Inhibition of HI and NA activity and virus replication by BCX 2798 and BCX 2855.
We first evaluated the ability of BCX 2798 and BCX 2855 to inhibit attachment (HA activity), release (NA activity), and replication of the parainfluenza viruses hPIV-1, hPIV-2, hPIV-3, and rSV(hHN) in vitro. The results of the HI assays showed that both compounds inhibited the binding of the three hPIVs, although some differences in anti-HA binding activity were observed between the two agents (Table ). The IC50 values for BCX 2798 in HI assays with hPIV-1, hPIV-2, or hPIV-3 were within a wide range: 0.1 to 4.8 μM. BCX 2798 was ca. 20 times more effective in inhibiting the binding of hPIV-1 and rSV(hHN) than that of hPIV-2 and hPIV-3. The IC50 values for BCX 2855 (2.0 to 6.0 μM) varied less than those for BCX 2798. Although both compounds were equally effective against hPIV-2 and hPIV-3 (range of IC50 values, 2.0 to 4.8 μM), BCX 2798 was 50 times more potent in inhibiting the binding of hPIV-1 and rSV(hHN) than was BCX 2855. BCX 2798 and BCX 2855 compounds were highly specific for parainfluenza viruses: both agents failed to inhibit the HA activity of influenza viruses A/New Caledonia/20/99 and B/Yamanashi/166/98, even when the concentrations of the compounds were as high as 1000 μM (data not shown).
Inhibitory effects of BCX 2798 and BCX 2855 in vitro
The ability of BCX 2798 and BCX 2855 to inhibit NAs of hPIVs was evaluated in NI assays (Table ). Both compounds effectively inhibited the NA activity of the test viruses: the IC50 values for BCX 2798 ranged from 0.02 to 20 μM, and those for BCX 2855 ranged from 1.2 to 4.3 μM. Similar to the results obtained in HI assays with hPIVs, the IC50 values determined in NI assays were more constant for BCX 2855 than for BCX 2798. BCX 2798 was 80 to 1,000 times more effective against of the NA activity of hPIV-1 and rSV(hHN) than against that of hPIV-2 and hPIV-3. The lowest anti-NA activity was observed when BCX 2798 was tested against hPIV-3 (IC50 value, 20 μM). BCX 2855 demonstrated equal effectiveness against the NA activity of hPIV-1 and hPIV-2 but was slightly less effective against the NA activity of hPIV-3. As observed in the HI assays, both compounds were equally effective in inhibiting the NA activity of hPIV-2. BCX 2855 was about five times more active against hPIV-3 than was BCX 2798, but BCX 2798 was 30 to 90 times more effective in the NI assay against hPIV-1 and rSV(hHN) than was BCX 2855. Neither compound effectively inhibited the NAs of the influenza viruses A/New Caledonia/20/99 and B/Yamanashi/166/98 or the NA of the bacterium Clostridium perfringens, even when concentrations of the compounds as high as 1,000 μM were used (data not shown).
Because the results of HI and NI assays indicated that BCX 2798 and BCX 2855 inhibited the ability of the parainfluenza viruses to bind to and be released from the cell, we hypothesized that the life cycle of the viruses was disrupted by the inhibition of these crucial activities. To test this hypothesis, we evaluated the abilities of BCX 2798 and BCX 2855 to inhibit the growth of parainfluenza viruses in LLC-MK2 cells. The trypan blue vital staining of uninfected cells treated with either compound showed that neither BCX 2798 nor BCX 2855 were cytotoxic at concentrations as high as 100 μM (data not shown).
Both agents were effective in inhibiting the growth of parainfluenza viruses in cell culture: the EC50 values for BCX 2798 ranged from 0.7 to 11.3 μM, and those for BCX 2855 ranged from 1.8 to 11.5 μM (Table ). BCX 2798 was 6 to 16 times more effective in inhibiting the growth of hPIV-1 and rSV(hHN) than that of hPIV-2 and hPIV-3. BCX 2855 was more active against hPIV-2 and hPIV-3 than against hPIV-1 and rSV(hHN). BCX 2798 was at least 10 times more effective in inhibiting the growth of hPIV-1 and rSV(hHN) than was BCX 2855, but BCX 2855 was better than BCX 2798 at inhibiting the growth of hPIV-2 and hPIV-3 (1.8 to 2.4 μM compared to 7.0 to 11.3 μM). In general, the results of the growth inhibition study were consistent with those of the HI and NI assays and indicated a high sensitivity of tested viruses to BCX 2798 and BCX 2855 compounds.
