Long-term management of some chronic hepatitis B patients might require combination therapy using drugs with distinct resistance profiles to sustain viral suppression and to reduce the resistance-associated failure. Tenofovir disoproxil fumarate (TDF), approved for hepatitis B virus (HBV) and HIV-1 treatment, is active against wild-type HBV and HBV containing YMDD mutations, which confer resistance to emtricitabine (FTC), lamivudine (3TC) and telbivudine (LdT) and contribute to entecavir (ETV) resistance. We therefore evaluated the in vitro anti-HBV activity of tenofovir (TFV), the active parent drug of TDF, combined with FTC, 3TC, ETV, LdT and adefovir (AFV).
Methods:
The anti-HBV activities of the compounds were tested using the AD38 cell line that expresses wild-type HBV from a tetracycline-controllable promoter. Intracellular HBV DNA levels were quantified using real-time PCR assay and cytotoxicities were assessed with XTT assays. The antiviral data of the drug combinations were evaluated using MacSynergy analyses on the basis of the Bliss independence model as well as isobologram analyses on the basis of the Loewe additivity theory.
Results:
All drug combinations tested, FTC+TFV, 3TC+TFV, ETV+TFV, LdT+TFV and AFV+TFV, showed additive antiviral interactions as analysed by MacSynergy. Isobologram analyses revealed that these combination pairs were additive, with the exception of FTC+TFV, which demonstrated slight synergistic activity. No cytotoxic or antagonistic effects were observed with any of the combinations tested.
Conclusions:
The combination of TFV with FTC, 3TC, ETV, LdT or AFV had additive to slightly synergistic anti-HBV effects in vitro. These results support the use of TDF as a component in combination regimens with currently available anti-HBV nucleoside analogues.
References
1.
LavanchyD. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat2004; 11: 97–107.
2.
LeeWM. Hepatitis B virus infection. N Engl J Med1997; 337: 1733–1745.
3.
WongDKHCheungAMO'RourkeKNaylorCDDetskyASHeathcoteJ. Effect of alpha-interferon treatment in patients with hepatitis B e antigen-positive chronic hepatitis B. A meta-analysis. Ann Intern Med1993; 119: 312–323.
4.
LauGKKPiratvisuthTLuoKX. Peginterferon alfa-2a, lamivudine, and the combination for HBeAg-positive chronic hepatitis B. N Engl J Med2005; 352: 2682–2695.
5.
MarcellinPLauGKBoninoF. Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B. N Engl J Med2004; 351: 1206–1217.
6.
ZoulimFPerrilloR. Hepatitis B: Reflections on the current approach to antiviral therapy. J Hepatol2008; 48Suppl 1: S2–S19.
LaiC-LDienstagJSchiffE. Prevalence and clinical correlates of YMDD variants during lamivudine therapy for patients with chronic hepatitis B. Clin Infect Dis2003; 36: 687–696.
9.
GishRGTrinhHLeungNW. Safety and antiviral activity of emtricitabine (FTC) for the treatment of chronic hepatitis B infection. A two year study. J Hepatol2005; 43: 60–66.
10.
LaiCLGaneELiawYF. Telbivudine versus lamivudine in patients with chronic hepatitis B. N Engl J Med2007; 357: 2576–2588.
11.
YangHQiXSabogalAMillerMXiongSDelaneyWEIV. Cross-resistance testing of next-generation nucleoside and nucleotide analogues against lamivudine-resistant HBV. Antivir Ther2005; 10: 625–633.
12.
TenneyDJLevineSMRoseRE. Clinical emergence of entecavir-resistant hepatitis B virus requires additional substitutions in virus already resistant to lamivudine. Antimicrob Agents Chemother2004; 48: 3498–3507.
13.
ColonnoRJRoseRBaldickCJ. Entecavir resistance is rare in nucleoside naive patients with hepatitis B. Hepatology2006; 44: 1656–1665.
14.
PerrilloRHannHWMutimerD. Adefovir dipivoxil added to ongoing lamivudine in chronic hepatitis B with YMDD mutant hepatitis B virus. Gastroenterology2004; 126: 81–90.
15.
PetersMGHannHWMartinP. Adefovir dipivoxil alone or in combination with lamivudine in patients with lamivudine-resistant chronic hepatitis B. Gastroenterology2004; 126: 91–101.
