Efficacy of the carbocyclic 2'-deoxyguanosine nucleoside BMS-200475 in the woodchuck model of hepatitis B virus infection

Abstract

Daily oral treatment with the cyclopentyl 2'-deoxyguanosine nucleoside BMS-200475 at doses ranging from 0.02 to 0.5 mg/kg of body weight for 1 to 3 months effectively reduced the level of woodchuck hepatitis virus (WHV) viremia in chronically infected woodchucks as measured by reductions in serum WHV DNA levels and endogenous hepadnaviral polymerase activity. Within 4 weeks of daily therapy with 0.5 or 0.1 mg of BMS-200475 per kg, endogenous viral polymerase levels in serum were reduced about 1,000-fold compared to pretreatment levels. Serum WHV DNA levels determined by a dot blot hybridization technique were comparably decreased in these treated animals. In the 3-month study, the sera of animals that had undetectable levels of WHV DNA by the dot blot technique were further analyzed by a highly sensitive semiquantitative PCR assay. The results indicate that BMS-200475 therapy reduced mean WHV titers by 10(7)- to 10(8)-fold, down to levels as low as 10(2) to 10(3) virions/ml of serum. Southern blot hybridization analysis of liver biopsy samples taken from animals during and after BMS-200475 treatment showed remarkable reductions in the levels of WHV DNA replicative intermediates and in the levels of covalently closed circular viral DNA. WHV viremia in BMS-200475-treated WHV carriers eventually returned to pretreatment levels after therapy was stopped. These results indicate that BMS-200475 should be evaluated in clinical trials for the therapy of chronic human hepatitis B virus infections.

FIG. 1

Chemical structure of BMS-200475.

FIG. 2

Serum WHV levels in animals treated for 1 month with BMS-200475 as measured by EPA. Data for individual animals are presented in panels A to C. Animals were treated per os with the following daily dosages of BMS-200475: 0.5 (A), 0.1 (B), and 0.02 (C) mg/kg/day. (D) Mean EPA values obtained for each of the three groups treated with BMS-200475 at 0.02 (□), 0.1 (■), and 0.5 (▵) mg/kg/day. End. Pol. Act., endogenous polymerase activity.

FIG. 3

Serum WHV DNA levels following daily oral administration of BMS-200475 or 3TC for 3 months. Shown are WHV DNA dot blot data. The treatment regimens and the individual animals treated were as follows: (A) 0.1 mg of BMS-200475/kg per day; animals 94-053 (□), 94-141 (■), 94-313 (▵), 95-153 (▴), 94-146 (○), and 95-321 (•); (B) 0.02 mg of BMS-200475/kg per day; animals 94-163 (□), 95-341 (■), 95-204 (▵), 95-323 (▴), 95-422 (○), and 95-012 (•); (C) 5 mg of 3TC/kg per day; animals 95-093 (□), 95-181 (▴), 95-182 (▵), 95-423 (○), 95-411 (■), and 95-414 (▵); (D) placebo-treated animals; animals 95-111 (□), 94-144 (▴), 95-152 (▵), and 94-423 (○). See text for further details.

FIG. 4

Mean serum WHV DNA levels following daily oral administration of BMS-200475 for 3 months. Data reflect combined results of dot blot and PCR analyses of serum samples from the 10 woodchucks that responded to BMS-200475 treatment with undetectable levels of WHV DNA as determined by the dot blot assay. Not included are data for two animals receiving 0.02 mg of BMS-200475 per kg/day, in which reductions in WHV DNA levels were about 2 log units. ■, 0.02 mg/kg per day; □, 0.1 mg/kg per day. See text for further details.

FIG. 5

Analysis of liver WHV DNA species. Nucleic acid samples enriched for either viral cccDNA (A) or replicative intermediates (B) were prepared from liver biopsy samples. The nucleic acids were electrophoresed in parallel through 1% agarose and analyzed by Southern hybridization. See text for further details. The dosing regimens and individual animals represented in the study were as follows: (A1 and B1) BMS-200475 (0.02 mg/kg/day); animals 95-422 (lanes 1 and 5), 95-341 (lanes 2 and 4), 95-012 (3 and 6), and 95-163 (lane 7); (A2 and B2) BMS-200475 (0.1 mg/kg/day); animals 94-313 (lane 8), 94-141 (lanes 9 and 12), 95-053 (lanes 10, 14, and 15), 95-253 (lane 11), and 95-321 (lanes 13 and 16); (A3 and B3) 3TC (5 mg/kg/day); animals 95-414 (lanes 17 and 23), 95-423 (lane 18, 21, and 22), and 95-181 (lanes 19 and 20). The biopsy times (in weeks) are indicated above the autoradiogram (PT, pretreatment). The positions of a 3.3-kb WHV DNA marker (lane M) and a 2.0-kb DNA size marker are indicated at the right of the panels; the cccDNA and replicative intermediate species are indicated at the left.