Pathogenicity of rSV(hHN) virus in mice.
The hPIVs are strict pathogens of humans and cause no disease in mice. To establish an animal model that can be used for the evaluation of the efficacy of BCX 2798 and BCX 2855 in vivo, we rescued rSV(hHN), in which the HN gene of SV was replaced with that of hPIV-1. Before we tested the compounds in vivo, we determined whether rSV(hHN) is pathogenic in 129x1/SvJ mice. Animals were infected with doses of rSV(hHN) ranging from 105 to 107 TCID50 per mouse, and the number of mice that survived (Table ) and the changes in body weight were determined (Fig. ). All mice infected with the highest dose of virus (107 TCID50 per mouse) lost >35% of their initial weight and died within 9 days after the start of infection. Only 1 of 15 mice survived infection when an inoculation dose of 106.5 TCID50 per mouse was used. In contrast, 80% of the mice infected with 106 TCID50 of rSV(hHN) survived. Infection with no more than 105.5 TCID50 killed no mice, although weight loss (≤15% of initial weight) and signs of infection were observed.
Infectivity of rSV(hHN) in micea
To further examine the pathogenicity of rSV(hHN) in mice, we determined the growth of the recombinant virus in lungs of mice infected with different doses (105, 106, and 107 TCID50; Fig. ). Titers of virus from lungs collected on days 1, 3, 5, 7, and 9 were determined. The virus titers and the clearance of virus from mouse lungs were dependent on the dose of virus. The highest virus titer (~6 log10 TCID50/ml) was recovered on days 1, 3, and 5 from mice infected with 107 TCID50. Titers of virus from lungs of mice infected with 105 or 106 TCID50 were ca. 10 times lower than those from the lungs of mice infected with 107 TCID50 at the same time points. The virus titers decreased in all groups after day 7, but the decrease was more rapid and significant in groups of mice infected with the lower doses than in those infected with the higher doses. Thus, no virus was detected at day 9 in mice infected with 105 TCID50, whereas detectable levels of infectious virus (3.5-log10 TCID50/ml) were present in the lungs of mice infected with 106 TCID50. Despite a reduction on day 7 in the titers of virus from lungs of animals infected with 107 TCID50, all mice in this group died by day 9.
Because the virus titers reached their peak in the lungs within 24 h after infection, we examined whether replication or the dose of virus contribute to the peak titers on day 1. Mice were infected with 106.5 TCID50 of rSV(hHN), and the titers of virus in the lungs were determined 6, 12, 18, and 24 h after infection. The virus titer 6 h after infection was ca. 100 times lower (104 TCID50/ml) than the administered dose and increased steadily every 6 h until a peak titer was reached 24 h after the start of infection (data not shown).
We also examined the lungs of mice for histopathologic changes caused by rSV(hHN) at day 9 after infection (Fig. ). Animals infected with a sublethal dose of virus (105.5 TCID50) (Table ) experienced pathological changes in their airways and interstitium. Alveolar spaces were filled with moderate inflammatory infiltrates of lymphocytes, macrophages, and neutrophils, and alveolar edema was present. Fibrin deposition and alveolar necrosis were also observed. The mucosal epithelium in bronchi was hyperplastic, and there were focal areas of mucosal necrosis with sloughing of degenerate cells into the bronchial lumen. Mononuclear cuffing of vessels was prominent. No signs of lung inflammation were seen in mice in the control group that received only PBS (Fig. ).
FIG. 3. Histopathologic changes in the lungs of mice infected with rSV(hHN). (A) Mice (three per group) were infected with a dose of 105.5 TCID50. Lungs were removed 9 days after infection, fixed, and cut into 5-μm sections that were later stained with (more ...)
The results described in this section indicated that the severe illness characterized by weight loss and death reflected the replication of the rSV(hHN) and pathological changes in mouse lungs. We have therefore established a mouse model for the evaluation of the antiviral activity of BCX 2798 and BCX 2855, as well as that of other potential antiviral compounds.