16.
AngusPVaughanRXiongS. Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase. Gastroenterology2003; 125: 292–297.
17.
LocarniniSQiXArterburnS. Incidence and predictors of emergence of HBV mutations associated with ADV resistance during 4 years of ADV therapy for patients with chronic hepatitis B. 40th Annual Meeting of the European Association for the Study of the Liver. 13–17 April 2005, Paris, France. Abstract 36.
18.
HeathcoteEJGaneEDeManR. Randomized, double-blind comparison of tenofovir DF (TDF) versus adefovir dipivoxil (ADV) for the treatment of HBeAg-positive chronic hepatitis B (CHB): Study GS-US-174-0103. Hepatology2007; 46: 861A.
19.
MarcellinPButiMKraslevZ. Randomized, double-blind comparison of tenofovir DF (TDF) versus adefovir dipivoxil (ADV) for the treatment of HBeAg-negative chronic hepatitis B (CHB): Study GS-US-174-0102. Hepatology2007; 46: 290A–291A.
20.
Panel on Antiretroviral Guidelines for Adults and Adolescents.Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. US Department of Health and Human Services. (Updated 3 November 2008. Accessed 20 February 2009.) Available from http://www.aidsinfo.nih.gov/ContentFiles/AdultandAdolescentGL.pdf.
21.
HeijtinkRAKruiningJde WildeGABalzariniJDe ClercqESchalmSW. Inhibitory effects of acyclic nucleoside phosphonates on human hepatitis B virus and duck hepatitis B virus infections in tissue culture. Antimicrob Agents Chemother1994; 38: 2180–2182.
22.
YingCDe ClercqENicholsonWFurmanPNeytsJ. Inhibition of the replication of the DNA polymerase M550V mutation variant of human hepatitis B virus by adefovir, tenofovir, L-FMAU, DAPD, penciclovir and lobucavir. J Viral Hepat2000; 7: 161–165.
23.
DelaneyWEIVRayASYangH. Intracellular metabolism and in vitro activity of tenofovir against hepatitis B virus. Antimicrob Agents Chemother2006; 50: 2471–2477.
24.
VilletSPichoudCVilleneuveJPTrepoCZoulimF. Selection of a multiple drug-resistant hepatitis B virus strain in a liver-transplanted patient. Gastroenterology2006; 131: 1253–1261.
25.
LacombeKOllivetAGozlanJ. A novel hepatitis B virus mutation with resistance to adefovir but not to tenofovir in an HIV-hepatitis B virus-co-infected patient. AIDS2006; 20: 2229–2231.
26.
BrunelleMNLuciforaJNeytsJ. In vitro Activity of 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]-pyrimidine against multidrug-resistant hepatitis B virus mutants. Antimicrob Agents Chemother2007; 51: 2240–2243.
27.
VilletSOllivetAPichoudC. Stepwise process for the development of entecavir resistance in a chronic hepatitis B virus infected patient. J Hepatol2007; 46: 531–538.
28.
SheldonJCaminoNRodesB. Selection of hepatitis B virus polymerase mutations in HIV-coinfected patients treated with tenofovir. Antivir Ther2005; 10: 727–734.
29.
ClavelFHanceAJ. HIV drug resistance. N Engl J Med2004; 350: 1023–1035.
30.
LamperticoPViganoMManentiELavaroneMLunghiGColomboM. Adefovir rapidly suppresses hepatitis B in HBeAg-negative patients developing genotypic resistance to lamivudine. Hepatology2005; 42: 1414–1419.
31.
LamperticoPViganoMManentiELavaroneMSablonEColomboM. Low resistance to adefovir combined with lamivudine: A 3-year study of 145 lamivudine-resistant hepatitis B patients. Gastroenterology2007; 133: 1445–1451.
32.
LadnerSKOttoMJBarkerCS. Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: A novel system for screening potential inhibitors of HBV replication. Antimicrob Agents Chemother1997; 41: 1715–1720.
33.
QiXXiongSYangHMillerMDelaneyWEIV. In vitro susceptibility of adefovir-associated hepatitis B virus polymerase mutations to other antiviral agents. Antivir Ther2007; 12: 355–362.
34.
PrichardMNShipmanCJr. Analysis of combinations of antiviral drugs and design of effective multidrug therapies. Antivir Ther1996; 1: 9–20.
35.
