different day. Controls thymidine kinase can result in a loss of mtDNA copy were incubated in medium without NRTIs. number in some tissues [19,20]. Alternatively, it is conceivable that NRTIs might induce mtDNA deple- Lactate production tion by interacting with intramitochondrial nucleoside A 1.5 ml aliquot of supernatant was collected immedi- pools at the level of their mitochondrial import ately before trypsinization and L-lactate determined [21,22], phosphorylation or degradation [23]. The enzymatically in an automated analyzer unpredictable and potentially synergistic interactions (Roche/Hitachi 917) as described [5]. Production of L- of NRTI combinations prompted us to evaluate the lactate was normalized to cell count and expressed as a mitochondrial cytotoxicity of the newer nucleoside percentage of untreated HepG2 control cells. analogues (TFV, FTC and ABC) in combination with other NRTI partners. Intracellular lipids Intracellular lipid contents were evaluated at day 20 or Materials and methods at the last harvest of viable cells. Cells were freeze-dried and lipids extracted using methanol/chloroform/water Materials 10:5:4. After sonication and vortexing, lipids were The human hepatoma HepG2 cell line was provided by extracted with a water/chloroform mixture (1:1). Lipids the American Type Culture Collection (ATCC HB-8065). were quantified using a sulpho-phosphovanillin reac- Cell culture flasks (75 cm2) were from Becton Dickinson tion on pure olive oil standards, as described [28]. Lipid (Beiersdorf, Germany) and 10% fetal bovine serum was content was calculated in nanograms per 106 cells. from PAA Laboratories (Linz, Germany). TFV and FTC were supplied by Gilead Sciences (Foster City, CA, USA). Quantification of mtDNA AZT, ddI and d4T were from Sigma (Taufkirchen, Total DNA was extracted with the QIAamp DNA isola- Germany) and CBV, the active metabolite of ABC and tion kit (Qiagen, Hilden, Germany). The mtDNA and

Toxicity of NRTI combinations

nuclear DNA (nDNA) copy numbers were determined discernable effects on cell count, lactate production, by quantitative PCR using the ABI 7700 sequence detec- mtDNA content, COXII expression and lipids. With

tion system (Applied Biosystems, Foster City, CA, USA) 10-fold Cmax concentrations of TFV, mild changes were as described [29]. Briefly, the mtDNA ATP6 gene was found in lactate production. These changes did not amplified between nucleotide positions 8,981 and 9,061 increase over time. A slightly reduced COXII content and quantified with a FAM-fluorophore-labelled probe. was found on days 20 and 25. For the detection of nDNA we selected exon number 8 3TC showed a slight effect on lactate production in of the glyceraldehyde-3-phosphate dehydrogenase both concentrations. The increase in lactate release was (GAPDH) gene between nucleotide positions 4,280 to of borderline significance and did not increase over 4,342 and used a VIC-fluorophore-labelled probe. Each time. There were no significant changes in the other 25 μl reaction contained 25 ng of genomic DNA. All parameters. FTC induced a slight and dose-dependent samples were run in triplicate. Absolute mtDNA and impairment of cell proliferation, whereas lactate, nDNA copy numbers were calculated using serial dilu- mtDNA, COXII and lipids showed no difference tions of plasmids with known copy numbers [30]. compared with the control in both concentrations. The decrease in cell proliferation was not time-dependent.

Quantification of the COXII respiratory chain subunit ddI in Cmax concentrations showed no signs of Subunit II of cytochrome c oxidase (COXII) is encoded mitochondrial toxicity. By contrast significant time- by mtDNA, whereas subunit IV of cytochrome c and dose-dependent effects were found with 10-fold

oxidase (COXIV) is encoded by nDNA. COXII was Cmax concentrations. This concentration of ddI rapidly quantified by immunoblot and normalized to the signal reduced cell division and limited the observation of a simultaneously used antibody against COXIV as period to 20 days. This ddI concentration was also described [5]. The intensities of the signals were densit- associated with increases in lactate release (275% of ometrically quantified using Scionimage (Scion control values) and increased intracellular lipid Corporation, Frederick, MD, USA). The relative content (sixfold of normal), whereas mtDNA levels cellular content of COXII was normalized to the and COXII expression were drastically reduced (13% content of the COXIV subunit and expressed as a and 28% of control values, respectively). d4T induced COXII/COXIV ratio. time- and dose-dependent toxicities in all parameters in a pattern similar to ddI. In comparison with ddI, Data analysis the toxic effects were more pronounced and

The parameters of interest (cell count, lactate concen- detectable with Cmax concentrations of d4T. CBV in

tration, mtDNA copy number, COXII/COXIV ratio Cmax concentrations slightly enhanced lactate produc-