Efficacy of BCX 2798 and BCX 2855 in mice infected with a lethal dose of rSV(hHN).
To determine the efficacy of BCX 2798 and BCX 2855 in a mouse model, we administered the compounds intranasally to mice at dosages of 1.0 to 50 mg/kg per day twice daily for 5 consecutive days. Administration began 4 h before or 24 h after lethal challenge with rSV(hHN) (106.5 TCID50). At a dosage as high as 50 mg/kg per day, neither compound showed toxicity in uninfected mice in terms of weight change and survival during the observation period (data not shown).
The duration of survival of infected animals and changes in weight were assessed in our evaluation of the effectiveness of the two compounds in vivo. Both parameters were monitored for 21 days after infection. Changes in body weight on days 5, 7, and 9 after infection underwent statistical analysis. Table shows the results of our analysis of the survival duration and weight changes for groups of mice pretreated with different dosages of either compound 4 h before virus infection.
Efficacy of pretreatment with BCX 2798 or BCX 2855 on rSV(hHN) infection in micea
Comparison of the survival curves of rSV(hHN)-infected mice treated or untreated with novel compounds showed that treatment with either BCX 2798 (5 mg/kg per day) or BCX 2855 (50 mg/kg per day) was protective against lethal infection (P < 0.05). BCX 2798 at a dosage of 5 mg/kg per day protected 66.7% of infected mice from death at day 21 after viral infection, whereas 86% of the mice in the control group (infected but treated with PBS only) died. The mean day to death for treated mice was 17 days. In contrast, the mean day to death of mice in the control group was 11.3 days. Complete protection against lethal challenge was observed when mice were treated with 10 mg/kg per day of BCX 2798. Unlike mice in the control group, which lost a maximum of 28.6% of their initial weight by day 9, mice treated with 5 or 10 mg/kg per day of BCX 2798 lost a maximum of 22.8 or 15.9%, respectively, of their initial weight by day 7 and began to regain weight by day 9. Analysis of survival curves of mice treated with different dosages of BCX 2798 and BCX 2855 showed that BCX 2855 was less effective than BCX 2798 in protecting mice against lethal infection by rSV(hHN) (P < 0.05). Only treatment with 50 mg of BCX 2855/kg per day was sufficient to protect 83.3% of infected mice from death 21 days after infection by a lethal dose of virus and increased the mean day to death to 19.3 days. Mice from this group lost a maximum of 17.3% of initial weight.
To determine the effect of the test compounds on virus replication in lungs, we assayed the virus titers from the lungs of infected mice given protective concentrations of either BCX 2798 (10 mg/kg per day) or BCX 2855 (50 mg/kg per day). Treatment began 4 h before inoculation and continued twice daily for 5 days (Fig. ). The virus titers in lungs of animals treated with either agent were significantly lower than those of the control mice (infected but treated with PBS only) (P < 0.05). The virus titers in the lungs of control mice on days 1, 3, and 7 were ca. 10 times greater than those of treated mice on the same days. On day 7, the titers of virus in the lungs of treated mice were more than 50 times less than they were on day 5. Our results indicated that the antiviral effects of both compounds were associated with the inhibition of virus replication in lungs.
FIG. 4. Effect of pretreatment with BCX 2798 or BCX 2855 on virus titers from lungs of mice infected with rSV(hHN). BCX 2798 (10 mg/kg per day ) and BCX 2855 (50 mg/kg per day [•]) were intranasally administered to 129x1/SvJ mice for 5 days; (more ...)
To assess the potential therapeutic usefulness of BCX 2798 and BCX 2855 against parainfluenza virus infection, we examined the effectiveness of the agents when they were given 24 h after inoculation. The number of survivors and weight changes in the treated groups did not differ from those of the control group (mice that were infected but treated with only PBS) even when the highest tested dosage as 50 mg/kg per day of either compound was used (data not shown). Mice infected with a lethal dose of rSV(hHN) and treated with either compound consistently lost weight, and all died 7 to 10 days after inoculation.
Thus, the results of our in vivo experiments indicated that BCX 2798 and BCX 2855 were effective for prophylactic, but not for therapeutic, purposes.