Antiviral effects of lamivudine, emtricitabine, adefovir dipivoxil, and tenofovir disoproxil fumarate administered orally alone and in combination to woodchucks with chronic woodchuck hepatitis virus infection

Abstract

Adefovir dipivoxil (ADV) and tenofovir disoproxil fumarate (TDF) are nucleotide analogs that inhibit the replication of wild-type hepatitis B virus (HBV) and lamivudine (3TC)-resistant virus in HBV-infected patients, including those who are coinfected with human immunodeficiency virus. The combination of ADV or TDF with other nucleoside analogs is a proposed strategy for managing antiviral drug resistance during the treatment of chronic HBV infection. The antiviral effect of oral ADV or TDF, alone or in combination with 3TC or emtricitabine (FTC), against chronic woodchuck hepatitis virus (WHV) infection was evaluated in a placebo-controlled study in the woodchuck, an established and predictive model for antiviral therapy. Once-daily treatment for 48 weeks with ADV plus 3TC or TDF plus FTC significantly reduced serum WHV viremia levels from the pretreatment level by 6.2 log(10) and 6.1 log(10) genome equivalents/ml serum, respectively, followed by TDF plus 3TC (5.6 log(10) genome equivalents/ml), ADV alone (4.8 log(10) genome equivalents/ml), ADV plus FTC (one survivor) (4.4 log(10) genome equivalents/ml), TDF alone (2.9 log(10) genome equivalents/ml), 3TC alone (2.7 log(10) genome equivalents/ml), and FTC alone (2.0 log(10) genome equivalents/ml). Individual woodchucks across all treatment groups also demonstrated pronounced declines in serum WHV surface antigen, characteristically accompanied by declines in hepatic WHV replication and the hepatic expression of WHV antigens. Most woodchucks had prompt recrudescence of WHV replication after drug withdrawal, but individual woodchucks across treatment groups had sustained effects. No signs of toxicity were observed for any of the drugs or drug combinations administered. In conclusion, the oral administration of 3TC, FTC, ADV, and TDF alone and in combination was safe and effective in the woodchuck model of HBV infection.

FIG. 1.

Antiviral effects of oral 3TC, FTC, ADV, and TDF administration alone and in combination on serum WHV DNA levels in chronic WHV carrier woodchucks. Treatment groups were as follows: placebo (A), 3TC (B), FTC (C), ADV (D), ADV plus 3TC (E), ADV plus FTC (F), TDF (G), TDF plus 3TC (H), and TDF plus FTC (I). Horizontal bars indicate the 48-week treatment period. Log₁₀ changes in serum WHV DNA levels from baseline at week 0 prior to drug administration for individual woodchucks in each treatment group are displayed. WHVge, WHV genomic equivalents (virion- or WHV DNA-containing virus particles).

FIG. 2.

Comparison of antiviral effects of oral 3TC, FTC, ADV, and TDF administration alone and in combination on serum WHV DNA levels in chronic WHV carrier woodchucks. (A) Monotherapy with ADV or 3TC and combination therapy with ADV and 3TC. The placebo control group is displayed for comparison. (B) Monotherapy with TDF or 3TC and combination therapy with TDF and 3TC. (C) Monotherapy with ADV or FTC and combination therapy with ADV and FTC. (D) Monotherapy with TDF or FTC and combination therapy with TDF and FTC. Horizontal bars indicate the 48-week treatment period. Mean log₁₀ changes in serum WHV DNA levels from baseline at week 0 prior to drug administration for each treatment group are displayed. Vertical lines indicate standard deviations. Each group contained five woodchucks at the start of treatment. Differences in geometric mean serum WHV DNA concentrations from pretreatment values were significant for the following treatment groups and time points (P < 0.05): 3TC, days 1 to 5 and weeks 1 to 8; FTC, days 2 to 5 and weeks 1 to 12 and 28 to 52; ADV, days 1 to 5 and weeks 1 to 44; TDF, days 2 to 5 and weeks 1 to 6 and 28 to 36; ADV plus 3TC, days 1 to 5 and weeks 1 to 36; ADV plus FTC, days 2 to 5 and weeks 1 to 24; TDF plus 3TC, days 2 to 5 and weeks 1 to 8; and TDF plus FTC, days 1 to 5 and weeks 1 to 32 of the study. Differences in geometric mean serum WHV DNA concentrations from those in the placebo control group were significant for the following treatment groups and time points (P < 0.05): 3TC, days 2 to 5 and weeks 1 to 2; FTC, days 2 to 5 and weeks 1 to 2; ADV, days 2 to 5 and weeks 1 to 3; TDF, days 2 to 5 and weeks 1 to 3; ADV plus 3TC, days 1 to 5 and weeks 1 to 3; ADV plus FTC, days 2 to 5 and weeks 1 to 3; TDF plus 3TC, days 2 to 5 and weeks 1 to 3; and TDF plus FTC, days 2 to 5 and weeks 1 to 3 of treatment. WHVge, WHV genomic equivalents (virion- or WHV DNA-containing virus particles).

FIG. 3.