and lipid levels) were collected at days 5, 10, 15, 20 and tion and intracellular lipids, whereas 10-fold Cmax 25 and analysed as a percentage of the mean value of concentrations were highly toxic with respect to cell untreated control cells. count (10 ±2% of control values), lactate production The statistics were computed using SPSS 13.0 (SPSS (198 ±72% of control values) and intracellular lipid Inc. Chicago, IL, USA) for Windows. A univariate vari- concentrations (sevenfold higher than control values). ance analysis model was used to evaluate the effects of Cell survival was limited to only 10 days, mtDNA dose and treatment over time. The test used a two- depletion was not observed. tailed alpha of 0.05. Statistics for lipids were AZT, as described previously [5], impaired cell performed using Wilcoxon rank sum tests on the Sigma proliferation and stimulated lactate production in a Stat™, version 3.1 (Jandel Scientific, San Rafael, CA, pattern similar to CBV. Intracellular lipid levels also USA) package for Windows. increased in a dose-dependent fashion. Similar to CBV, there was no mtDNA depletion. CBV showed more Results pronounced effects than AZT. Control HepG2 cells Additive and synergistic effects of NRTIs in dual and HepG2 cells proliferated rapidly in medium without triple combinations NRTIs. At each 5-day interval the 2.7×106 seeded cells The effects of dual combinations of NRTIs in had multiplied on average by a factor of 6.3, resulting comparison with their single use are presented in in 16.3×106 cells (SD 3.8×106). Untreated cells were Tables 1 and 2; the comparison of dual and triple characterized by a basal lactate production of 0.127 combinations is summarized in Tables 3 and 4. No ±0.038 mmol/l/105 cells, a mtDNA content of 562 ±117 toxicity was observed in the combination of TFV with copies/cell and a COXII/COXIV ratio of 0.62 ±0.14. 3TC. In the combinations containing ddI, d4T, AZT or CBV, the toxicity was generally associated with these Effects of single NRTIs substances. The specific toxicity pattern of these drugs The results of NRTI monotherapy are summarized in and their time and/or dose dependency remained

Tables 1 and 2. TFV in Cmax concentrations had no mostly unchanged when combined with a second

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N Venhoff et al. 38 , 20 , 26 , 9 28 30 34 36 14 14 16 24 10 , , , , , , , , , , 6 7 8 3 5 7 7 8 3 4 7 5 2 5 6 2 15 10 14 2 6 , , , , , , , , , , , , , , , , , , , , , -value P 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 24 11 24 22 12 24 42 54 31 17 15 – 16 60 95 4 24 – 12 45 – 7– 11 – 15 – 8– 9– 14 – 13 – ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.001; italic values, 0.001; < P 3 * 48 417 48 417 40 170 37 212 37 212 38 124 40 245 1 141 1 141 43 148 36 139 30 137 41 137 48 178 81 137 16 167 7 167 109 * 109 31 100 96 185 246 665 6 228 20 92 21 97 27 120 2 76 3 94 8 80 16 92 44 80 9 96 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± -value column indicate a statistically P 79 152 * 15 210 15 210 76 380 17 216 17 216 107 * – 31 197 31 6 270 6 270 50 143 81 289 81 22 * – 31 154 31 39 148 37 135 150 507 150 507 31 192 31 27 150 59 200 92 154 36 183 14 275 20 223 * 36 100 91 17791 226 61 261 24 113 68 * – 19 118 32 147 12 101 970 970 5 79 19 73 5 83 30 93 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 6 65 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Lactate, % of control 36 191 36 191 23 209 26 116 187 190 44 202 44 202 163 223 71 285 71 52 177 182 226 24 174 53 281 53 281 43 172 28 154 28 182 42 217 54 332 90 401 90 401 72 * – – 79 157 49 745 * – 43 139 11 186 11 29 200 12 327 37 245 96 430 32 100 17 * – – 53 * – – 20 102 24 108 3 165 25 76 13 71 15 88 21 105 6 92 7 92 16 89 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 21 221 23 168 29 130 59 207 * – – 57 277 45 195 43 265 25 203 * – – 27 315 27 315 22 235 56 151 56 151 15 188 50 156 30 149 8 154 21 205 43 189 51 154 32 141 41 179 41 64 198 19 192 24 224 38 217 26 100 15 132 19 94 92 201 92 201 13 286 71 374 71 32 105 31 346 31 19 103 9 144 14 134 1 85 14 77 12 98 13 79 11 83 6 98 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 166 129 139 220 220 146 168 170 170 174 141 141 180 210 108 108 167 81 136 93 122 120 133 213 213 151 151 202 202 81 126 231 231 91 84 134 41 40 38 40 tide reverse transcriptase inhibitor; 3TC, lamivudine; TFV, tenofovir. inhibitor; 3TC, transcriptase tide reverse , , , , experiment using lower NRTI doses or only one combination partner. Bold values, experiment using lower NRTI doses or only one combination partner. 