INTRODUCTION

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Respiratory syncytial virus (RSV) is the most prevalent viral cause of lower respiratory tract disease in infants and young children (15) and is associated with significant morbidity in children with underlying cardiopulmonary disease (20). Ribavirin is recommended for use as a small-particle aerosol by RSV-infected children who are at high risk of having serious sequelae (2). When administered as a small particle, aerosol ribavirin has also proved to be effective in the treatment of naturally occurring RSV infection in children (11, 26). The long treatment schedules, cost of therapy, and potential for environmental contamination during treatment have discouraged the use of ribavirin in many situations. Prolonged aerosol therapy may also prevent parents and health care personnel from monitoring therapy. On the other hand, RSV is increasingly being recognized as a cause of serious pneumonia following marrow transplantation (3, 13, 16). Recently, Whimbey et al. (29, 30) described an outbreak of RSV in 42 patients at a marrow transplant center; 16 (38%) of the patients developed pneumonia, and of these, 42% died. It was reported that intravenous ribavirin treatment of bone marrow transplant recipients with RSV pneumonia did not improve the rate of mortality compared with that for historical controls treated with the aerosol form of the drug (19). Furthermore, to date, efforts to develop a vaccine have failed (18). On the other hand, a role for circulating antibody in the protection against RSV infection has been demonstrated repeatedly by passive immunization with monoclonal antibodies (28, 33) and high-titer anti-RSV immune globulin (9). The high-titer anti-RSV immune globulins from pooled human serum (RespiGam) have now been approved by the U.S. Food and Drug Administration. However, they have prophylactic but not therapeutic efficacy. Until an effective vaccine against RSV can be developed, effective protection of high-risk infants against serious RSV disease may require treatment with combinations of currently available therapeutic agents.

RD3-0028, a benzodithiin compound, has been reported to have antiviral activity against RSV in tissue culture, and its activity is superior to that of ribavirin. RD3-0028 inhibited all RSV strains of subgroups A and B and clinical isolates; however, it did not inhibit the replication of influenza A virus, measles virus, herpes simplex virus types 1 and 2, or human cytomegalovirus (27). In an effort to increase the therapeutic effectiveness of this compound, adjunctive aerosol administration of RD3-0028 was tested. Previous studies with ribavirin or SP-303 delivered by aerosol administration in order to target the drug to the infected respiratory epithelium have demonstrated the effectiveness of this route (5, 7, 36, 38). The present study demonstrates the effectiveness of aerosol administration of RD3-0028 in reducing the pulmonary titer of RSV and an improvement of pathologic changes of pulmonary tissues from RD3-0028-treated, RSV-infected mice.

	MATERIALS AND METHODS

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Animals, cells, and viruses. Pathogen-free, 10-week-old female BALB/c mice were purchased from Charles River Laboratories. All mice were housed in cages covered with barrier filters and were fed mouse chow and water ad libitum. HeLa cells were maintained in Eagle's minimal essential medium supplemented with glutamine, gentamicin, penicillin G, and 10% fetal bovine serum. The A2 strain of RSV was obtained from the American Type Culture Collection.

Compounds. 1,4-Dihydro-2,3-benzodithiin (RD3-0028) (molecular weight, 168) was synthesized at Iwate University, Morioka, Japan. 1-(-D-Ribonuranosyl)-1,2,4-triazole-3-carboxamide (ribavirin) (molecular weight, 244) was provided by H. Machida (Yamasa Corp., Choshi, Japan). RD3-0028 was dissolved in 10% dimethyl sulfoxide (DMSO)-saline containing 1% Tween 80. Aerosols were generated from reservoirs containing 0.3 to 7.0 mg of RD3-0028 per ml. Solutions of ribavirin were prepared in saline containing 2.5 to 60 mg/ml.

Aerosol characteristics. The aerosol was generated with a head-exposure chamber, mono-position, with a mist generator (Sibata Scientific Technology Ltd., Tokyo, Japan). The particle size distribution of the RD3-0028 aerosol was determined with an Andersen-type air sampler (AN-200; Sibata Scientific Technology Ltd., Tokyo, Japan) and glass fiber filters, PTFE binding (model T60A20). The concentration of RD3-0028 generated in the aerosol was measured by sampling onto glass fiber filters with a low-volume air sampler (model L-15P; Sibata Scientific Technology Ltd.). The samples collected were eluted from the filters by soaking the filters in 10 ml of absolute methanol for 1 h. Quantification of RD3-0028 was performed with a high-performance liquid chromatography (HPLC) system (HITACHI, Tokyo, Japan). For the HPLC we used a Superspher RP-18(e) column (4 µm; Merck, Darmstadt, Germany) with a mobile phase which consisted of methanol and water. A 50 to 100% methanol gradient was generated over a 12-min period with a flow rate of 1.0 ml/min, and the absorbance was measured at 220 nm.