ElionGBSingerSHitchingsGH. Antagonists of nucleic acid derivatives. VIII. Synergism in combinations of biochemically related antimetabolites. J Biol Chem1954; 208: 477–488.
36.
Borroto-EsdoaKVelaJEMyrickFRayASMillerMD. In vitro evaluation of the anti-HIV activity and metabolic interactions of tenofovir and emtricitabine. Antivir Ther2006; 11: 377–384.
37.
SellesethDWTalaricoCLMillerTLutzMWBironKKHarveyRJ. Interactions of 1263W94 with other antiviral agents in inhibition of human cytomegalovirus replication. Antimicrob Agents Chemother2003; 47: 1468–1471.
38.
MoraledaGSaputelliJAldrichCEAverettDCondreayLMasonWS. Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus. J Virol1997; 71: 9392–9399.
39.
ZhuYYamamotoTCullenJ. Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis. J Virol2001; 75: 311–322.
40.
SummersJMasonWS. Residual integrated viral DNA after hepadnavirus clearance by nucleoside analogue therapy. Proc Natl Acad Sci U S A2004; 101: 638–640.
41.
LocarniniSMasonWS. Cellular and virological mechanisms of HBV drug resistance. J Hepatol2006; 44: 422–431.
42.
KorbaBE. In vitro evaluation of combination therapies against hepatitis B virus replication. Antiviral Res1996; 29: 49–51.
43.
KorbaBECotePHornbuckleWSchinaziRGerinJLTennantBC. Enhanced antiviral benefit of combination therapy with lamivudine and famciclovir against WHV replication in chronic WHV carrier woodchucks. Antiviral Res2000; 45: 19–32.
44.
ColledgeDCiviticoGLocarniniSShawT. In vitro antihepadnaviral activities of combinations of penciclovir, lamivudine, and adefovir. Antimicrob Agents Chaemother2000; 44: 551–560.
45.
SeigneresBMartinPWerleB. Effects of pyrimidine and purine analogue combinations in the duck hepatitis B virus infection model. Antimicrob Agents Chaemother2003; 47: 1842–1852.
46.
DelaneyWEIVYangHMillerMDGibbsCSXiongS. Combinations of adefovir with nucleoside analogs produce additive antiviral effects against hepatitis B virus in vitro. Antimicrob Agents Chaemother2004; 48: 3702–3710.
47.
SungJJYLaiJYZeuzemS. A randomized double-blind phase II study of lamivudine (LAM) compared to lamivudine plus adefovir dipivoxil (ADV) for treatment naive patients with chronic hepatitis B (CHB): Week 52 analysis. J Hepatol2003; 38: 25–26. Abstract 69.
48.
LauGKCooksleyHRibeiroRM. Impact of early viral kinetics on T-cell reactivity during antiviral therapy in chronic hepatitis B. Antivir Ther2007; 12: 705–718.
49.
PrichardMNPrichardLEShipmanCJr. Strategic design and three-dimensional analysis of antiviral drug combinations. Antimicrob Agents Chaemother1993; 37: 540–545.
50.
BerenbaumMC. What is synergy?Pharmacol Rev1989; 41: 93–141.
51.
TanJDegertekinBWongSNHusainMOberhelmanKLokAS. Tenofovir monotherapy is effective in hepatitis B patients with antiviral treatment failure to adefovir in the absence of adefovir-resistant mutations. J Hepatol2008; 48: 391–398.
52.
BenhamouYFleuryHTrimouletP. Anti-hepatitis B virus efficacy of tenofovir disoproxil fumarate in HIV-infected patients. Hepatology2006; 43: 548–555.
53.
PetersMGAndersenJLynchP. Randomized controlled study of tenofovir and adefovir in chronic hepatitis B virus and HIV infection: ACTG A5127. Hepatology2006; 44: 1110–1116.
54.
SchmutzGNelsonMLutzT. Combination of tenofovir and lamivudine versus tenofovir after lamivudine failure for therapy of hepatitis B in HIV-coinfection. AIDS2006; 20: 1951–1954.
55.
Bani-SadrFPalmerPScieuxCMolinaJM. Ninety-six-week efficacy of combination therapy with lamivudine and tenofovir in patients coinfected with HIV-1 and wild-type hepatitis B virus. Clin Infect Dis2004; 39: 1062–1064.