20 36 37 , , , 9 10 13 30 34 13 22 18 38 26 12 9 , , , , , , , , , , , , 8 3 28 6 6 7 7 6 7 5 3 2 3 10 14 5 3 6 3 2 5 4 36 , , , , , , , , , , , , , , , , , , , -value Day 5 Day 10 Day 15 Day 20 Day 25 , , , , 1 1 1 1 1 1 1 1 P 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 20 – 103 9 – 105 27 – 100 44 94 38 – 124 28 – 143 39 83 11 – 117 6 – 177 24 – 121 4 31 – 180 19 13 30 28 17 49 16 27 27 12 2 20 6107 6107 ± ± ± ± ± ± ± ± ± ± 10 11 12 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ) of untreated control HepG2 cells (#1). Experiments were performed in triplicate. The numbers in the performed in triplicate. The numbers HepG2 cells (#1). Experiments were control ) of untreated 25 115 13 134 39 114 22 100 29 104 22 125 30 115 14 118 12 60 22 74 16 90 39 105 13 80 14 6 1424 110 59 8 7 2 * 13 85 26 74 38 87 9 61 9 61 27 55 21 75 15 91 7 74 16 79 13 32 ± ± ± ± SD ± ± ± ± 7 * 13 * 6 * 14 6 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 27 113 21 100 25 133 33 92 28 107 31 118 17 83 23 103 18 30 87 14 8 6 8 23 83 6 92 9 16 3 12 24 82 14 72 30 73 11 57 11 13 120 870 870 25 74 18 41 18 41 27 113 40 94 7 12 12 59 4 52 ± 8 82 34 73 ± ± ± ± ± 7 *9 3 – 5 * 10 * – 7 * – ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 27 98 21 100 30 94 37 122 24 115 28 106 10 17 11 66 18 22 24 16 27 97 15 8 17 9 4 20 71 97 25 22 18 27 45 91 30 21 10 6 7 77 2 *25 75 – – 23 68 21 6 26 94 3 61 3 61 45 65 20 103 11 44 11 41 51 11 109 13 93 60 71 23 73 2* – – 11 8 ± 16 86 ± ± ± ± ± 5 * – – 8 * – – 2 * – – ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 18 84 46 127 27 100 26 75 21 38 27 75 35 32 25 69 30 110 26 102 36 43 5 3 7 36 23 92 18 25 12 4 30 18 4 46 14 28 31 76 50 46 24 70 10 18 10 22 112 22 78 2 13 25 79 10 83 10 810 810 37 115 3 5 7 21 7 21 43 22 27 90 15 71 37 77 28 54 6 25 9 89 ± ± 37 70 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ) 74 ) 76 ) 100 ) 104 ) 39 ) 84 )110 )110 ) 45 ) 54 ) 66 ) 127 ) 98 ) 80 × × × × × × × × × × × × × ) Day 5 Day 10 Day 15 Day 20 Day 25 ) 80 ) 76 ) 101 ) 88 ) 68 ) 95 ) 97 max × × × × × × × The effect of single NRTIs and double combinations on cell proliferation and lactate production The effect of single NRTIs and double combinations on cell proliferation ) 84 ) 13 )) 42 20 ) 105 ) 54 ) 33 ) 26 ) 53 ) 25 ) 16 ) 43 ) 124 ) 62 ) 55 +15 60 )71 )71 ) 100 ) 65 ) 55 ) 97 × × × × × × × × × × × × × × × × × × × × NRTI Cell count, % of control 0.05. *Cell death. AZT, zidovudine; CBV, carbovir; ddI, didanosine; d4T, stavudine; FTC, emtricitabine; NRTI, nucleoside/nucleo stavudine; FTC, zidovudine; CBV, carbovir; ddI, didanosine; d4T, 0.05. *Cell death. AZT, < 1 Control 100 22 TFV+CBV (1 30 (1 FTC+CBV 32 (1 FTC+AZT 33 (10 34 (1 FTC+d4T # (fold C 10 CBV (1 18 (1 TFV+FTC 29 (10 31 (10 3637 (1 ddI+3TC (10 26 TFV+d4T (1 41 (10 14 d4T (1 38 (1 CBV+3TC 35 (10 2 TFV (1 6FTC (1 6FTC 2324 (10 TFV+AZT (1 19 (10 11 (10 20 TFV+ddI (1 39 (10 27 (10 3(10 15 (10 40 (1 AZT+3TC 7(10 12 AZT (1 25 (10 16 (1 TFV+3TC 28 (1 FTC+ddI 8 ddI (1 21 (10 43TC (1 43TC 13 (10 9(10 17 (10 5(10 Cell proliferation and lactate production were calculated as a percentage of the mean (± calculated as a percentage were and lactate production Cell proliferation significant difference with the experiment for which a number is indicated, that is, with either control HepG2 cells, the same with the experiment for which a number is indicated, that is, either control significant difference P Table 1. Table

