siR-7-EM / KK-46 complexes show strong anti-SARS-CoV-2 effectsin vivo
In order to investigate the therapeutic potential of the siR-7-EM/KK-46 complex in vivo , female Syrian hamsters were infected with SARS-CoV-2 at dose of 105 PFU/animal and then given a single inhalation exposure of 0.7, 1.96 and 5.6 mg/kg of siR-7-EM/KK-46 per day for 6 days. This schedule was thought as an early topical post-exposure treatment. Half of the animals were sacrificed two and six days post infection, respectively. Viral loads were evaluated in the lungs by virus titration and expressed as plaque forming units (PFU) per 1 mL. We found that progeny virus production was significantly and dose-dependently decreased. Up to a 1.5-3 times reduction, in the lungs of the siR-7-EM/KK-46-treated animals on day two (5.0 ± 0.5, 4.7 ± 0.2, 4.5 ± 0.3 in log10 PFU/mL for 0.7, 1.96 and 5.6 mg/kg of siR-7-EM/KK-46, respectively), compared to infected and untreated animals was found (6.1 ± 0.2), P<0.01. (Fig. 5a). Similar results were obtained for the other half of animals which were analyzed on day six. Again, treatment with siR-7-EM/KK-46 significantly and dose-dependently decreased viral loads in the lungs (2.2 ± 0.2, 2.0± 0.2, 1.1 ± 0.1 in log10 PFU/mL for 0.7, 1.96 and 5.6 mg/kg of siR-7-EM/KK-46, respectively) as compared with the infected and untreated animals (3.5 ± 0.2), (P<0.01). The estimated ED50 was ∼ 3.453 mg/kg/day (GraphPad Prism8, non-linear fit, R2 = 0.88; Fig. 5b).
We also observed a reduction of viral load for the control group treated with oral administration of Hydroxychloroquine which was in the range of the siR-7-EM/KK-46-treated animals on day 2 and on day 6. However, effects of treatment with siR-7-EM/KK-46 on lung pathology were strikingly different from those observed with Hydroxychloroquine (Fig. 5c). Histological analysis (Fig. 5c) revealed significantly (p < 0.05 and p < 0.01, at day two and six, respectively) decreased lung inflammation as assessed by macroscopic indicators of disease in animals exposed to 0.7, 1.96 and 5.6 mg/kg of siR-7-EM/KK-46 as compared to SARS-CoV-2 infected, untreated animals group (Fig. 5c). By contrast, no significant improvement of lung pathology was found in the Hydroxychloroquine treated animals as compared to the infected and untreated animals (Fig. 5c). Taken together these results suggested that inhalation of siR-7-EM/KK-46 at a dose of 3.5 mg/kg significantly and dose-dependently attenuates pathological effects in vivo by hindering viral replication.
To further investigate alternative dosing schemes of siR-7-EM/KK-46, a multiple dose study with repeated low doses of siR-7-EM/KK-46 was conducted. In this second set ofin vivo experiments Syrian hamsters were infected with SARS-CoV-2 and treated with 0.175, 0.35 and 1.0 mg/kg (yielding daily doses of 0.35, 0.7 and 2.0 mg/kg, respectively) of siR-7-EM/KK-46 inhalation exposure twice a day with two hours interval for 6 days. Again, viral load was evaluated in the lungs by virus titration and expressed as plaque forming units (PFU) per 1 mL. The SARS-CoV-2 viral titers were found to be significantly reduced by the siR-7-EM/KK-46 compared to the untreated virus-infected animals (P<0.01 for all dose levels) at days two and six (Fig. 6a). In particular, viral titers were reduced a 1.3 (26) and 2.5 (54) times (%) on day two and six, respectively, in the lungs of the siR-7-EM/KK-46-treated animals on day two (5.1 ± 0.1, 4.9 ± 0.2, 4.9 ± 0.1 in log10 PFU/mL for 0.35, 0.7 and 2.0 mg/kg, of siR-7-EM/ KK-46, respectively), compared with results for the untreated, virus-infected animals (6.7 ± 0.03), P<0.01. Also, on day six viral titers in the siR-7-EM/KK-46-treated animals were significantly reduced (1.7 ± 0.1, 1.6± 0.2, 1.3 ± 0.1 in log10 PFU/mL for 0.35, 0.7 and 2.0 mg/kg of siR-7-EM/KK-46, respectively) as compared with the untreated, virus-infected animals (3.3 ± 0.1), (P<0.01). Oral administration of Favipiravir reduced the viral titer approximately 1.2-times on day two (5.4± 0.1) compared to SARS-CoV-2–infected and untreated animals (P<0.01).
Again, we performed scoring of lung pathology and found significantly (P<0.01) decreased lung inflammation in animals exposed to 2 mg/kg of siR-7-EM/KK-46 aerosol compared to SARS-CoV-2 infected group at day six but not in the animals treated with lower doses of siR-7-EM/KK-46 or with Favipiravir as compared to the infected but untreated animals. However, this significant reduction was only found after 6 days suggesting that the dose should be 2 mg/kg or higher.