Abstract
Background:
HIV–HBV-coinfected individuals who need to be treated only for their HBV infection have limited therapeutic options, since most approved anti-HBV agents have a risk of selecting for drug-resistant HIV mutants. In vivo data are inconclusive as to whether telbivudine (LdT) may exert antiviral effects against HIV. Thus, we investigated in further detail the antiviral activity and the biochemical properties of LdT against HIV-1.
Methods:
To investigate the activity of LdT against HIV-1 in humans we analysed viral dynamics and genotypic and phenotypic resistance development in two HIV–HBV-coinfected individuals with no prior antiviral exposure. To investigate the activity of LdT against HIV-1 in vitro, LdT susceptibility for HIV-1 wild-type strains as well as drug-resistant strains was determined. Furthermore, we studied whether the 5′-triphosphate form of LdT (LdT-TP) can act as a substrate for wild-type HIV-1 RT.
Results:
In the two patients studied, LdT treatment did not result in a significant decline of HIV-1 RNA load nor in selection of genotypic or phenotypic resistance in HIV-1 RT. In vitro virological analyses demonstrated that LdT had no activity (50% effective concentration >100 μM) against wild type HIV and drug-resistant variants. Biochemical analyses demonstrated that LdT-TP is not incorporated by wild-type HIV-1 RT.
Conclusions:
Based on the in vivo and in vitro evidence obtained in this study, we conclude that LdT has no anti-HIV-1 activity and is currently the only selective anti-HBV agent among the five FDA-approved nucleoside/nucleotide analogues for treatment of HBV infections in HIV-infected individuals.
Introduction
Treatment of chronic HBV infections in patients coinfected with HIV who are not yet eligible for HIV treatment is challenging due to the fact that most approved HBV agents also have an antiviral effect against HIV-1 [1]. HBV treatment in this group of subjects elicits a risk for suboptimal HIV-1 inhibition, and thereby the selection of resistance-associated mutations in HIV-1 reverse transcriptase (RT). Based on in vitro experiments, it was initially thought that entecavir (ETV) had no significant antiviral activity against HIV-1. However, in vivo studies demonstrated that ETV monotherapy in HIV–HBV-coinfected patients could lead to a significant reduction in HIV-1 RNA load and selection of resistance mutations in HIV RT [2–5]. Later on, detailed in vitro testing under modified culture conditions did show activity of ETV against HIV-1 [6]. Furthermore, biochemical studies also demonstrated that the 5′-triphosphate (TP) form of ETV (ETV-TP) can act as substrate for HIV-1 RT [7]. Interestingly, ETV acts as a delayed chain-terminator that blocks DNA synthesis three nucleotides after its incorporation [8].
Telbivudine (LdT), also known as l-thymidine, was approved for the treatment of chronic HBV infection in 2006. LdT in its 5′-TP form (LdT-TP) was shown previously to be a specific inhibitor of the HBV polymerase and did not demonstrate in vitro activity against HIV-1 [9–11]. This latter finding would make LdT a suitable candidate for the treatment of HIV–HBV-coinfected individuals without risking the selection of resistance-associated mutations in HIV RT. However, recently two in vivo case reports hinted that LdT may possess activity against HIV-1 [12,13]. Low et al. [12] reported a case of an HIV–HBV-coinfected individual who received dual therapy with adefovir and LdT, which resulted in a reduction in HIV-1 RNA load. After withdrawal of LdT the HIV viral load increased, followed by a decline in HIV-1 viral load after re-initiation of LdT therapy. Furthermore, Milazzo et al. [13] described two HIV–HBV-coinfected individuals who showed a transient reduction in HIV-1 RNA load during LdT monotherapy. Despite the (transient) reductions in HIV-1 RNA load, the development of resistance-associated mutations in HIV-1 RT was not observed.
Since these in vivo data are inconclusive as to whether LdT may exert an antiviral effect against HIV-1, we performed a detailed analysis of the activity of LdT against HIV-1 both in vivo and in vitro.