Toxicity of NRTI combinations cells 6 † † † † † † † † † † † † † † † † † † † † † † † 0.06 0.30 0.31 0.03 0.1 0.15 0.04 0.07 0.11 0.07 0.07 0.06 0.27 0.12 0.09 0.24 0.03 0.03 0.02 0.13 0.03 0.09 0.04 0.02 0.17 0.04 0.05 0.04 0.40 0.04 0.08 0.02 0.01 0.05 0.01 0.01 0.001; italic 0.001; ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± † < P Lipids, ng/10 0.46 0.70 0.70 0.55 1.23 0.54 0.74 0.63 0.44 0.77 0.80 0.14 0.18 0.30 0.16 0.16 Day 20 –value column indicate a 37 P , 20 , 9 34 26 28 9 36 , , , , , , 3 14 7 2 3 8 6 7 5 16 12 8 2 -value , , , , , , , , , , , , , P 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 27 – 0.19 10 – 0.12 33 – 0.29 22 – 0.10 9 – 0.10 26 – 0.17 23 – 0.11 25 – 0.24 26 – 0.14 38 – 0.21 2217 – – 0.22 0.29 23 – 0.11 22 – 0.11 39 – 0.17 36 7 17 – 0.10 28 – 0.14 18 –5 0.22 30 11 9 35 – 0.12 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 21 100 38 113 10 114 30 114 15 101 24 127 10 114 27 124 11 60 11 21 105 3 * 19 112 112 21 79 17 86 14 * – 0.68 3229 93 111 20 102 14 76 2 105 32 72 20 102 33 93 9110 9110 12 75 26 94 13 33 7 84 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 25 94 21 100 2616 100 109 10 82 24 113 21 116 34 87 23 103 20 89 16 90 36 37 8 * – 8 * – 4 28 32 84 94 23 80 5 83 22 31 27 88 13 93 29 90 15 82 22 79 36 86 6 79 21 77 34 97 18 54 30 23 81 17 83 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 32 115 40 * – – – 0.39 22 100 31 97 25 131 19 104 1522 99 114 26 117 32 110 3 104 13 21 *24 8 – * – – 9 13 19 102 11 93 7 79 38 117 19 44 9 31 9 31 7 90 13 93 20 81 6 76 21 39 * – 3,2 96 16 66 14 81 25 95 14 50 30 100 11 28 11 24 20 10 90 ± 27 64 ± ± ± ± ± ± ± ± ± ± COXII/COX IV ratio, % of control COXII/COX IV ratio, ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± oxidase; ddI, didanosine; d4T, stavudine; FTC, emtricitabine; nd, not determined; NRTI, stavudine; FTC, oxidase; ddI, didanosine; d4T, c 49 nd * – – – 0.64 21 110 13 111 36 102 100 nd * – 0.3 38 108 35 103 21 97 49 100 25 99 25 100 15 96 5 98 23 97 20 86 51 69 27 99 19 105 43 106 11 97 25 104 33 71 20 124 23 131 lls, the same experiment using lower NRTI doses, or only one combination partner. Bold values, lls, the same experiment using lower NRTI doses, or only one combination partner. 33 19 45 75 14 29 16 18 19 51 19 51 39 46 30 27 22 105 99 73 * – 0.17 27 87 27 68 691 691 22 87 22 52 60 41 11 83 ± ± ± ± ± ± ± 36 44 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 54 91 56 100 38 63 nd122 * – – – nd 60 71 124 nd * – – – nd 68 118 118 65 75 32 , 36 20 , , 34 30 13 9 28 9 ) of untreated control HepG2cells (#1). Experiments were performed in triplicate. The numbers in the performed in triplicate. The numbers HepG2cells (#1). Experiments were control ) of untreated , , , , , , 7 8 22 3 7 2 7 6 5 7 3 SD 38 -value Day 5 Day 10 Day 15 Day 20 Day 25 , , , , , , , , , , , , P 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12 – 100 27 – 118 4 – 120 12 – 91 10 – 115 36 – 118 27 – 95 7 – 105 21 – 100 19 –15 – 138 110 24 – 122 11 – 104 30 – 101 13 – 109 9– 107 9– 107 15 – 93 9– 107 9– 107 9 – 113 9 28 – 93 17 – 94 18 – 118 24 – 127 14 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 25 133 11 127 22 100 16 126 14 119 5 137 23 118 12 110 21 79 11 * – 124 7 142 24 134 14 117 14 91 6110 4 * 6110 16 89 21 22 21 120 35 105 105 20 65 23 97 20 136 21 98 15 90 12 31 12 31 1 13 3 114 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.05. AZT, zidovudine; CBV, carbovir; COXII, subunit II of cytochrome zidovudine; CBV, carbovir; COXII, subunit II of cytochrome 0.05. AZT, < 31 97 31 101 24 122 41 105 33 86 44 106 22 100 13 97 8 101 10 118 28 95 11 106 24 102 10 98 691 691 P 26 80 * – 88 20 106 6102 4 13 6102 18 79 11 88 8 10 8 10 19 22 24 90 149 84 84 16 28 25 23 94 20 77 12 29 10 67 10 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 6 59 * – 132 ± 0 * – 1 * – 1 * – ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 13 95 38 79 103332 * 100 113 – – 13 134 33 120 18 109 22 100 15 136 22 94 29 127 35 89 10 101 12 105 13 87 30 82 18 * – – – 93 47 103 56 122 21 106 38 134 9107 9107 5 15 83 5 97 * – 7 13 3 24 20 85 2923 88 88 13 104 16 51 16 51 3 5 13 22 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3 25 ± ± ± ± ± 1 2 1 2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 59 153 30 135 10 175 *51 – 163 32 121 * – – – 43 104 13 177 * – – 10 118 37 118 18 110 17 100 89 107 8 95 21 110 11 125 22 89 19 95 21 106 14 102 7 108 21 135 11 152 10 95 9 108 26 142 27 97 7 19 5 12 13 128 7 3 18 26 100 6 12 20 96 11 11 22 57 24 25 21 108 30 52 2 6 174 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Increase of lipids versus controls, controls, of lipids versus Increase ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± † ) 103 ) 106 ) 119 ) 117 ) 125 ) 105 ) 93 ) 27 ) 38 ) 105 ) 103 ) 86 ) 104 × × × × × × × × × × × × × ) Day 5 Day 10 Day 15 Day 20 Day 25 ) 112 ) 95 ) 120 ) 52 ) 116 )91 )91 ) 104 max × × × The effect of single NRTIs and double combinations on lipid content, mtDNA copy number COXII expression × × × × 0.05. *Cell death. )151 ))151 ) 33 ) 111 32 )))10 178 166 )) 156 ) 17 ) 125 150 ) 159 ) 116 ) 115 ) 55 ) 127 ) 43 ) 133 ) 87 ) 63 < × × × × × × × × × × × × × × × × × × × × P NRTI mtDNA copies/cell, % of control 1 Control 100 # (fold C 9(10 10 CBV (1 11 (10 12 AZT (1 13 (10 14 d4T (1 15 (10 16 (1 TFV+3TC 18 (1 TFV+FTC 22 TFV+CBV (1 23 (10 24 TFV+AZT (1 25 (10 26 TFV+d4T (1 27 (10 28 (1 FTC+ddI 31 (10 35 (10 36 (1 ddI+3TC 38 (1 CBV+3TC 39 (10 40 (1 AZT+3TC 41 (10 32 (1 FTC+AZT 33 (10 34 (1 FTC+d4T 5(10 2 TFV (1 19 (10 30 (1 FTC+CBV 37 (10 17 (10 6FTC (1 6FTC 29 (10 3(10 20 TFV+ddI (1 7(10 8 ddI (1 43TC (1 43TC 21 (10 statistically significant difference with the experiment for which a number is indicated, that is, with either control HepG2 ce with the experiment for which a number is indicated, that is, either control statistically significant difference values, Lipid content, mtDNA copy number and COXII expression were calculated as a percentage of the mean (± calculated as a percentage were Lipid content, mtDNA copy number and COXII expression nucleoside/nucleotide reverse transcriptase inhibitor; 3TC, lamivudine; TFV, tenofovir. inhibitor; 3TC, transcriptase nucleoside/nucleotide reverse Table 2. Table

