Dual nucleoside reverse transcriptase inhibitor (NRTI) combinations form the backbone of all currently recommended HIV treatment regimens . Despite preclinical evaluations to identify competing metabolic pathways, unexpected treatment failures have occurred, particularly among regimens containing dual purine nucleoside [abacavir (ABC), tenofovir disoproxil fumarate (TDF), didanosine (DDI)] combinations [2–8]. In study ES30009 , 194 treatment-naive patients were randomized to ABC/TDF/lamivudine (3TC) vs. efavirenz (EFV)/ABC/3TC. After 12 weeks of treatment, virologic failure occurred in 49% of patients receiving triple NRTI therapy compared with 5% receiving EFV. Considering potent antiviral responses have been associated with ABC/3TC-containing  and TDF/3TC-containing  regimens, a potential pharmacologic interaction between purine analogues had been proposed . However, multiple in-vitro [12–14] and two clinical studies [15,16] have failed to demonstrate a mechanism to explain the high rates of virologic failures observed with these combinations. This highlights the need for a greater understanding of the complexity of in-vivo pharmacodynamic/pharmacokinetic interactions between NRTIs as well as a reevaluation of how these drugs are combined to create new treatment regimens.
A central principle of combination therapy is that antiviral activity of individual drugs is at least additive when co-administered. Initiation of antiretroviral therapy results in a rapid decline in plasma HIV-1 RNA, referred to as the phase I viral decay . The more negative the slope of viral decline, the greater the antiviral efficacy the agent is considered to have [18,19]. On the basis of this premise, the relative activity of many HIV medications has been estimated in HIV-1-infected individuals given monotherapy for short durations [18,20–22]. Indeed, combining drugs with greater relative activity does augment the overall potency of a treatment regimen . Conversely, relative efficacy can also be used to test pharmacodynamic compatibility of NRTIs to determine whether certain combinations are additive, synergistic or antagonistic [13,24].
Another consideration is pharmacologic compatibility. Nucleoside analogues are prodrugs that require multiple phosphorylation steps within the cell to generate an active dideoxynucleotide triphosphate (ddNTP) that competes with endogenous nucleotides (dNTPs) for HIV-1 reverse transcriptase (RT). Combining NRTIs with shared phosphorylation pathways could result in reduced ddNTP formation and treatment antagonism [25,26]. Clinically, this has been confirmed for zidovidine (ZDV)/stavudine (D4T) , although a common-pathway mechanism may also explain antagonism between newer pyrimidine analogues, such as apricitabine and 3TC . Alternatively, an increase in endogenous dNTP pools could result in a relative dilution of active drug and adversely affect efficacy. This is supported by findings that higher intracellular ratios of ddNTP and dNTP have been correlated with greater antiviral activity [29,30].
The purpose of study was to evaluate a potential in-vivo drug interaction between ABC and TDF. We conducted a randomized trial that compared 7 days of ABC or TDF monotherapy to 7 days of ABC + TDF dual-therapy in treatment-naive, HIV-1-infected patients. A pharmacodynamic interaction was evaluated by comparing the slope of the phase I viral decay between monotherapy and dual-therapy. Intracellular ddNTP levels and corresponding endogenous dNTP levels were also measured after dual-therapy and were comparable to the levels obtained when ABC or TDF were given alone.
Study population and design
Participants were HIV-1-infected men and women at least 18 years of age who had not received antiretroviral therapy. All patients had an HIV-1 RNA level of 5000 copies/ml or more, a CD4 cell count above 200 cells/μl and genotype resistance test without evidence of primary HIV-1 drug resistance mutations at screening. An institutional review board at each site approved the study, and all patients provided written informed consent.
This multicenter, open-label trial randomized patients to ABC (600 mg once daily) or TDF (300 mg once daily) for 7 days. Randomization was stratified on screening level of plasma HIV-1 RNA (<100 000 vs. >100 000 copies/ml). After completion of monotherapy, all patients stopped antiretrovirals for at least 35 days before initiating ABC + TDF for 7 days. After completion of dual-therapy, genotypic resistance testing was repeated and treatment was intensified with EFV (600 mg once daily) for an additional 14 days. HIV therapy was then changed to EFV + ABC/3TC (600/300 mg once daily) for 44 weeks of follow-up.