Methods
In vivo analysis
Viral dynamics
To investigate the activity of LdT against HIV-1 in vivo, we analysed viral dynamics in two HIV–HBV-coin-fected individuals. From both patients, plasma HBV DNA load was quantified using an in-house real-time PCR assay described by Pas et al. [14], whereas plasma HIV-1 RNA load was quantified using the commercial COBAS® TaqMan HIV assay (version 2.0; Roche Diagnostics, Almere, the Netherlands).
Genotypic and phenotypic resistance testing
From both patients, a pretherapy sample and the latest sample on LdT monotherapy were used for HIV-1 RNA isolation and population-based sequencing of HIV-1 RT using the ViroSeq HIV-1 Genotyping System (Celera Diagnostics, Alameda, CA, USA; Figure 1). In addition, phenotypic resistance was performed by generating recombinant viruses containing patient-derived RT, essentially as described elsewhere [15]. Using these recombinant viruses an in vitro drug susceptibility analysis for LdT was performed in MT-2 cells, as previously described [16].
Figure 1.
Viral dynamics and HIV-1 phenotypic resistance analysis in two HIV-HBV coinfected individuals on LdT monotherapy
(A) Analysis of HBV and HIV-1 viral load prior to and during telbivudine (LdT) monotherapy and (B) phenotypic resistance testing results for patient 1. (C) Analysis of HBV and HIV-1 viral load prior to and during LdT monotherapy and (D) phenotypic resistance testing results for patient 2. The numbers in the graphs (A&C) indicate the time points that were used for HIV-1 genotypic and phenotypic resistance testing. EC50′ 50% effective concentration.
Quantification of LdT levels in human plasma samples
HPLC-grade methanol and acetonitrile were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Formic acid was purchased from Acros Organics (Bridgewater, NJ, USA). Ultrapure water was filtered and deionized from ELGA Ultrapure system equipped with US filters (ELGA LabWater, Woodridge, IL, USA).
The HPLC system was a Dionex Packing Ultimate 3000 modular LC system (Dionex, Sunnyvale, CA, USA) comprising of a ternary pump, vacuum degasser, thermostated autosampler and thermostated column compartment. A TSQ Quantum Ultra Triple Quadrupole mass spectrometer (Thermo Scientific, Waltham, MA, USA) was used for detection. Thermo Scientific Xcalibur software version 2.0 (Thermo Fisher Scientific, Waltham, MA, USA) was used to operate HPLC, the mass spectrometer and to perform data analyses.
A quantity of 100 μl of human plasma was incubated with 1 unit of thymidine phosphorylase for 2 h at 37°C, and 10 μl of lamivudine (3TC; 10 μg/ml) was added before incubation as internal standard. Proteins were then precipitated using 400 μl of methanol. The supernatant was evaporated and reconstituted in 400 μl of water before being injected into the LC-MS/MS system.
Chromatographic separation was performed on a Hypersil GOLD column (100×1.0 mm) with a 3 mm particle size (Thermo Electron, Waltham, MA, USA). The mobile phase A consisted of 0.1% formic acid in water and the mobile phase B consisted of acetonitrile. The flow rate was maintained at 50 μl/min and a 20 μl injection was used. The proportion of B was 4% for the first 3 min and was increased to 90% in 5 min. The column was equilibrated with 4% B for 14 min.
The first 0.85 min of the analysis was diverted to waste. The mass spectrometer was operated in positive ionization mode with a spray voltage of 3.5 KV, sheath gas at 45 (arbitrary units), ion sweep gas at 0.3 (arbitrary units), auxiliary gas at 0 (arbitrary units) and a capillary temperature of 300°C. The collision cell pressure was maintained at 1.5 mTorr. The precursor and product ion transitions and collision energies were m/z 230 → m/z 112 (15 V) for 3TC and m/z 243 → m/z 127 (15V) for LdT.