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N Venhoff et al.

compound. For example, the toxicity of d4T was not data and are intended as reference charts. We try to affected when combined with TFV or FTC, neither in discuss the most important aspects, but it should be dual nor in triple combination. The dose-dependent emphasized that the interested reader may extract reduction of cell proliferation observed with FTC additional information. Aside from confirming monoexposure was also replicated in the combination previous findings [5], we detected significant and of FTC with TFV, but cotreatment with TFV had no unanticipated interactions. added effect. NRTIs have a differential capacity to inhibit isolated Some interactions, however, became apparent upon polymerase-γ in vitro. The active metabolites of ddI and the addition of substances that were found to be harm- d4T, for example, are strong inhibitors of the mtDNA less in monotherapy. These interactions include addi- replication enzyme, whereas the diphosphate of TFV and tive effects, multiplicative effects and antagonistic the triphosphates of 3TC, FTC, AZT and CBV (the active effects and are detailed as follows. With ddI, all combi- form of ABC) exhibit much weaker interactions, as indi-

nations showed the typical time- and dose-dependent cated by their Ki values [31]. In keeping with its Ki value, toxicity described for the monosubstance. Interestingly, FTC had little effect on mtDNA and COXII expression,

however, in the combinations of 10-fold Cmax concen- but induced an unanticipated dose-dependent reduction trations of ddI with either TFV or 3TC, the latter of cell proliferation. substances appeared to attenuate the ddI-associated Among the NRTI partners we found combinations effects: as the addition of these NRTIs reduced consis- with no influence on each other, combinations with tently and significantly (TFV P=0.05; 3TC P=0.001) additive or multiplicative toxicity and combinations the toxicity of ddI monotherapy in terms of cell count, with antagonistic effects. The combination of TFV and mtDNA content and COXII expression. 3TC also had 3TC was the only combination tested without any a significant effect (P=0.05) when added to the dual additive or synergistic toxic effects. combination of ddI plus TFV, whereas TFV had no Synergistic effects were observed with FTC, which significant effect when it was added to the dual exacerbated the toxicity of CBV and of ddI. FTC also combination of ddI plus 3TC. enhanced the toxic effects of AZT. Similarly, 3TC was The AZT-induced cytotoxicity was increased in found to increase the cytotoxicity of AZT synergisti- combinations with 3TC or FTC, despite the fact that cally in HepG2 cells [5,32] and to increase the rate of both of these cytidine analogues alone showed no or point mutations in mouse hearts [33]. A competitive only mild toxicity. FTC revealed slightly reduced cell mechanism was suggested by the protective effect of counts in monotherapy, but when combined with AZT uridine on the combination of AZT plus 3TC [34]. significantly impaired cell division in both concentra- Interestingly, FTC and 3TC are both cytidine

tions (Cmax P=0.05; 10-fold Cmax P=0.001), thus analogues. Several interactions have been found in showing stronger cytotoxic effects than 3TC. The vitro and in vivo to occur between NRTIs sharing the mtDNA content was increased during the first days in same metabolic pathways, that is, between AZT and

combinations of 10-fold Cmax concentrations of AZT d4T for thymidine analogues, and 3TC and ddC for with FTC, 3TC and also TFV (P=0.001 for all combi- cytidine analogues [35,36]. nations). Neither concentration of TFV had an effect Additive toxic effects were observed whenever ddI, on the toxicity of AZT. Likewise, no effect was d4T, AZT or CBV were combined with each other in observed when TFV was added to the dual combina- dual or triple combination. The issue of drug–drug tion of AZT with FTC or 3TC. Similar to AZT, the interactions is complex because NRTIs might interact cytotoxicity of CBV was increased, when the latter not only at the level of systemic drug absorption, distri- substance was combined with 3TC or FTC. Again FTC bution and elimination [37,38], but also at the cellular had a stronger effect on the cell count than 3TC. This level through compartmentation, membrane transfer

effect was observed mainly in the combination of Cmax and compartmental metabolism. NRTIs are trans- concentrations of FTC with CBV (P=0.001), possibly ported into the cytoplasm and mitochondria and might

because of the high toxicity of 10-fold Cmax concentra- interact in their complex activation by kinases [39]. tions of CBV. Also similar to AZT, CBV appeared to NRTIs also interact and exert toxic effects at the increase mtDNA content. It is remarkable, however, nuclear level [40]. At all steps NRTIs could, in addi- that a decreased COXII expression was observed in the tion, be subject to competitive interactions with their combination of the high concentrations of CBV with natural nucleoside and nucleotide counterparts [23]. FTC, despite increased mtDNA levels (P=0.001). CBV at high concentrations reduced cell growth, enhanced lactate production and increased intracellular Discussion lipids, but had no effect on mtDNA and COXII levels. The toxicity of CBV was also increased in combination We examined the long-term mitochondrial hepato- with cytidine analogues. The toxicological profile of CBV toxicity of NRTI combinations in an established in is, therefore, very similar to AZT [41]. AZT interferes vitro system. The tables present a huge amount of with the production of cellular energy in many different

