Treatment of late stage disease in a model of arenaviral hemorrhagic fever: T-705 efficacy and reduced toxicity suggests an alternative to ribavirin

Abstract

A growing number of arenaviruses are known to cause viral hemorrhagic fever (HF), a severe and life-threatening syndrome characterized by fever, malaise, and increased vascular permeability. Ribavirin, the only licensed antiviral indicated for the treatment of certain arenaviral HFs, has had mixed success and significant toxicity. Since severe arenaviral infections initially do not present with distinguishing symptoms and are difficult to clinically diagnose at early stages, it is of utmost importance to identify antiviral therapies effective at later stages of infection. We have previously reported that T-705, a substituted pyrazine derivative currently under development as an anti-influenza drug, is highly active in hamsters infected with Pichinde virus when the drug is administered orally early during the course of infection. Here we demonstrate that T-705 offers significant protection against this lethal arenaviral infection in hamsters when treatment is begun after the animals are ill and the day before the animals begin to succumb to disease. Importantly, this coincides with the time when peak viral loads are present in most organs and considerable tissue damage is evident. We also show that T-705 is as effective as, and less toxic than, ribavirin, as infected T-705-treated hamsters on average maintain their weight better and recover more rapidly than animals treated with ribavirin. Further, there was no added benefit to combination therapy with T-705 and ribavirin. Finally, pharmacokinetic data indicate that plasma T-705 levels following oral administration are markedly reduced during the latter stages of disease, and may contribute to the reduced efficacy seen when treatment is withheld until day 7 of infection. Our findings support further pre-clinical development of T-705 for the treatment of severe arenaviral infections.

Figure 1. Serum and tissue virus burden during the course of PICV infection in hamsters.

All daily sacrifice groups had 4 hamsters/group except for the day-0 control group, which had 3 animals. Due to death prior to time of sacrifice, tissue and serum samples could only be obtained for 2 and 1 hamsters for the day 9 and 10 groups, respectively. The serum sample was unobtainable from 1 of the day-8 hamsters due to death just prior to sacrifice. (A) Serum, (B) liver, (C) spleen, (D) kidney, (E) lung, (F) intestinal, and (G) brain virus titers were determined by infectious virus cell culture assay. Data points represent viral loads for individual hamsters with the group mean represented by the respective horizontal bar.

Figure 2. Systemic ALT, AST and type I IFN levels during the course of PICV infection in hamsters.

Serum samples assayed for viral burden, as described for Figure 1, were also assayed for (A) ALT, (B) AST, and (C) type I IFN. Data points represent levels for individual hamsters with the group mean represented by the respective horizontal bar.

Figure 3. Effect of delayed T-705 and ribavirin therapy on PICV infection outcome in hamsters.

Drugs were given orally twice per day for 7 days. Treatments were initiated on day 4, 5 or 6. T-705 was administered as a daily dose of 100 mg/kg. Ribavirin was dosed at 40 mg/kg/day. Ten animals per group (20 for the placebo) were observed daily for 4 weeks for (A) survival and every third day for (B) mean group weight determination of survivors. ***P<0.001 compared to 0.4% CMC placebo-treated hamsters.

Figure 4. Reduction of viral load and liver disease in PICV-challenged hamsters following delayed treatment with T-705 and ribavirin.

Five hamsters per group were treated as described in Figure 3, and sacrificed on day 7 of the infection to assess (A) systemic and (B) hepatic viral burden, and liver disease as determined by (C) ALT measurement. Data points represent values for individual animals and mean virus titers and ALT levels for each group are represented by horizontal lines. *P<0.05; **P<0.01 compared to 0.4% CMC placebo-treated hamsters. ^a P<0.05 compared to hamsters receiving ribavirin treatment starting on the same day.

Figure 5. Effect of delayed treatment with equitoxic doses of T-705 and ribavirin on PICV infection in hamsters.

Drugs were given orally twice per day for 7 days starting on day 5, 6 or 7 for the drug-treated groups and day 7 for the placebo group. T-705 was dosed at 160 mg/kg/day on the first day and 80 mg/kg/day thereafter. Ribavirin was given at 22 mg/kg/day on the first day and 11 mg/kg/day thereafter. Hamsters were observed 28 days for (A) survival and weights and standard deviations of survivors are shown for treatments initiated on day (B) 5, (C) 6 or (D) 7. The 0.4% CMC-treated placebo and sham-infected, untreated groups are included in B–D for comparison. For the survival analysis, *P<0.05; ***P<0.001 compared to placebo-treated hamsters by log-rank test. ^a P<0.05 compared to hamsters receiving ribavirin treatment starting on the same day.

Figure 6. Reduction of viral load and liver disease in PICV-challenged hamsters following delayed treatment with equitoxic doses of T-705 and ribavirin.

Five hamsters per group were treated as described in Figure 5, and sacrificed on day 7 of the infection to assess (A) systemic and (B) hepatic viral burden, and (C) ALT. The day-7 treatment initiation group only received the morning dose, with animals sacrificed 3-4 h after treatment. Data points represent values for individual animals and mean virus titers and ALT levels for each group are represented by horizontal lines. *P<0.05; **P<0.01 compared to 0.4% CMC placebo-treated hamsters. ^a P<0.05 compared to hamsters receiving ribavirin treatment starting on the same day.

Figure 7. Survival outcome following high-dose treatment of advanced PICV infection in hamsters.

T-705 or placebo was given orally twice per day for 8 days starting on day 6 or 7. T-705 was dosed at 320 mg/kg/day on the first day and 100 mg/kg/day thereafter. Hamsters were observed 28 days for survival. ***P<0.001 compared to respective placebo-treated hamsters by log-rank test.

Figure 8. Effect of delayed high-dose T-705 therapy on viral burden and liver disease in PICV-challenged hamsters.

Five hamsters per group for the treatment groups initiated on day 6 were treated as described in Figure 7, and sacrificed on day 7 of the infection to assess (A) systemic, (B) splenic, and (C) hepatic viral loads, and (D) ALT. Data points represent values for individual animals and mean virus titers and ALT levels for each group are represented by horizontal lines.

Figure 9. Reduced systemic absorption of orally administered T-705 in PICV-infected hamsters.

Infected or sham-infected animals were treated with 50 mg/kg of T-705 on day 7 of infection and plasma samples were taken from animals sacrificed at 0.25, 0.5, 1, 2 or 4 h after treatment. Samples were processed and analyzed by HPLC for T-705 and its primary metabolite, T-705M1, as described in the methods section. Data represent the mean and standard error of the mean from 3 hamsters (n = 3) per time point.