In vitro analyses
Reagents
ETV was purchased from Moravek Biochemicals Inc. (Brea, CA, USA). 3TC was purchased from Sigma Aldrich (Zwijndrecht, the Netherlands). LdT was purchased from Ribio Biotech (Beijing, China). The 5′-TP forms of ETV, 3TC, clevudine (CLV; L-FMAU), and LdT were synthesized as previously described [8].
Cells
MT-2 and 293T cells were obtained from the NIH AIDS Research and Reference Reagent Program. MT-2 cells were cultured in RPMI 1640 medium, supplemented with l-glutamine (Lonza, Verviers, Belgium), 10% heat-inactivated fetal bovine serum (FBS; Lonza) and 10 μg/ml gentamicin (Gibco, Breda, the Netherlands) and passaged twice per week. The 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated FBS and 10 μg/ml gentamicin and passaged twice per week.
Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of five HIV-negative human donors using Ficoll-Paque gradient centrifugation (GE Heathcare, Diegem, Belgium). Aliquots of pooled cells were frozen at −130°C in RPMI 1640 medium supplemented with l-glutamine, 10% FBS, 10% DMSO and 10 μg/ml gentamicin. At 3–4 days before use, cells were thawed and stimulated with 2 μg/ml phytohemagglutinin (PHA) in RPMI 1640 medium with l-glutamine, 10% FBS and 10 μg/ml gentamicin.
Viruses
Five recombinant viruses, containing patient-derived subtype B wild type RT (AA 25–314) in an HXB2 backbone were constructed as described [15]. RT sequences of the viruses (1B, 4B, 8B, 9B and 10B) are shown in Table 1.
Table 1.
In vitro activity of LdT, ETV and 3TC against wild type and drug-resistant HIV-1
| Virus | RT amino acid changes compared to consensus Ba | MOI | LdT EC50, μM | ETV EC50, μM | 3TC EC50, μM |
|---|---|---|---|---|---|
| HXB2 | K122E and F214L | 0.01 | >100 | 5.52 ±3.50 | 1.70 ±0.82 |
| 0.02 | >100 | 103.40 ±13.87 | 4.85 ±0.99 | ||
| 0.03 | >100 | 160.60 ±19.93 | 5.90 ±0.78 | ||
| 1B | V35I/V and V60I/V | 0.006 | >100 | ND | ND |
| 4B | V35T, V60I, R83K, T200A and T215D | 0.006 | >100 | ND | ND |
| 8B | K101R | 0.006 | >100 | ND | ND |
| 9B | V35I, E44E/A, R83K, K122E, Q207E, R211K, L228L/F and L283I | 0.006 | >100 | ND | ND |
| 10B | V35L, R83K, G196E, T200A, R211A and P225P/S | 0.006 | >100 | ND | ND |
| M184V | M184V | 0.005 | >100 | >200 | ND |
| 0.0075 | ND | ND | >200 | ||
| 0.01 | >100 | >200 | ND | ||
| 0.0125 | ND | ND | >200 | ||
| M184I | M184I | 0.005 | >100 | >200 | >200 |
| 0.01 | >100 | >200 | >200 | ||
| DR1 | V35T, M41L, V60I, D67N, R83K, V179V/I, M184V, G190G/E, T200A, L210W, R211K and T215Y | 0.002 | >100 | >200 | >200 |
| 0.006 | >100 | >200 | >200 | ||
| DR2 | K20R, K32E, V35R, V60I, D67G, S68D, T69N, 69 insertion, K70R, R83K, K101Q, V118I, K122E, T139L, D177E, R211K,T215F and K219Q | 0.002 | >100 | 6.6 | 21 |
| 0.006 | >100 | 96 | 85 |
Data presented as mean ±sd unless otherwise indicated.