Toxicity of NRTI combinations 42 52 , , 48 22 50 29 23 , , , , , 30 34 19 18 18 19 24 26 38 16 28 36 21 20 16 17 21 -value , , , , , , , , , , , , , , , , , P 0.001; 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 < P 22 54 11 14 12 20 42 24 4 95 24 – 51 60 45 – 18 – 11 – 29 – 14 – 13 – 7– ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 38 124 30 137 32 132 37 212 37 212 135 232 40 170 40 245 19 264 79 181 36 139 48 417 48 417 16 167 109 * 109 96 185 37 93 31 100 6 228 20 92 3 94 7 99 44 80 2 76 9 96 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± -value column indicate a statistically P 79 152 * 31 192 31 22 * – 50 159 22 * – 31 197 31 40 * – 33 227 * 91 297 91 15 210 15 210 6 270 6 270 107 * – 81 289 81 86 235 37 135 76 380 36 183 50 143 91 17791 226 76 243 * 68 * – 20 223 * 61 261 54 * – 22 121 36 100 12 101 30 173 32 147 970 970 5 79 29 75 30 93 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 6 65 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 23 209 79 157 53 281 28 184 33 213 33 213 147 454 56 162 36 189 50 299 26 116 26 116 187 190 107 * – – 182 226 90 401 90 401 36 191 36 191 42 217 71 285 71 33 334 28 171 28 171 28 182 29 200 24 174 52 177 17 * – – 96 430 68 170 68 170 32 100 37 245 12 327 53 * – – 24 108 25 76 18 104 15 88 25 93 16 89 13 71 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 2959 207 130 * – – 25 203 * – – 56 151 56 151 43 189 15 188 27 315 27 315 30 159 55 203 31 335 31 2445 178 189 58 182 243 * * – – – 8 157 57 277 81 294 81 23 168 8 154 16 201 16 201 48 194 20 168 30 149 21 221 41 179 41 22 235 19 192 13 286 24 93 24 224 26 100 38 217 92 201 92 201 32 105 15 132 31 346 31 5 186 12 98 1 85 8 97 14 77 13 79 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± tide reverse transcriptase inhibitor; 3TC, lamivudine; TFV, tenofovir. inhibitor; 3TC, transcriptase tide reverse 119 119 139 220 174 167 126 136 180 269 169 139 187 176 123 151 151 84 220 282 210 93 170 170 129 120 152 141 141 187 125 122 166 213 213 123 108 108 81 40 50 , , experiment using lower NRTI doses, or one additional or fewer combination partner. Bold values, experiment using lower NRTI doses, or one additional fewer combination partner. 25 48 18 22 52 37 24 42 17 , , , , , , , , , 18 40 41 30 22 34 17 19 24 19 19 18 26 36 17 40 18 28 38 16 29 20 38 36 21 16 -value Day 5 Day 10 Day 15 Day 20 Day 25 , , , , , , , , , , , , , , , , , , , , , , , , , , P 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 – 100 9 – 105 27 – 100 39 – 83 6 – 177 24 – 121 49 27 4 30 7 4 28 10 12 19 13 31 – 180 20 36 – 90 ± ± ± ± ± ± 10 10 12 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ) of untreated control HepG2 cells (#1). Experiments were performed in triplicate. The numbers in the performed in triplicate. The numbers HepG2 cells (#1). Experiments were control ) of untreated 18 139 13 134 22 100 15 91 30 115 14 118 SD 14 110 38 87 9 61 9 61 21 75 12 60 8 7 2 * 24 59 14 63 24 57 13 85 20 89 16 90 13 80 22 74 10 6 13 32 ± ± ± ± ± ± 14 6 6 * 4 * 13 * 13 * ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 21 100 25 133 31 118 37 123 6 92 6 10 6 10 6 8 30 73 11 57 11 25 74 9 16 11 2 3 12 16 8 22 114 14 8 6 * – 26 74 10 56 10 24 82 14 63 11 * – 18 41 18 41 30 87 27 113 23 83 17 83 8 82 34 73 ± ± ± ± 9 3 5 * – 5 * – 5 * – 7 * – 7 * – ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 21 100 29 76 31 111 24 115 28 106 4 97 17 16 17 9 24 16 25 75 10 6 10 6 23 68 20 3 2 * – – 25 22 32 18 18 27 18 25 15 8 20 71 20 71 18 22 9 15 26 60 12 46 15 49 21 12 21 11 44 11 11 66 13 93 27 97 26 94 11 8 60 71 23 73 16 86 ± ± ± ± ± 5 * – – 7 * – – 8 * – – 1 * – – 2 * – – ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 18 84 27 100 21 38 17 96 33 62 35 32 26 75 27 75 31 118 42 104 30 110 3 35 30 18 5 3 7 36 25 79 10 18 10 6 18 10 83 10 7 21 7 21 2 13 15 71 4 46 12 4 2 5 36 47 14 28 31 53 18 25 36 43 20 28 37 115 24 70 34 59 36 35 3 5 9 89 23 92 35 12 17 4 ± 37 70 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ) 95 ) 85 ) 50 ) 58 ) 52 ) 115 ) 113 × × × × × × × ) 74 ) 76 ) 100 ) 104 ) 39 ) 84 )110 )110 ) 45 ) 54 ) 66 ) 127 ) 80 ) 98 × × × × × × × × × × × × × ) Day 5 Day 10 Day 15 Day 20 Day 25 0.05. *Cell death. AZT, zidovudine; CBV, carbovir; ddI, didanosine; d4T, stavudine; FTC, emtricitabine; NRTI, nucleoside/nucleo stavudine; FTC, zidovudine; CBV, carbovir; ddI, didanosine; d4T, 0.05. *Cell death. AZT, < max P )41 )41 )31 ) 13 ) 33 ) 36 ) 53 ) 26 )71 )71 ) 20 ) 16 ) 49 ) 54 ) 105 ) 42 ) 43 ) 100 ) 100 ) 100 ) 16 ) 97 The effect of double and triple NRTI combinations on cell proliferation and lactate production The effect of double and triple NRTI combinations on cell proliferation × × × × × × × × × × × × × × × × × × × × NRTI Cell count, % of control Lactate, % of control 32 (1 FTC+AZT 36 (1 ddI+3TC 50 (1 TFV+ddI+3TC 45 (10 4748 (10 (1 TFV+FTC+d4T 3334 (10 (1 FTC+d4T 24 TFV+AZT (1 26 TFV+d4T (1 35 (10 # (fold C 46 (1 TFV+FTC+CBV 23 (10 28 (1 FTC+ddI 25 (10 31 (10 1 Control 100 53 (10 55 (10 41 (10 42 (1 TFV+FTC+ddI 49 (10 37 (10 2930 (10 (1 FTC+CBV 27 (10 54 (1 TFV+3TC+AZT 51 (10 20 TFV+ddI (1 43 (10 38 (1 CBV+3TC 16 (1 TFV+3TC 52 (1 TFV+3TC+CBV 44 (1 TFV+FTC+AZT 2122 (10 TFV+CBV (1 39 (10 40 (1 AZT+3TC 1718 (10 (1 TFV+FTC 19 (10 significant difference with the experiment for which a number is indicated, that is, with either control HepG2 cells, the same with the experiment for which a number is indicated, that is, either control significant difference italic values, Cell proliferation and lactate production were calculated as a percentage of the mean (± calculated as a percentage were and lactate production Cell proliferation Table 3. Table