Mouse infection and harvest. Mice were treated intraperitoneally with 100 mg of cyclophosphamide (CYP; Nacalai Tesque, Tokyo, Japan) per kg of body weight 5 days before virus inoculation. The mice were weighed, anesthetized with sodium pentobarbital (50 mg/kg), and inoculated intranasally with approximately 10⁵ PFU of RSV A2 in 50 µl (day 0). From day 1 through day 3, the mice were exposed to the RD3-0028 or ribavirin aerosol. Placebo consisted of 10% DMSO-saline containing 1% Tween 80. On day 4, the day on which untreated mice had the maximum RSV pulmonary titer, all animals were killed and the lungs of each mouse were removed.

Virus quantification. The removed lungs were homogenized with glass homogenizers with a Teflon pestle (Ikemoto Scientific Technology Co., Ltd., Tokyo, Japan) in 4 ml of Hanks balanced salt solution supplemented with 0.218 M sucrose, 4.4 mM glutamate, 3.8 mM KH₂PO₄, and 3.2 mM K₂HPO₄ as described previously (21). The resulting suspensions were stored at 70°C prior to assay. HeLa cells were seeded into a 24-well tissue culture plate (Falcon 3074; Becton Dickinson, Lincoln Park, N.J.) at approximately 2 × 10⁵ cells/well, and the plate was incubated at 37°C in 5% CO₂. Lung homogenates from mice inoculated with strain A2 were diluted (10-fold) with Eagle's minimal essential medium supplemented with 2% fetal calf serum (Cell Culture Laboratories, Cleveland, Ohio), 100 U of penicillin G per ml, and 100 µg of streptomycin per ml. Each dilution of the homogenate was tested for the virus titer in confluent HeLa cells. After incubation for 5 days at 35°C, 80% methanol was added to the cell monolayer. The virus titers were assayed by plaquing. The wells were first incubated with 5% Fraction V in phosphate-buffered saline (PBS) for 30 min and then with horseradish peroxidase-conjugated anti-RSV serum (Virostat, Portland, Maine) diluted (20-fold) with 1% Fraction V in PBS for 1 h at 37°C. After washing twice with 5% Fraction V in PBS, the wells were then incubated with a 4 CN membrane peroxidase substrate (no. 50-73-00; Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.) at room temperature for optimal color development. The numbers of RSV plaques were counted.

Histologic methods and evaluation. Lungs were removed for histologic examination and were placed in buffered formalin for a minimum of 24 h. The staining and evaluation of tissue was carried out by A. Ichikawa (Fuji Biomedix, Yamanashi, Japan). The tissue was then embedded in low-melting-point paraffin, sectioned at a 5-µm thickness, and stained with hematoxylin and eosin. The stained sections were coded by number and were evaluated blind as to the previous treatment. To determine lung condition, the lungs were assigned a score ranging from 0 (no pathology) to 4 (maximal pathology).

Statistical analysis. The geometric mean virus titers for the experimental groups were compared with those for the control groups by a Mann-Whitney U test. A P value of 0.05 or less was considered significant.

	RESULTS

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Mouse model of RSV infection. Mice were treated intraperitoneally with 100 mg of CYP per kg of body weight. After 5 days, the mice were anesthetized with sodium pentobarbital (50 mg/kg) and were inoculated intranasally with approximately 10⁵ PFU of RSV A2 in 50 µl (day 0). On day 2 after infection, the lungs contained 1.1 log PFU/g of lung. On day 3, the titers increased slightly, reaching a maximum on day 4 and day 5 (3.93 ± 0.10 and 3.97 ± 0.21 log PFU/g of lung, respectively). On day 6, the titers began to decline and only a few titers were detected on day 7 (Fig. 1). In all subsequent drug studies described here, the mice were exposed to the RD3-0028 aerosol on day 1 through day 3 and were then killed 4 days after infection to measure the effect of the drug on the maximal levels of virus in tissue. The lungs of each animal were removed, weighed, and assayed for virus levels.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Growth curves for RSV (A2 strain) in lungs of CYP-treated mice. CYP-treated mice were inoculated with 105 PFU of RSV and were killed at 1 to 7 days. Each point represents the geometric mean virus titer (log10) for five to eight animals. Capped bars indicate standard deviations.

RD3-0028 aerosol particle characteristics. RD3-0028 suspended in 10% DMSO solution containing 1% Tween 80 at 1.25 mg/ml was characterized in an aerosol-generating system with a head-exposure chamber. The mass median aerodynamic diameter (MMAD) of the aerosol particle was determined to be 2.10 µm, with a geometric standard deviation of 1.86. Increasing the concentration of RD3-0028 in the reservoir did not significantly alter these characteristics.