Nucleoside/nucleotide reverse transcriptase inhibitor resistance-associated mutations are indicated in bold. EC50′ 50% effective concentration; ETV, entecavir; LdT, telbivudine; MOI, multiplicity of infection; ND, not determined; RT, reverse transcriptase; 3TC, lamivudine.
In addition, four recombinant viruses containing either the M184V or the M184I change in HIV-1 RT or patient-derived RT (AA 25–314) with several thymidine analogue resistance-associated mutations were constructed as described [15]. RT sequences of the viruses with thymidine analogue resistance (DR1 and DR2) are shown in Table 1. For all viruses the median tissue culture infective dose (TCID50) was determined using end point dilutions in MT-2 cells.
Drug susceptibility analysis
Drug susceptibility was determined in MT-2 cells using the multiple cycle 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and/or in PBMC, as previously described [16,17]. Briefly, MT-2 cells were infected with ≥2 different multiplicities of infection (MOI) in the presence of increasing drug concentrations. After 5 days of infection, MTT was added to determine the amount of cell killing. The 50% effective concentration (EC50) values were calculated using logistic regression analysis. In parallel, cytotoxicity assays were performed by exposing uninfected cells to increasing drug concentrations. After 5 days of incubation, the amount of cytotoxicity was determined by adding MTT.
PHA-stimulated PBMC were infected at an MOI of 0.001 in a total volume of 1 ml. After 2 h, infected cells were washed twice and resuspended in RPMI 1640 medium with l-glutamine, 10% FBS, 10 μg/ml gentamicin and 5 U/ml interleukin (IL)-2 and seeded into 96-wells microtitre plates (0.2×106/well) containing serial drug dilutions. After 7 days of infection, cell-free viral supernatant was harvested and the amount of virus production was determined using an HIV-1 p24 ELISA.
In vitro selection experiments
To investigate if LdT can select mutations in HIV-1 RT, multiple in vitro selections (n=5) were performed; SupT1 cells were infected with HXB2 (MOI 0.01). After 1h incubation at 37°C, LdT was added at the highest non-toxic concentration of 50 μM. All selection experiments were monitored daily for syncytia formation and, when full-blown syncytia were observed, cell-free virus was used to initiate the next cell culture passage (50 μM LdT). After five passages, viral RNA was isolated and the entire HIV-1 RT gene was amplified and sequenced.
As a control experiment, in vitro selections were performed with increasing concentrations of tenofovir (starting concentration 4 μM and final concentration 32 μM).
LdT-MP incorporation assays
Heterodimeric wild-type HIV-1 RT p66/p51 was expressed and purified as described [18]. For single turnover experiments with dTTP, LdT-TP and CLV-TP, 5′-radio labelled PBS-21 DNA primer was heat-annealed to a threefold molar excess of PBS-52 DNA substrate (Figure 2). Subsequently, 50 nM of DNA/DNA template-primer hybrid was incubated with 250 nM RT in a buffer containing 50 mM Tris-HCl pH 7.8, 50 mM NaCl and 0.1 mM EDTA pH 8.0 at 37°C, in the presence of increasing concentrations of dTTP, LdT-TP or CLV-TP (0.4–100 μM in a series of twofold dilutions). Nucleotide incorporation was initiated by the simultaneous addition of MgCl2 and heparin to a final concentration of respectively 6 mM and 4 mg/ml. Reactions were allowed to proceed for 10 min, after which the reactions were stopped. Products were then separated on a 12% polyacrylamide gel and visualized by phosphorimaging. Similar experiments were performed for dGTP and ETV-TP with the exception that a different DNA primer was used (PBS-22 instead of PBS-21; Figure 2A).
Figure 2.