Antiviral effects of oral 3TC, FTC, ADV, and TDF administration alone and in combination on serum WHsAg concentrations in chronic WHV carrier woodchucks. Treatment groups were as follows: placebo (A), 3TC (B), FTC (C), ADV (D), ADV plus 3TC (E), ADV plus FTC (F), TDF (G), TDF plus 3TC (H), and TDF plus FTC (I). Horizontal bars indicate the 48-week treatment period. Serum WHsAg concentrations for individual woodchucks in each treatment group are displayed.

FIG. 4.

Antiviral effects of oral 3TC, FTC, ADV, and TDF administration alone and in combination on hepatic WHV replication in chronic WHV carrier woodchucks. Treatment groups were as follows: placebo (hepatic tissue was not available from woodchuck M7006 at week 60) (A), 3TC (hepatic tissue was not available from woodchuck F3121 at week 48) (B), FTC (C) ADV (hepatic tissue was not available from woodchuck F6554 at weeks 48 and 60 and from woodchuck F7028 at week 48) (D), ADV plus 3TC (hepatic tissue was not available from woodchuck M7039 at weeks 48 and 60) (E), ADV plus FTC (hepatic tissue was not available from woodchucks F3122, M3306, M6501, and F7007 at weeks 36, 48, and 60) (F), TDF (hepatic tissue was not available from woodchucks M3003 and F7030 at weeks 48 and 60) (G), TDF plus 3TC (hepatic tissue was not available from woodchuck F3143 at weeks 12, 36, 48, and 60 and from woodchuck M3082 at week 60) (H), and TDF plus FTC (hepatic tissue was not available from woodchuck F7004 at weeks 36, 48, and 60 and from woodchuck M7040 at week 60) (I). Levels of hepatic cellular DNA were quantified by hybridization to a woodchuck-specific β-actin gene probe by the Southern blot hybridization technique.

FIG. 5.

Antiviral effects of oral 3TC, FTC, ADV, and TDF administration alone and in combination on hepatic WHV RNA levels in chronic WHV carrier woodchucks. Treatment groups were as follows: placebo (A), 3TC (B), FTC (C), ADV (D), ADV plus 3TC (E), ADV plus FTC (F), TDF (G), TDF plus 3TC (H), and TDF plus FTC (I). See the legend to Fig. 4 for data on the availability of hepatic tissue of individual woodchucks from the experimental groups. Levels of hepatic cellular RNA were quantified by hybridization to a woodchuck-specific 18S gene probe by the Northern blot hybridization technique.

FIG. 6.

Antiviral effects of oral 3TC, FTC, ADV, and TDF administration alone and in combination on hepatic expression of WHcAg in chronic WHV carrier woodchucks. Treatment groups were as follows: placebo (A), 3TC (B), FTC (C), ADV (D), ADV plus 3TC (E), ADV plus FTC (F), TDF (G), TDF plus 3TC (H), and TDF plus FTC (I). See the legend to Fig. 4 for data on the availability of hepatic tissue of individual woodchucks from the experimental groups.

FIG. 7.

Antiviral effects of oral 3TC, FTC, ADV, and TDF administration alone and in combination on hepatic expression of cytoplasmic WHsAg in chronic WHV carrier woodchucks. Treatment groups were as follows: placebo (A), 3TC (B), FTC (C), ADV (D), ADV plus 3TC (E), ADV plus FTC (F), TDF (G), TDF plus 3TC (H), and TDF plus FTC (I). See the legend to Fig. 4 for data on the availability of hepatic tissue from individual woodchucks from the experimental groups. According to the number of hepatocytes, staining was scored on a scale of 0 to 4 (1 is staining of up to 1% of hepatocytes, 2 is staining of up to 2% of hepatocytes, 3 is staining of up to 5% of hepatocytes, and 4 is staining of 10% or more hepatocytes).

FIG. 8.

Antiviral effects of oral 3TC, FTC, ADV, and TDF administration alone and in combination on portal hepatitis in chronic WHV carrier woodchucks. Treatment groups were as follows: placebo (A), 3TC (B), FTC (C), ADV (D), ADV plus 3TC (E), ADV plus FTC (F), TDF (G), TDF plus 3TC (H), and TDF plus FTC (I). See the legend to Fig. 4 for data on the availability of hepatic tissue from individual woodchucks from the experimental groups. According to their severities, the specific lesions were graded on a scale of 0 to 4 (representing an absent lesion to the most severe lesion, respectively).

FIG. 9.

Antiviral effects of oral 3TC, FTC, ADV, and TDF administration alone and in combination on lobular hepatitis in chronic WHV carrier woodchucks. Treatment groups were as follows: placebo (A), 3TC (B), FTC (C), ADV (D), ADV plus 3TC (E), ADV plus FTC (F), TDF (G), TDF plus 3TC (H), and TDF plus FTC (I). See the legend to Fig. 4 for data on the availability of hepatic tissue from individual woodchucks from the experimental groups. According to their severity, the specific lesions were graded on a scale of 0 to 4 (representing an absent lesion to the most severe lesion, respectively).