Plasma for HIV-1 RNA viral dynamics was collected during monotherapy (screen and days 1, 2, 3, 5 and 8) and dual-therapy visits (days 37, 42, 43, 44, 46 and 49). Premonotherapy and predual-therapy baseline viral loads were calculated as the geometric mean of plasma HIV-1 RNA levels at screening + day 1 and day 37 + day 42, respectively. All HIV-1 RNA load measurements for viral dynamics were performed at University California San Diego using Amplicor HIV-1 Monitor Assay (by Roche Molecular Systems, Pleasanton, California, USA).
Samples for ABC and tenofovir (TFV) plasma levels were collected after monotherapy (days 7 and 8) and dual-therapy (days 48 and 49) at the following times: predose, 30 min, 1, 2, 3, 6 and 24 h postdose. The steady-state area under the plasma concentration vs. time curve from predose to 24 h postdose (AUC0–24 h) was calculated using WinNonlin (version 4.2). Intracellular concentrations of carbovir triphosphate (CBV-TP), tenofovir triphosphate (TFV-DP), deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) were measured in peripheral blood mononuclear cells (PBMCs) collected after monotherapy and dual-therapy at predose, 3, 6 and 24 h postdose. All measurements for CBV-TP and TFV-DP were performed at Taylor Laboratories and Gilead Sciences Inc., respectively, using liquid chromatography with tandem mass spectrometry (LC/MS/MS) based on techniques previously described [15,31]. Endogenous nucleotides (dATP and dGTP) were done by Dr Louie's laboratory at the University of Southern California using a validated LC/MS/MS technique (described in Supplemental Data, http://links.lww.com/QAD/A18). The average intracellular ddNTP and dNTP concentration for each patient was calculated as the mean of all four time points.
Adherence to study medication was assured by witnessed doses during monotherapy (days 1, 2, 3, 5 and 8) and dual-therapy visits (days 42, 43, 44, 46 and 49) and assessed by standardized self-report for other visits .
The co-primary endpoints of the study were the slope of viral decay and intracellular CBV-TP and TFV-DP concentrations during monotherapy and dual-therapy. A sample size of 10 patients per arm resulted in 80% power to detect a 30% difference in viral decay rates and a 30% difference in intracellular ddNTP between monotherapy and dual-therapy. Viral dynamics and pharmacokinetic data were analyzed descriptively. The Wilcoxon signed-rank test was used to analyze within-patient differences in drug concentrations between monotherapy and dual-therapy for all patients and separately for participants randomized to ABC or TDF arms. Spearman's correlation determined the association between drug concentration and viral decay rates for all participants and separately for participants randomized to ABC or TDF arms.
Linear mixed effects models assessed differences in viral decay rates (log10 copies/ml per day) between the monotherapy and dual-therapy for all participants and separately for participants randomized to ABC or TDF arms. Repeated HIV-1 RNA measurements were treated as outcomes in the mixed-effects model. The primary fixed effects included time, treatment sequence (monotherapy vs. dual-therapy) and treatment sequence-by-time interaction. The random effects were both the intercept and slope allowing each participant to have an individual baseline viral load and viral decay rate. The statistical software package R (version 2.9.0, http://www.r-project.org) was used.
Twenty-three patients were enrolled from 2004 to 2008. During the mono/dual-therapy sequences, two participants were unable to complete the viral dynamics and were replaced and intracellular drug levels in one participant were not obtainable due to insufficient sampling. The baseline characteristics of the 21 participants who completed both monotherapy and dual-therapy sequences are provided in Table 1. The majority of participants were white men of similar body mass index and calculated creatinine clearance. Baseline plasma HIV-1 RNA values and CD4 cell counts were not significantly different between treatment groups. However, participants randomized to TDF were older (median 48 vs. 32 years; P = 0.014). After completion of monotherapy, all participants discontinued HIV medications for at least 35 days. Predual-therapy plasma HIV-1 RNA levels and CD4 cell counts were comparable to premonotherapy measurements and between treatment arms.