Single nucleotide incorporation of various nucleotide analogues under single turnover conditions
(A) DNA/DNA primer-template hybrids used in the single turnover assays. DNA primer PBS-21 was used to incorporate dTTP, the 5′-triphosphate form of telbivudine (LdT-TP) and of clevudine (CLV-TP), while DNA primer PBS-22 was used to incorporate dGTP and the 5′-triphosphate form of entecavir (ETV-TP). Both primers were hybridized separately to DNA substrate PBS-52. (B) Single nucleotide incorporation in the presence of increasing concentrations of either dGTP, ETV-TP, dTTP, CLV-TP and LdT-TP. Lane C represents the control reaction where MgCl2 was omitted. Lanes 1–9 represent nucleotide concentrations of 0.4, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 μM. (C) Graphic representations of the data shown in (B).
Results
In vivo analysis of the activity of LdT against HIV-1
We investigated the activity of LdT against HIV-1 in two HIV–HBV-coinfected individuals with no prior antiviral exposure. Patient 1, a 41-year-old male, was diagnosed with HIV-1 in August 2008 and diagnosed with chronic HBV 1 month later. Based on his relatively high CD4+ T-cell counts (>350 cells/μl) and a gradually decreasing HIV-1 RNA load, antiviral therapy was not required for his HIV-infection (Figure 1A). The patient could not be motivated to initiate HAART to allow simultaneous treatment of his HIV and HBV infection and in July 2009 LdT monotherapy was initiated. After 27 weeks on treatment a 4.7 log decrease in HBV load (IU/ml) was observed. Unfortunately, at week 47 HBV DNA load rebounded with 3.9 log to 2.62×107 IU/ml. LdT plasma levels were analysed retrospectively on week 13, 27 and 47 and in all three samples LdT could be detected in a range from 449–7,011 ng/ml. HBV genotypic resistance testing at week 47 revealed the selection of the M204I, L80L/V and V207V/M/I changes in HBV RT. For M204I it has been clearly demonstrated that it confers LdT resistance, whereas the L80V is considered to be a secondary mutation and the role of the V207M and V207I is not yet understood [19].
Soon after HBV genotypic resistance testing was performed, LdT monotherapy was stopped and HAART consisting of Truvada® (Gilead Sciences, Inc., Foster City, CA, USA) combined with efavirenz was initiated. Within 3 months after HAART initiation, the HIV RNA load decreased to a level below the limit of detection (<50 copies/ml) and the HBV DNA load decreased with 4.3 log.
During LdT monotherapy, the HIV-1 RNA load had fluctuated within a 0.8 log range and the CD4+ T-cell count varied between 484 and 612 cells/μl. Although no significant changes in HIV-1 RNA load were observed during LdT monotherapy, HIV-1 genotypic and phenotypic resistance testing was performed. Population sequencing of HIV-1 RT on baseline, week 27 and 47, showed no selection of resistance-associated mutations according to the International AIDS society (IAS) [20]. In addition, phenotypic resistance testing demonstrated that LdT showed no antiviral activity against these viruses even though concentrations up to 100 μM were used (Figure 1B).
Patient 2, a 48-year-old male, was diagnosed with HIV-1 and HBV in May 2009. His CD4+ T-cell counts were >800 cells/μl and the HIV-1 RNA load was 7.57×103 copies/ml. Based on these parameters, the patient chose to postpone initiation of HAART, and LdT treatment for his HBV-infection was started in June 2009. After 23 weeks on treatment, HBV DNA load decreased with >6.0 log to a level below the limit of detection (<200 IU/ml; Figure 1C).
During LdT treatment, HIV-1 RNA load fluctuated within a 0.9 log range, whereas the CD4+ T-cell count increased from 1,067 cells/μl to 1,295 cells/μl at week 37. Despite the fact that no significant changes in HIV-1 RNA load were observed, HIV-1 genotypic and phenotypic resistance testing were performed. Population sequencing of HIV-1 RT at baseline and week 37 showed no selection of resistance-associated mutations according to IAS during LdT treatment. In addition, phenotypic resistance testing demonstrated that LdT exerted no antiviral activity (Figure 1D).
