Development of a novel mouse model to evaluate drug candidates against hepatitis B virus

Abstract

Woodchuck hepatitis virus (WHV)-infected woodchucks have been used for preclinical development of drugs against hepatitis B virus (HBV). However, there is no simple in vivo model to evaluate small amounts of compounds against HBV. To develop such a model, HepAD38 cells, in which HBV replication is regulated by tetracycline (tet), were grown as subcutaneous tumours in nude mice. Mice developing viraemia were then left untreated or given tet in the drinking water. In some of the mice given tet, it was removed and the mice were injected intraperitoneally with phosphate buffer saline (PBS), lamivudine (3TC), clevudine (CLV) or tenofovir dipivoxil fumarate (TDF). Virus DNA titres were measured by real-time PCR during and after drug treatment. In water-fed and PBS-injected mice, virus titres reached approximately 10(9) copies/ml serum within 35 days of HepAD38 injection, whereas in tet-treated mice, virus titres remained at 10(4)-10(5) copies/ml. HBV DNA levels were suppressed by 3TC, TDF and CLV, with the latter two drugs showing more sustained virus suppression compared with 3TC. Combination therapy with CLV plus TDF was much more effective than either drug alone in suppressing virus titre for at least 3 weeks after the end of treatment. There was no demonstrable toxicity to HepAD38 cells in drug-treated mice. Hence, a robust tet-controlled system for HBV replication in vivo was demonstrated, validated with monotherapies against HBV and shown to be useful in assessing combination therapy. This system will be useful for preclinical assessment of small amounts of single or multiple compounds against HBV in vivo.

Figure 1.

Protocol used for testing drugs in nude mice transplanted with AD38 cells *Each injection was given daily intraperitoneally from days 31–36. ^†Each injection was given intraperitoneally at 100 mg/kg per day from days 31–36. H&E, hematoxylin & eosin; PBS, phosphate buffered saline; TDF, tenofovir dipivoxil fumarate; tet, tetracycline; 3TC, lamivudine.

Figure 2.

HBV DNA titres (assayed by real-time PCR) in serum samples from nude mice injected subcutaneously with AD38 cells Tumours and virus in the blood were detected by day 21 after injection of AD38 cells. Mice were on drinking water for 35 days (curve 1), on tetracycline (tet; 2.5 mg/ml) for 35 days (curve 2), or on tet for 10 days (in their drinking water for days 21–31), and then injected daily for 6 days with phosphate buffered saline (curve 3), lamivudine (3TC; curve 4), or tenofovir dipivoxil fumarate (TDF; curve 5). 3TC and TDF were used at doses of 100 mg/kg per day. Each point is the mean titre of HBV DNA from five mice in each group. *Day 21 is the first day of treatment. Treat, treatment period.

Figure 3.

Tumour weights and ALT levels from nude mice injected subcutaneously with AD38 cells at day 56 (A) Tumour weights. (B) Alanine aminotransferase (ALT). From the left, mice were treated with water only (group 1), water plus tetracycline (tet; 2.5 mg/ml) for 35 days (group 2), or water plus tet for 10 days, then phosphate buffered saline (group 3), lamivudine (3TC; group 4) or tenofovir dipivoxil fumarate (TDF; group 5). 3TC and TDF were used at doses of 100 mg/kg per day.

Figure 4.

Dose–response curves for CLV-, 3TC- and TDF-treated mice and for control mice (A) Clevudine (CLV), (B) lamivudine (3TC), (C) tenofovir dipivoxil fumarate (TDF) and (D) control. In (D), mice were fed with water only or with tetracycline (tet) in water, followed by phosphate buffered saline (PBS). Each point represents the average value from five mice in each group. In panels A–C, the average values shown varied by no more than ±6%.

Figure 5.

Treatment of mice with different doses of TDF, CLV or with TDF + CLV combination therapy The protocol outlined in Figure 1 was followed. Five mice per group were treated with tenofovir dipivoxil fumarate (TDF), clevudine (CLV) or with TDF + CLV combination therapy. Doses were administered at (A) 300 mg/kg per day, (B) 100 mg/kg per day, (C) 33.3 mg/kg per day, (D) 11.1 mg/kg per day or (E) 3.7 mg/kg per day for 6 consecutive days (days 31–36). Mice were then bled over the next 30 days as indicated. (F) Curves reflecting virus DNA titres in serum resulting from different concentrations of combination therapy shown in panels A–E were compared with each other and to phosphate buffered saline (PBS)-fed mice. *All concentrations in F are measured as mg/kg per day. Treat, treatment period.