Effect of RD3-0028 aerosol treatment on RSV-infected mice. The RD3-0028 (0.625 to 7 mg/ml) aerosol, which was administered for 2 h twice daily for 3 consecutive days, significantly reduced the pulmonary titer of RSV-infected mice (Table 1). Mice given 10% DMSO containing 1% Tween 80 showed no significant reduction in virus titer compared to those for untreated, infected mice. The efficacy of RD3-0028 treatment was dose dependent in groups given between 0.3 and 2.5 mg of RD3-0028 per ml. In the mice given 2.5 or 7 mg of RD3-0028 solution per ml, the reduction was about 65%. With ribavirin, the same efficacy was observed (reduction, 60.8%) when the concentration was 60 mg/ml. At a concentration of 10 mg of RD3-0028 per ml, the reduction in the pulmonary RSV titer was 40.3%, although the efficacy was not significant (P = 0.089). In contrast, 2.5 mg of ribavirin per ml had no effect on the pulmonary RSV titer.

View larger version (28K): [in this window] [in a new window]   FIG. 2.   Output of RD3-0028 and ribavirin aerosol in the head-exposure chamber. The graph presents mean data from two separate experiments.

Histologic findings. In sections of lung from RSV-infected mice collected on day 4 after virus inoculation, evidence of interstitial pneumonia was observed in nearly all microscopic fields (Fig. 3a). Mononuclear cells were detected in the peribronchiolar and perivascular spaces. There was thickening of the alveolar walls and arteritis followed by monocyte infiltration in the artery. Neutrophils and giant cells were identified in the alveolar walls. Alveolar edema was also determined. Moreover, eosinophil infiltration was also detected in the peribronchiolar and perivascular spaces. In contrast, sections of lung collected on day 4 from an infected mouse treated with 7 mg of RD3-0028 per ml showed improvement to the level of the lungs of uninfected controls. RD3-0028 aerosol administration appeared to protect against tissue damage, as evidenced by preservation of the lung architecture and a reduction in pulmonary inflammatory infiltrates (Fig. 3b). There was no improvement in sections of lung collected on day 4 from an infected mouse treated with 10% DMSO-saline containing 1% Tween 80 (data not shown). The pathologic changes in the pulmonary tissues were scored (0, no pathology; 4, maximal pathology). The histologic changes in RSV-infected mice were monitored daily from day 2 to day 7, and those in RD3-0028-treated, infected mice were monitored from day 2 to day 4 (Table 2). Each score was totaled, and the average of the total scores for RD3-0028-treated mice was compared with that for untreated, RSV-infected mice (Fig. 4). In the untreated, infected mice, a high score was shown immediately after RSV infection (day 2). The score increased slightly, reaching a maximum on day 4 and day 5. Although the score began to decline on day 6, evidence of interstitial pneumonia lasted until day 7. On the other hand, in the case of the RD3-0028-treated mice, the total score decreased gradually with each day that the aerosol was administered. It is clear that RD3-0028 aerosol treatment significantly improved the pathologic changes in the pulmonary tissues of RSV-infected mice.

View larger version (87K): [in this window] [in a new window]   FIG. 3.   (a) Interstitial pneumonia; mononuclear cell filtration in peribronchiolar and perivascular spaces and thickness of alveolar walls in lungs from an untreated, RSV-infected, CYP-treated mouse, day 4, with hematoxylin and eosin staining. B, bronchiole; A, artery; V, vein. Magnification, ×25. (b) Reduced interstitial pneumonia in lungs from an RSV-infected, CYP-treated mouse treated twice daily on days 1 through 3 for 2 h with 7 mg of RD3-0028 per ml aerosol, day 4, with hematoxylin and eosin staining. B, bronchiole, A, artery. Magnification, ×25.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Comparison of total scores for RD3-0028-treated, RSV-infected mice (----) and untreated infected mice (). This graph represents the total score for each pathology in Table 2.

	DISCUSSION

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RD3-0028 has antiviral activity against RSV in cell culture. By the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay developed in our laboratories, the 50% effective concentration and the 50% cytotoxic concentration of this compound were 4.5 and 271.0 µM, respectively (27). To evaluate RD3-0028 for its antiviral efficacy in vivo, mice were inoculated with RSV and were then treated with different concentrations of RD3-0028 small-particle aerosols. RD3-0028 reduced the pulmonary RSV titers in infected, CYP-treated mice given aerosols generated from reservoirs containing 0.625 to 7 mg of RD3-0028 solution per ml. These results were in general agreement with those obtained in in vitro studies (27). The reductions in pulmonary virus titers in animals given these doses ranged from 50 to 65% compared to the pulmonary titers in untreated controls.