In vitro assessment of LdT activity against HIV-1
To analyse the possible in vitro activity of LdT against wild type HIV-1, a multiple cycle MTT assay was performed in MT-2 cells in quadruplicate using a wild type HIV-1 laboratory strain (HXB2). Irrespective of the MOI used, LdT did not demonstrate in vitro activity against HXB2, even though concentrations up to 100 μM were used (Table 1). As a control, similar experiments were performed with 3TC and ETV resulting in EC50 values ranging from 1.7 to 5.9 μM for 3TC and 5.5 to 160 μM for ETV depending on the MOI used (Table 1). As previously demonstrated by Lin et al. [6], the antiviral activity of ETV was strongly MOI dependent. Assessment of LdT activity in PBMC also failed to demonstrate any activity (HIV-1 BaL; EC50>100 μM). Furthermore, LdT did not show antiviral activity against five different recombinant HIV-1 viruses containing patient-derived subtype B wild-type RT (Table 1).
Clinical trials in chronically HBV-infected individuals have shown that LdT treatment may result in selection of resistance mutations at codon 204 in HBV RT (rtM204I or rtM204V [21]). Because codon 204 in HBV RT is analogous to codon 184 in HIV-1 RT, LdT activity was determined against two recombinant HIV-1 variants containing either the M184V or the M184I change in RT. No LdT activity was observed in MT2 cells and in PBMCs (EC50>100 μM) and both 184 variants were resistant to ETV and 3TC (with EC50 values >200 μM).
Because LdT is related to thymidine, we also analysed its activity against two recombinant viruses containing patient-derived subtype B RT harbouring several thymidine analogue resistance-associated mutations (DR1 and DR2). Also here, LdT did not demonstrate any antiviral activity. As expected, virus DR1 harbouring an M184V change was resistant to 3TC and ETV, whereas virus DR2 with wild type codon 184 was susceptible to ETV and resistant to 3TC due to several other resistance mutations in HIV-1 RT (Table 1).
Furthermore, we investigated if LdT can select for mutations in HIV-1 RT. Therefore multiple in vitro selection experiments were performed using the highest non-toxic LdT concentration (50 μM). After five passages in the presence of LdT no changes were observed in the HIV-1 RT, whereas in the control experiments using tenofovir the selection of K65R was observed indicating that tenofovir indeed displayed activity against HIV-1.
LdT-MP incorporation into HIV-1 RT in vitro
A biochemical analysis was performed to assess the possible inhibitory effects of LdT on wild-type HIV-1 RT. We studied whether LdT-TP is accepted as a substrate in the context of single nucleotide incorporations. The reactions were performed under defined single turnover conditions to assess whether the inhibitor can be incorporated before the RT–primer/template complex dissociates. It was found that wild type HIV-1 RT cannot incorporate LdT-MP, even though concentrations of LdT-TP up to a 100 μM were used (Figure 2B and 2C). Under similar assay conditions, the natural substrates dGTP and dTTP were incorporated at submicromolar concentrations (Figure 2B and 2C). Also ETV-MP was incorporated by wild type HIV-1 RT, although higher ETV-TP concentrations were required (approximately 3 μM). In contrast, CLV-MP was not incorporated by wild type HIV-1 RT.
Discussion
In this study we have used three different approaches to investigate in detail the activity of LdT against HIV-1. First, we analysed the effect of in vivo administration of LdT monotherapy in two HIV–HBV-coinfected individuals. Despite high HBV DNA loads prior to treatment, both patients initially responded well to LdT treatment. During LdT treatment, no significant changes in HIV-1 RNA load were observed. Although in both patients the HIV-1 RNA fluctuated slightly above the interassay variation of 0.5 log, it seems unlikely that this is related to a direct anti-HIV effect of LdT, as no resistance-associated mutations in HIV-1 RT appeared, despite the selection of resistance associated mutations in HBV RT in one of the patients. Furthermore, phenotypic resistance testing using recombinant viruses containing patient-derived HIV-1 RT demonstrated no LdT activity.