No participant reported having missed a dose and no treatment-limiting toxicities were observed during the mono/dual-NRTI treatment sequences. After completion of dual-therapy, no primary HIV-1 drug-resistance mutations were detected on repeat genotypic testing. Five of 21 participants did not complete the 44-week follow-up portion of the study: incarceration 1; withdrew consent 2; moved away 2. In addition, four participants developed a treatment-limiting toxicity requiring a change in regimen and two experienced virologic failure (only one had HIV-1 drug resistance mutations). Both episodes of virologic failure occurred in participants randomized to initial ABC monotherapy. Lastly, 19 (90%) participants had plasma HIV-1 RNA levels below the limits of detection at last study visit.
Early viral decay dynamics
The relative efficacy of ABC + TDF combination therapy were evaluated by comparing the slopes of phase I viral decay during monotherapy and dual-therapy and are displayed in Fig. 1. For participants randomized to TDF, the viral decay rate during dual-therapy was significantly faster by −0.04 log10 per day than during monotherapy (P = 0.005). In contrast, the addition of TDF to previously ABC-treated participants did not increase viral decline (difference −0.008 log10 per day; P = 0.68). Further, dual-therapy viral decay rates for participants randomized to ABC [median −0.16 log10 per day (range −0.05 to −0.26)] were nearly identical to dual-therapy viral decay rates in participants initially treated with TDF [median (range) −0.16 log10 per day (range −0.07 to −0.20)].
This disparity in antiviral response between treatment arms was not due to initial NRTI exposure. Rather, the apparent additive antiviral effect in the TDF arm appears to be driven by relatively slower rates of viral decline during monotherapy. Viral decay rates during ABC monotherapy [median (range) −0.15 log10 per day (range −0.04 to −0.25)] were slightly faster than rates observed with TDF [median (range) −0.11 log10 per day (range −0.03 to −0.16)]. Although not statistically significant in this comparison (P = 0.13), the magnitude of this difference is the same that was observed in viral decay rates between TDF monotherapy and ABC + TDF dual-therapy (i.e., −0.04 log10 per day). Indeed, viral decay rates during ABC monotherapy (n = 11) were similar to rates observed for all participants during dual-therapy (n = 21) (−0.15 vs. −0.16 log10 per day), suggesting a nonadditive antiviral effect between ABC and TDF.
To adjust for initial delays in viral load decline (i.e., shoulder effect in HIV-1 RNA decay curve) , slopes were estimated with and without HIV-1 RNA measurements at day 0. In addition to mixed-effects models, viral decay slopes were also calculated using linear regression models. In separate analyses, all four methods of calculating slope of viral decay resulted in similar findings (data not shown).
Pharmacokinetics of abacavir and tenofovir
Plasma NRTI and intracellular ddNTP concentrations were measured 7 days after treatment with ABC or TDF alone and were comparable to levels obtained after 7 days of treatment with both drugs (Table 2). Median plasma AUC0–24 h were 12.54 μg/ml h for ABC and 3.82 μg/ml h for TFV during monotherapy. Following co-administration, there were no significant changes in the plasma exposure of either ABC or TFV.
Similar observations were made for intracellular ddNTP concentrations. Intracellular concentrations during monotherapy were similar to those previously reported for patients receiving long-term combination therapy for CBV-TP [median (range) 72.2 (19.3–503.8) fmol/106 cells] and TFV-DP [median (range) 49.3 (25.2–916.8) fmol/106 cells] . The addition of a second NRTI did not adversely affect the phosphorylation of either ABC [median CBV-TP (range) 80.9 (26.8–376) fmol/106 cells] or TFV [median TFV-DP (range) 108.1 (42.4–508.4) fmol/106 cells]. Although TFV-DP levels appeared to increase by two-fold in participants re-treated with TDF (P = 0.08), these concentrations were not significantly different than those in patients initially randomized to ABC monotherapy [median TFV-DP (range) 76.9 (20.4–255.5) fmol/106 cells].