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N Venhoff et al. cells 6 † † † † † † † † † † † † † † † † † † † † † † † † † 0.13 0.30 0.1 0.24 0.04 0.11 0.02 0.02 0.03 0.03 0.15 0.06 0.03 0.02 0.38 0.01 0.28 0.09 0.20 0.02 0.07 0.02 0.08 0.12 0.13 0.03 0.02 0.17 0.04 0.04 0.04 0.02 0.01 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± † nd Lipids, ng/10 0.46 0.74 0.50 0.14 0.16 1.23 1.29 1.00 0.54 0.16 0.80 0.18 0.29 0.30 0.16 Day 20 -value column indicate a 42 50 , , P 48 37 29 37 54 , , , , , 20 26 34 19 18 28 19 17 16 19 41 36 25 , , , , , , , , , , , , , -value 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 P 27 – 0.16 10 – 0.12 22 – 0.10 9 – 0.10 25 – 0.24 22 – 0.22 17 – 0.29 34 – 0.48 39 – 0.17 36 18 – 0.22 5 28 – 0.14 3 – 0.15 17 – 0.10 5 30 11 35 – 0.12 10 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 21 100 38 113 21 122 10 114 15 101 24 127 14 – * 0.68 14 76 29 111 5 22 25 110 32 93 17 86 10 41 9 39 32 72 33 93 2 105 26 94 11 60 11 7 84 13 33 79 10 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 10 82 21 100 17 67 26 100 3 * – – 0.78 16 109 24 113 34 87 21 116 8 * – 29 90 8 * – 6 * – 5 * – 12 16 22 31 22 31 27 88 691 29 105 28 79 * – 0.18 5 83 19 57 23 50 22 79 6 79 15 82 34 97 36 37 23 81 18 54 30 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± oxidase; ddI, didanosine; d4T, stavudine; FTC, emtricitabine; nd, not determined; NRTI, stavudine; FTC, oxidase; ddI, didanosine; d4T, c 19 104 19 104 22 114 22 100 22 100 40 * – – – 0.39 15 99 14 94 25 131 25 131 31 108 39 105 3 104 9 13 13 13 93 10 13 29 62 20 81 6 28 19 44 19 102 7 74 9 31 9 31 16 96 11 93 18 66 21 39 * – 38 117 19 52 16 66 6 76 25 95 14 50 27 64 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 49 nd * – – – 0.64 38 108 36 102 100 nd * – 0.3 21 97 13 111 35 103 33 104 52 * – – – – 0.62 6 99 25 100 49 100 25 99 15 96 20 86 27 99 43 106 33 71 11 97 23 131 6 116 24 118 16 18 11 nd * – – 33 19 22 15 29 53 9 95 39 46 24 93 nn*– 6ndnd* 30 27 22 105 99 73 * – 0.17 27 57 27 68 22 87 22 52 ± ± ± ± ± ± ± 36 44 17 62 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± lls, the same experiment using lower NRTI doses, or one additional or fewer combination partner. Bold values, lls, the same experiment using lower NRTI doses, or one additional fewer combination partner. 104 104 63 nd122 * – – – nd 38 67 100 100 100 100 54 71 44 107 124 nd * –65 – – nd 77 71 68 62 65 118 118 86 75 42 50 38 , , , 46 48 29 21 22 , , , , , 30 38 19 22 34 19 32 18 18 19 33 28 36 17 20 16 , , , , , , , , , , , , , , , -value Day 5 Day 10 Day 15 Day 20 Day 25 , P 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ) of untreated control HepG2 cells (#1). Experiments were performed in triplicate. The numbers in the performed in triplicate. The numbers HepG2 cells (#1). Experiments were control ) of untreated SD 26 4 – 120 12 – 100 10 – 115 15 – 110 21 – 100 19 – 138 30 – 101 11 – 104 17 – 120 18 14 9 – 113 9– 107 9– 107 9 22 28 – 93 17 – 94 24 – 127 14 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 22 100 22 100 11 127 11 11 * – 124 18 124 6 112 7 142 5 105 21 10 14 117 14 91 21 22 21 120 3 105 1714 27 86 16 89 20 65 21 52 21 23 97 21 98 12 31 12 31 3 114 21 79 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.05. AZT, zidovudine; CBV, carbovir; COXII, subunit II of cytochrome zidovudine; CBV, carbovir; COXII, subunit II of cytochrome 0.05. AZT, < P 31 101 22 100 13 97 81 01 27 * – 26 80 * – 88 10 118 28 95 10 98 691 691 28 82 * – 92 15 116 9 84 6 59 * – 132 5 19 19 22 24 90 14 84 2024 106 7 33 87 10 116 16 * – – 111 16 