These data are in contrast with the case reports from Low et al. [12] and Milazzo et al. [13], who both reported a (transient) decline in HIV-1 RNA load during LdT monotherapy. Since the HIV-1 RNA decline reported in the study from Milazzo et al. [13] was only observed in the first 2 weeks of treatment, this decline may have been missed in our study due to less frequent sampling. However, consistent with our results, resistance-associated mutations in HIV-1 RT could not be detected in both studies, which strongly argues against a direct anti-HIV activity of LdT, although the number of patients in our study and also in the study of Milazzo et al. [13] are small.
The cases described in our study, as well as in the studies of Low et al. [12] and Milazzo et al. [13] differ considerably from the cases described for ETV, where ETV monotherapy for chronic HBV in HIV-coinfected individuals frequently results in selection of the M184V change in HIV-1 RT. The possibility that the observed effect of LdT on HIV-1 viral load in vivo is the result of indirect immunomodulatory effects as a consequence of LdT blocking HBV replication or other (indirect) interactions between HBV and HIV-1 cannot be excluded.
In addition to the in vivo approach, we also investigated the activity of LdT against HIV-1 in vitro by performing both virological and biochemical analyses. It was clearly demonstrated that, in contrast to ETV, LdT does not exert an antiviral effect against wild-type as well as drug-resistant HIV strains. These virological data are in agreement with the data from Lin et al. [10], who also reported no in vitro anti-HIV activity for LdT using the single-cycle PhenoSense™ assay (Monogram Biosciences, South San Francisco, CA, USA). Finally, we clearly demonstrate that, in contrast to ETV-MP, LdT-MP is not incorporated by wild-type HIV-1 RT. Our data show that the complex composed of enzyme and nucleic acid substrate dissociates before LdT-MP can be linked to the primer.
The biochemical and virological in vitro data obtained in this study support the in vivo finding that it is highly unlikely that LdT will exert selective pressure in vivo that may cause the emergence of resistance conferring mutations, particularly because the concentrations used in vitro (100 μM) were far above the physiologically relevant plasma concentrations in vivo (maximum concentration 13.2 ±4.5 μM at a dosage of 600 mg).
Based on our in vivo and in vitro observations, we conclude that LdT is currently the only selective anti-HBV agent among the five FDA-approved nucleoside/nucleotide analogues (that is, LdT, 3TC, ADV, TDF, ETV) for the treatment of HIV–HBV-coinfections.
Acknowledgements
The authors would like to thank Emilie Fromentin for excellent assistance.
Data were partly presented at HEP DART (6-10 December 2009, Kohala Coast, HI, USA), published as abstract in Globe Antiviral Journal (2009; 5 Suppl 1:59-60), partly presented at the International HIV & Hepatitis Drug Resistance Workshop and Curative Strategies (8-12 June 2010, Dubrovnik, Croatia) and published as an abstract in Antiviral Therapy (2010; 15 Suppl 2:A106).
AO and RFS provided the nucleoside 5′-TPs for the single-turnover experiments performed by GLB. Genotypic and phenotypic resistance testing was performed by NMM and DJ. In vitro selection experiments were performed by DJ and MP. ST and RFS were involved in the analysis of LdT plasma levels. JEA and AIMH were involved in patient follow-up and clinical data collection. NMM, AMJW, MG and MN were involved in data analysis. NMM, AMJW and MN designed the study and wrote the paper with helpful comments from JEA, AIMH, RFS and MG.
Funding was received from the Netherlands Organization for Scientific Research (NWO) VIDI (91796349) to MN and from the Canadian Institutes of Health Research (CIHR) to MG, and GLB is the recipient of a pre-doctoral stipend from the CIHR. This work was also supported in part by Public Health Service grants 5R37-AI-0419801, 5P30-AI-50409 (CFAR) and by the Department of Veterans Affairs (to RFS).
Footnotes
Disclosure statement
RFS receives royalties on the sales of LdT. All other authors declare no competing interests.
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