Endogenous deoxyadenosine triphosphate and deoxyguanosine triphosphate concentrations
Tenofovir monophosphate (TFV-MP) is likely a weak inhibitor of a catabolic enzyme of purines (i.e., purine nucleoside phosphorylase (PNP)] . Therefore, we anticipated greater endogenous purines pools among patients randomized to TDF. However, there were no differences in dATP (median 3238 vs. 3314 fmol/106 cells; P = 0.96) or dGTP (median 4026 vs. 2464 fmol/106 cells; P = 0.68) pools after 7 days of monotherapy with either TDF or ABC. Instead, we observed an overall increase in purines for all participants between monotherapy and dual-therapy (median dGTP 2798 vs. 4301 fmol/106 cells; P = 0.11 and dATP 3293 vs. 4638 fmol/106 cells; P = 0.08). Although this increase was statistically significant only for dATP and among participants randomized to TDF [median dATP (fmol/106 cells) 3238 vs. 4534; P = 0.047], a similar magnitude of increase (∼40%) during dual-therapy was observed for dGTP pools [median dGTP (fmol/106 cells) 4027 vs. 6516; P = 0.28] and among patients initially treated with ABC [median dATP 3314 vs. 4741 fmol/106 cells; P = 0.55 and dGTP 2462 vs. 3244 fmol/106 cells; P = 0.28].
Correlation between drug concentrations and viral decay
Depending on the NRTI evaluated, both plasma and intracellular pharmacokinetics have been correlated with antiviral activity [25,34–36]. Consistent with a previous report , increased ABC plasma concentrations were correlated with greater viral decline during monotherapy (ρ = 0.673; P = 0.04). However, neither the intracellular concentration of CBV-TP, dGTP or the ratio of CBV-TP and dGTP was correlated with viral decay (Fig. 2a). In contrast, higher dATP levels tended to be associated with slower viral decay during TDF monotherapy (ρ = 0.75; P = 0.07). Although this correlation was less pronounced among all participants during dual therapy (ρ = 0.407; P = 0.13), a lower ratio of TFV-DP and dATP was significantly associated with reduced viral decay for all patients during dual-therapy (ρ = −0.529; P = 0.045) (Fig. 2b). Of note, this observation was based on 15 participants due to insufficient sample for dATP quantitation in two participants from the ABC arm and three in the TDF arm.
This study evaluated the pharmacodynamic and pharmacologic interactions between ABC and TDF in a randomized clinical trial of HIV-1-infected, treatment-naive patients. A nonadditive antiviral effect between ABC and TDF was observed despite adequate intracellular ddNTP concentrations. Our data suggest that co-administration of ABC and TDF increased endogenous purine nucleotides, specifically dATP, resulting in a decrease in TDF antiviral activity. Considering that endogenous purine formation in lymphocytes is tightly regulated via competing dNTP-consuming and dNTP-regenerating pathways , HIV purine analogues (i.e., ABC and TDF) potentially alter this equilibrium. We propose a model of cellular resistance to HIV chemotherapy in which dual purine nucleoside analogues lead to an expansion of endogenous purine pools resulting in decreased antiviral activity.
The strongest evidence of a negative interaction between ABC and TDF are from clinical trials of ABC/TDF/3TC regimens in treatment-naive patients [2–4]. In these studies, treatment failure occurred rapidly and in nearly 50% of participants, a rate much greater than triple NRTI regimens containing ZDV with either ABC/3TC  or TDF/3TC [39–41]. The fact that other triple NRTI regimens work substantially better suggests that simply reduced overall potency of a triple NRTI regimen is not the primary explanation. Rather, our observation that a decreased TFV-DP-to-dATP ratio resulted in a nonadditive antiviral effect during dual-therapy is evidence of a pharmacodynamic interaction between ABC and TDF and is consistent with the mutational pattern observed during ABC/TDF/3TC treatment failures . In this scenario of triple NRTI therapy, reduced selective pressure from TFV-DP would have resulted in ‘effective dual-therapy’ leading to acquisition of M184V, from ABC and 3TC, followed by independent emergence of K65R from ABC selective pressure.
It should be noted that our finding of a nonadditive antiviral effect between ABC and TDF is in direct contrast to previous in-vitro studies demonstrating additive to synergistic activity using PBMCs and wild-type virus [12,13]. Differences in viral replication kinetics, cell types and/or drug concentrations might explain these discrepancies. Indeed, synergy studies in vitro can miss clinical interactions between NRTIs. For example, isobologram analyses of D4T and apricitabine demonstrated additive to synergistic antiviral activity with ZDV  and 3TC , respectively, but subsequent clinical studies found significant antagonism with large reductions in intracellular triphosphate concentrations of both combinations [27,28]. Although combination studies are useful during drug development, the precision of these systems is not high and a negative finding does not rule out a pharmacodynamic interaction in vivo.
