In Vitro Combination Studies of Tenofovir and Other Nucleoside Analogues with Ribavirin Against HIV-1 Nicolas a Margot and Michael D Miller*
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Antiviral Therapy 10:343–348 In vitro combination studies of tenofovir and other nucleoside analogues with ribavirin against HIV-1 Nicolas A Margot and Michael D Miller* Gilead Sciences, Inc, Foster City, CA, USA *Corresponding author: Tel: +1 650 522 5584; Fax: +1 650 522 5890; E-mail: [email protected] In patients coinfected and treated for both HIV-1 and didanosine (ddI). In contrast, low-level anti-HIV antago- hepatitis C virus (HCV), administration of ribavirin (RBV) nism was observed when RBV was combined with either may result in altered intracellular drug levels of nucleo- tenofovir or abacavir. Significantly stronger anti-HIV side reverse transcriptase inhibitors through inhibition of antagonism was observed when RBV was combined with inosine 5′-monophosphate dehydrogenase. Drug interac- either zidovudine, stavudine, emtricitabine or lamivudine. tions between tenofovir and RBV were studied in vitro in Thus, although tenofovir and ddI are both adenosine order to provide insights into the safety of co- analogues, their in vitro interactions with RBV are administration of tenofovir disoproxil fumarate (DF) and markedly different. These results suggest a low potential RBV in HCV/HIV-1-coinfected patients. In accordance for increased toxicity upon co-administration of teno- with previous in vitro studies, strongly increased anti-HIV fovir DF with RBV in patients. activity was observed when RBV was combined with Introduction Coinfection with hepatitis C virus (HCV) in HIV-1- vitro [8,9]. Alterations in levels of intracellular active infected patients is common. One study found that 16% metabolites of anti-HIV drugs can also be measured of HIV-1-infected patients were coinfected with HCV, indirectly through altered anti-HIV activity in vitro. with as many as 72% of patients coinfected in cohorts The purpose of this study is to investigate in vitro anti- of haemophiliacs or intravenous drug users [1]. The HIV drug combinations of RBV with nucleotide or primary treatment against HCV is a combination of nucleoside reverse transcriptase inhibitors (NRTIs) in pegylated interferon-α with ribavirin (RBV) that order to address the potential for intracellular drug HCV/HIV-1-coinfected patients receive in addition to interactions of RBV with these agents when co-admin- their antiretroviral therapy for HIV-1. The exact mode istered in HCV/HIV-1-coinfected patients. of action of RBV is not fully understood but is thought to be due in part to inhibition of inosine 5′-monophos- Methods phate dehydrogenase (IMPDH), which is an enzyme involved in de novo guanine nucleotide synthesis [2]. Drug combinations The US Food and Drug Administration (FDA) issued a Anti-HIV drug combination studies of RBV with warning that increased didanosine (ddI) toxicity has either tenofovir (TFV), emtricitabine (FTC), ddI, been observed in HCV/HIV-1-coinfected patients zidovudine (ZDV), lamivudine (3TC), stavudine receiving both ddI and RBV. Specifically, the US FDA (d4T), abacavir (ABC) or nelfinavir (NFV) were found a significant increase in the risk of developing performed. TFV and FTC were synthesized at Gilead mitochondrial toxicity in HCV/HIV-1-coinfected Sciences (Foster City, CA, USA); RBV, ZDV and ddI patients receiving RBV and ddI concomitantly [3]. This were purchased from Sigma (St Louis, MI, USA); 3TC finding was also documented by other groups [4–6], was obtained from Moravek Biochemicals (Brea, CA, leading to the conclusion that co-administration of RBV USA); ABC was supplied by GlaxoSmithKline and ddI should be avoided due to a high rate of clinically (Research Triangle Park, NC, USA); d4T was obtained significant toxicity [4]. These findings are in agreement from Bristol-Myers Squibb (New York, NY, USA); and with an earlier study demonstrating increased activity of NFV was obtained from Agouron Pharmaceuticals ddI in the presence of RBV [7] and with data showing (San Diego, CA, USA). The cytotoxic effect of RBV in that RBV increases the levels of the active metabolite MT-2 cells was measured using an XTT assay at RBV of ddI, dideoxyadenosine triphosphate (ddATP), in concentrations ranging from 500 nM to 3.2 mM. The © 2005 International Medical Press 1359-6535 343 NA Margot & MD Miller five concentrations of RBV tested in the combinations inhibit 50% of cell growth (CC50) was measured at ranged from 0.75–12 µM and were well below cyto- 70 µM (Figure 1) and was found to be approximately toxic levels. For the anti-HIV compounds, six sixfold above the highest concentration of RBV, concentrations of drug were tested, starting at a 12 µM, used in the combination experiments. The start maximum drug concentration of at least four times the of observed toxicity was found to be >12 µM in all of expected effective concentration to inhibit 50% of the CC50 experiments. viral replication (EC50) for each specific drug. In all The results for the combinations of RBV with the cases, the concentrations tested were well below cyto- eight HIV-1 inhibitors are shown in Figure 2, showing toxic levels. For each drug combination, the a representative experiment for each of the drug compounds were prepared separately by twofold combinations analysed. The values in Table 1 are the serial dilution and mixed in 96-well assay plates to average synergy and antagonism values from three to create a two-dimensional matrix of diluted drugs. five combination experiments and the standard devia- Each combination experiment was done in triplicate tions between experiments. RBV on its own did not and a minimum of three independent experiments exhibit any anti-HIV activity when tested at non- were conducted for each combination. A no-drug cytotoxic concentrations ranging from 0.75–12 µM in control was used for all the compounds tested. over 30 triplicate experiments. However, the presence of RBV had an effect on the anti-HIV activity of all the Antiviral assay and statistical analyses NRTIs analysed. The combination of RBV with ddI The antiviral effects of the drug combinations was showed strong enhancement of the anti-HIV activity of determined using an XTT assay in MT-2 cells as ddI, while the combinations with either ZDV, d4T, described earlier [10,11]. Briefly, 1.2 million MT-2 FTC or 3TC showed strong antagonism of the anti- cells were infected with wild-type HIV-1 (HXB2D) and HIV activity of these NRTIs. RBV combined with incubated in the assay plates for 5 days, starting at an either TFV or ABC resulted in moderate levels of anti- initial concentration of approximately 17 000 cells/well. HIV antagonism. The anti-HIV activity of the protease The antiviral effect of the combinations was measured inhibitor nelfinavir was not affected by RBV. by determining the HIV-1 cytopathic effect using the The effect of RBV on the anti-HIV EC50 values for vital dye XTT. The data were analysed with the soft- the NRTIs was also determined. The observed levels of ware MacSynergy II according to the method of RBV interaction in the drug combination studies corre- Prichard et al. [12,13]. The software uses the indepen- lated closely with the antiretroviral EC50 observed for dent effects definition of additive interactions. In this the NRTIs. Fold-changes in the anti-HIV EC50 values model, the inhibition observed in the drug combination for the antiretroviral drugs in the presence of RBV are is compared with the predicted theoretical inhibition shown in Figure 3. Combinations of RBV with ddI obtained by simply adding the inhibitory effect of each drug alone. Any deviation from predicted is scored as synergy (positive deviation) or antagonism (negative Figure 1. Cytotoxicity of RBV in MT-2 cells deviation). The software calculates confidence inter- vals to assess the statistical significance of the deviations observed. In this study, data were analysed at the 95% confidence level. Synergy volumes were 100 CC = 70 µM defined by the program as follows: values <25 µM2% 50 reflect insignificant synergy (neither synergy nor antag- 80 onism); values ≥25 to <50 µM2% indicate minor synergy/antagonism; values ≥50 to <100 µM2% indi- 60 cate moderate synergy/antagonism; and values ≥ 2 40 100 µM % indicate strong synergy/antagonism. Cell death, % Three-dimensional mesh plots and EC50 values were generated using SigmaPlot (SPSS, Inc, Chicago, IL, 20 USA). Statistical significance of EC50 fold-changes in the presence of RBV were calculated in Excel using 0 two-tailed paired t-tests. 0.0001 0.001 0.01 0.1 1 RBV concentration, mM Results The effect of ribavirin on cell viability was assayed using the vital dye XTT. The CC50 value was obtained by averaging the results of four independent experi- The cytotoxicity of RBV in MT-2 cells was analysed in ments. The standard error bars are shown for each averaged value. RBV, four independent experiments. The concentration to ribavirin. 344 © 2005 International Medical Press Drug combinations of nucleoside analogues with RBV Figure 2. Three-dimensional synergy plots A 100 B 30 90 80 20 70 60 10 50 0 40 30 –10 Synergy, % Synergy, Synergy, % Synergy, 20 –20 10 0 –30 24 5380 -10 16 12 53 10 8 8 5310 8 6 5327 6 4 2 0 4 2 0 0 µ 0 I, µM RBV, µ RBV, M dd M NFV, nM C D 0 0 53-10 53–10 53-20 53–20 53-30 53–30 gonism, % gonism, % a a 53-40 53–40 Ant Ant 53-50 53–50 53-60 16 53–60 1.8 10.7 5310 1.2 5310 8 6 5.3 8 6 4 0.6 4 2 0 0 µM 2 0 0 µM RBV, µM TFV, RBV, µM ABC, E F 0 0 53–10 53–10 53–20 53–20 53–30 53–30 gonism, % gonism, % a a 53–40 53–40 Ant Ant 53–50 53–50 53–60 12 3 53–60 2 5310 8 5310 8 6 4 8 6 4 1 4 2 0 0 µM 2 0 0 µM RBV, µM 3TC, RBV, µM FTC, G H 0 0 53–10 53–10 53–20 53–20 53–30 53–30 gonism, % gonism, % a a 53–40 53–40 Ant Ant 53–50 53–50 53–60 0.6 53–60 36 0.4 5310 24 5310 8 6 0.2 8 6 4 12 4 2 0 0 µM RBV, µ 2 0 0 µM RBV, µM ZDV, M d4T, The level of synergy or antagonism found at the 95% confidence intervals for each of the combinations tested is shown.