28 25 11 88 23 94 12 29 20 * – – 78 97 63 * – – 0.21 10 67 10 20 57 ± ± ± ± ± ± ± ± ± ± ± ± 1 * – 0 * – 1 * – 1 * – ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 103332 * 100 113 – – 13 95 9 106 18 98 38 79 18 109 29 127 22 100 35 89 33 72 8 114 13 87 6110 6110 47 103 21 106 33 70 23 88 5 15 9107 9107 3 24 29 88 1217 33 82 20 85 3 5 16 51 16 51 13 104 29 70 13 22 ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3 25 ± ± ± ± 1 2 1 2 1 2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Increase of lipids versus controls, controls, of lipids versus Increase † 5930 135 153 10 175 *51 32 121 – 163 * – 21 – 179,3 7 118 *27 – 18 143 * – – – – – 43 104 13 177 * – – 10 118 19 95 17 100 8 95 21 110 22 89 13 111 34 105 11 125 43 79 21 106 27 122 21 135 19 141 19 161 93 * – 26 142 10 95 20 12 6 12 7 3 930 39 89 9 3 20 96 11 11 24 25 13 128 22 57 30 52 2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.05. *Cell death. ) 130 ) 119 ) 101 ) 100 ) 34 < ) 103 ) 84 × × × P × × × × ) 103 ) 106 ) 119 ) 117 ) 125 ) 105 ) 93 ) 27 ) 38 ) 105 ) 103 ) 86 ) 104 × × × × × × × × × × × × × ) Day 5 Day 10 Day 15 Day 20 Day 25 max The effect of double and triple NRTI combinations on lipid content, mtDNA copy number COXII expression )10 )))10 178 166 )) 156 ) 17 )) 125 150 ) 44 ) 169 ) 17 ) 143 159 ) 159 ) 119 ) 43 ) 55 ) 115 ) 106 ) 127 ) 63 × × × × × × × × × × × × × × × × × × × × NRTI mtDNA copies/cell, % of control COXII/COX IV, % of control 0.001; italic values, 0.001; < 1 Control 100 48 (1 TFV+FTC+d4T # (fold C 18 (1 TFV+FTC 22 TFV+CBV (1 23 (10 24 TFV+AZT (1 25 (10 26 TFV+d4T (1 27 (10 28 (1 FTC+ddI 31 (10 35 (10 36 (1 ddI+3TC 38 (1 CBV+3TC 39 (10 40 (1 AZT+3TC 41 (10 42 (1 TFV+FTC+ddI 43 (10 44 (1 TFV+FTC+AZT 47 (10 49 (10 50 (1 TFV+ddI+3TC 52 (1 TFV+3TC+CBV 53 (10 54 (1 TFV+3TC+AZT 55 (10 32 (1 FTC+AZT 33 (10 45 (10 46 (1 TFV+FTC+CBV 34 (1 FTC+d4T 29 (10 16 (1 TFV+3TC 37 (10 19 (10 30 (1 FTC+CBV 51 (10 17 (10 20 TFV+ddI (1 21 (10 statistically significant difference with the experiment for which a number is indicated, that is, with either control HepG2 ce with the experiment for which a number is indicated, that is, either control statistically significant difference P Lipid content, mtDNA copy number and COXII expression were calculated as a percentage of the mean (± calculated as a percentage were Lipid content, mtDNA copy number and COXII expression nucleoside/nucleotide reverse transcriptase inhibitor; 3TC, lamivudine; TFV, tenofovir. inhibitor; 3TC, transcriptase nucleoside/nucleotide reverse Table 4. Table

Toxicity of NRTI combinations

ways. AZT binds to adenylate kinase and inhibits the References ADP–ATP translocator [42–44], effects that could 1. Brivet FG, Nion I, Megarbane B et al. Fatal lactic acidosis explain the comparatively early toxicity of AZT. and liver steatosis associated with didanosine and Furthermore, AZT was shown to inhibit intracellular stavudine treatment: a respiratory chain dysfunction? J Hepatol 2000; 32:364–365. thymidine phosphorylation in liver mitochondria [19]. 2. Chariot P, Drogou I, de Lacroix-Szmania I et al. The AZT-induced toxicity can be reversed by uridine, a Zidovudine-induced mitochondrial disorder with massive pyrimidine precursor, as shown in blood cell precursors, liver steatosis, myopathy, lactic acidosis, and mitochondrial DNA depletion. J Hepatol 1999; HepG2 cells and adipocytes [23,34,45,46]. It is therefore 30:156–160. conceivable that AZT interferes with the synthesis and 3. John M, Moore CB, James IR et al. 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