Our data also show that viral decay during ABC monotherapy was slightly faster than that during TDF. It is unlikely this was due to relatively lower TFV-DP concentrations. First, there are no rate-limiting steps for CBV-TP  or TFV-DP  formation and TFV appears to have faster phosphorylation kinetics in vitro than ABC . Second, TFV-DP and CBV-TP concentrations (median 49.33–108.09 and 72.2–93.9 fmol/106 cells, respectively) in our study were comparable to those from a previous study of patients receiving long-term TDF or ABC therapy (median 89.6 and 120 fmol/106 cells, respectively) . This suggests that steady-state concentrations were achieved for both drugs after 7 days. Rather, faster rates of viral decay during ABC monotherapy may be due to differences in intrinsic inhibitor potencies between CBV-TP and TFV-DP for HIV-1 RT. As CBV-TP has a 1.6–8.5-fold lower inhibitory constant (Ki) than TFV-DP [46,47], CBV-TP is a more potent inhibitor and would have faster viral decay than TFV-DP at similar concentrations.
Consistent with previous reports, plasma ABC/TFV  and intracellular CBV-TP/TFV-DP concentrations  did not change with co-administration. However, we did observe an approximately 40% increase in endogenous purines and a significant correlation between a reduced TFV-DP-to-dATP ratio and slower viral decay during dual-therapy. Together, these data suggest that increased endogenous purine pools, and not impaired ddNTP phosphorylation, resulted in decreased potency and a nonadditive antiviral effect. Interestingly, despite similar increases in dGTP pools, the ratio of CBV-TP and dGTP was not significantly associated with viral decay. Again, this may be due to intrinsic differences in the affinity of HIV-1 RT for endogenous substrate over inhibitor. Because of a relatively higher Ki, TFV-DP's potency may have been disproportionately affected despite similar increases in both dATP and dGTP.
In addition, endogenous purines increased to similar degrees in ABC and TDF arms. Potentially, anabolites of both nucleoside analogues are weak inhibitors of purine catabolism. To some extent, the rationale for this mechanism has already been proposed for TDF , as it is likely an inhibitor of PNP . Conversely, ABC metabolites have not been shown to inhibit purine catabolic enzymes. However, a minor pathway of ABC anabolism utilizes cytosolic 5′ nucleotidase , a multifunctional enzyme family that includes conversion of inosine monophosphate and adenosine monophosphate to inosine and adenosine, respectively. As inhibition of inosine monophosphate by mycophenolate acid increases CBV-TP formation by 75-fold , there may also be competitive inhibition between CBV and adenosine monophosphate resulting in decreased dNTP breakdown.
Moreover, the extent of immune activation may be an important modifier of this interaction. Under homeostatic conditions, T lymphocytes express a pro-inflammatory phenotype that is characterized by a predominance of ATP-generating and adenosine-eliminating pathways with relatively low expression of ATP-consuming pathways . During proliferation, increased production of endogenous dNTPs is primarily from the de-novo pathway [48,49]. As excessive intracellular levels of dATP/dGTP can induce apoptosis [50,51], compensatory dNTP-consuming pathways appear to be upregulated during uncontrolled HIV-1 infection . Thereby, it is reasonable to predict that weak inhibition of purine catabolic enzymes during periods of excessive T-lymphocyte activation could result in greater accumulation of endogenous purine nucleotides. In contrast, HIV-1 control results in less immune activation [53,54] and expression of dNTP-regenerating pathways is likely reduced and weak inhibition of catabolic enzymes may not significantly alter purine nucleotide pools. This may explain why Pruvost et al.  did not observe higher dGTP pools among patients with viral suppression and long-term treatment with single vs. dual purine analogue-based regimens.