It has been determined that particles with MMADs exceeding 5 µm are deposited primarily in the upper respiratory tract, whereas particles with MMADs of 3 µm or less penetrate throughout the respiratory tract (14). By using the Anderson-type air sampler and estimating the size distribution, it was determined that more than 90% of the particles of RD3-0028 generated in the aerosol machines used had MMADs of less than 5.0 µm and that the mean MMAD of the particles generated was 2.10 µm.

Aerosolized drugs are an alternative formulation that can be used to deliver drug directly to the pulmonary surface better than systemic regimens can. Such formulations may also achieve higher local concentrations of drug. This treatment regimen resulted in minimal systemic drug delivery, thus reducing possible toxicity. It has been reported that the administration of drugs as small-particle aerosols was effective in the treatment of lung disease (6, 35, 37). The aerosol route of administration used in present study was chosen for several reasons. First, no effect was observed when the drug was given by other routes such as the intraperitoneal, oral, or intravenous route. This result indicates that parenterally administered RD3-0028 may not reach the lung or nasal mucosa, which are the primary target areas of respiratory virus infection, in sufficient quantities. Second, another antiviral drug, ribavirin, has been successfully administered as a continuous small-particle aerosol to humans (4, 11, 12, 26) and cotton rats (17, 36) infected with RSV. Ribavirin was shown to be significantly more efficacious when it was delivered by this route than when it was given intraperitoneally (17). In contrast, it was reported that the intraperitoneal administrations of N-(phosphonoacetyl)-L-aspartate (34), SP-303, a naturally occurring polyphenolic polymer (32), and LY253963 (31) were effective in the treatment of RSV-infected cotton rats.

The immunosuppressed RSV-infected mouse model was chosen because it is the most practical model in which infection can take place throughout the life of the animal and because viral titers of approximately 10⁴ PFU per g of lung may be attained. Titers in tissues reached their maximum approximately 4 days after inoculation and rapidly diminished on day 6. Moreover, sections of lung from RSV-infected mice collected on day 4 after inoculation of the virus revealed histologic evidence of interstitial pneumonia: (i) mononuclear cell and eosinophil infiltration in peribronchiolar and perivascular spaces, (ii) thickening of alveolar walls, and (iii) arteritis followed by monocyte filtration in the artery. In vivo evaluation of RSV-infected animals has been carried out with the cotton rat model in previous studies (10, 21, 22, 31). In addition, an experimental model with ferrets (24) and primates (1, 25) has been reported, and Graham et al. (8) found that CYP-untreated, 8- to 10-month-old mice were more susceptible to RSV infection than 8-week-old mice. However, other than the cotton rat, the animal models reported previously are much more difficult to use for an antiviral drug evaluation. The cotton rat has been a singularly useful model and has provided data concerning pulmonary replication of viruses, protection, and circulating antibody response (23). However, unfortunately, cotton rats are no longer available from breeders. In contrast, although the mouse is viewed by us as the most desirable animal because of ease of handling, mice are not as susceptible to infection with RSV. Thus, we have developed a CYP-treated mouse model of RSV infection to evaluate antiviral activities in vivo. It seems unlikely that the pulmonary RSV titer in the CYP-treated mouse model is completely reduced. In the cotton rat model, ribavirin aerosol reduced the amount of virus in lung tissue by more than 90% (38). However, a 60-mg/ml solution of ribavirin showed about a 60% reduction in the present study. It is suspected that the 60 to 65% reduction of the pulmonary titer reached the limit of efficacy in the RSV-infected mouse. This limitation appears to be the reason why the reduction in the pulmonary titer is not dose dependent in groups given between 2.5 and 7 mg of RD3-0028 per ml. However, 7 mg of RD3-0028 per ml did improve the lung pathology more than a 2.5-mg/ml solution did.

We examined the toxic effect of RD3-0028 treatment in mice (data not shown). When RD3-0028 aerosol was administered at a concentration of 7 mg/ml in the reservoir twice a day for 10 consecutive days, no mortality was observed in the mice. However, both mouse groups treated with RD3-0028 and control aerosol showed a gradual reduction in their body weight, loosing in the range of 15.5 to 17.6% of their initial body weight by day 10. The weight loss in the aerosol-treated group might be due to the stress resulting from the restraint in the mouse holder during treatment. These preliminary studies indicate that at the therapeutic dose RD3-0028 is not toxic for mice.

In summary, the present study demonstrates that RD3-0028 is active against RSV infection in mice. The minimal effective dose of RD3-0028 for RSV-induced infection in CYP-treated mice is significantly less than that of ribavirin, the only compound currently available for use against RSV disease. However, further testing is necessary, and one major area of interest is the mechanism of action of RD3-0028 to allow the development of candidate drugs for chemotherapy of RSV infections.