This study has several limitations. First, the small sample size increases the likelihood of a type II error. Although our cohort is typical of most clinical phosphorylation studies (usually 5–15 participants) [25,26], intracellular nucleotide concentrations have a high degree of biologic variability and statistical comparisons are limited. This may explain why we observed a statistically significant difference only for dATP in participants randomized to TDF, despite a similar magnitude of increase (∼40%) for dGTP pools and among participants initially treated with ABC. Second, we were unable to analyze dATP concentrations in five participants due to insufficient amount of sample. Importantly, there were no significant differences in demographics, treatment arm assignment or viral decay kinetics between those with and without dATP data. Lastly, our study does not rule out the possibility that an enhanced mutational pathway resulted in ABC/TDF/3TC treatment failures. Although the absence of new mutations on population sequencing and subsequent viral response to antiretroviral therapy does suggest that accelerated drug resistance selection did not occur.
In summary, co-administration of ABC + TDF increased endogenous purine nucleotide pools that may be attributed to inhibition of dNTP-consuming pathways. In turn, increased dATP accumulation reduced the antiviral potency of TDF causing a nonadditive antiviral effect during co-administration with ABC (Fig. 3). Although current treatment guidelines  recommend against ABC/TDF-based combinations in treatment-naive patients, this study also raises concern whether ABC/TDF co-administration in treatment-experienced patients is efficacious.
We would like to thank the study coordinators and laboratory personnel of the California Collaborative Treatment Group for their hard work and dedication: Molly McClain (SCVMC); Angela Grbic, Mario Guerrero (Harbor-UCLA); Elizabeth Michel (UCI); Hulin Wu (U. Rochester); Leticia Muttera, Carol Mundy, Dee Dee Pacheco, Fang Wan, Andrew Rigby and Edward Seefried (UCSD).
M.G. receives research support from GlaxoSmithKline, Abbott Laboratories and Merck. B.H. is employed by GlaxoSmithKline Pharmaceuticals. J.F. is employed by Gilead Sciences. L.B. is employed by Covance. S.L. receives honorarium and/or is a consultant for GlaxoSmithKline Pharmaceuticals. E.D. receives research support from GlaxoSmithKline, Gilead Sciences, Merck, Abbott Laboratories and Pfizer and is a consultant and/or receives honorarium from Abbott Laboratories, Bristol Myers Squibb, GlaxoSmithKline, Gilead, Merck, Pfizer, Schering Plough and Tibotec. Richard Haubrich receives research support from GlaxoSmithKline and Abbott Laboratories and is a consultant and/or receives honorarium from Abbott, Bristol Myers Squibb, Gilead Sciences, GlaxoSmithKline, Merck, Pfizer, Progenics, Tibotec and Virco.
This project received funding and study medication through the GlaxoSmithKline Collaborative Studies Program. Laboratory support also provided by GlaxoSmithKline and Gilead Sciences. California HIVAIDS Research Program (CH05-SD-607-005), National Institute of Allergy and Infectious Diseases (K24 AI064086 and K23 AI066901), National Cancer Institute of (5 P30 CA 14089-27), University California San Diego Center for AIDS Research (5P30 AI 36214) and HRSA (HAB 6 H4A-HA00016-02). ClinicalTrials.gov number, NCT00214890.
Author's role. M.G., R.H. and D.R.: co-principle investigators. They are responsible for the design, conduct and analysis of the study. S.J. and S.S.: study statisticians. S.L. and L.B.: provided insight of cell purine metabolism and conducted LC/MS/MS measurements of endogenous purines and plasma TFV and CBV. B.H.: representative of GlaxoSmithKline and provided intellectual contributions of data interpretation and conducted LC/MS/MS measurements of CBV-TP. J.F.: representative of Gilead Sciences and provided intellectual contributions of data interpretation and conducted LC/MS/MS measurements of TFV-DP. E.D., C.D. and C.K.: this was a multicenter study and these authors are the site investigators and were responsible for recruitment, on-study evaluations and safety monitoring. In addition, all investigators contributed intellectually throughout the study from development to data analysis.
Presented in part at the 16th Conference on Retroviruses and Opportunistic Infections, (Abstract 703), Montreal, Canada, 8–11 February 2009 and 10th International Workshop on Clinical Pharmacology of HIV Therapy (Abstract O_08), Amsterdam, The Netherlands, 15–17 April 2009.
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