Therapeutic treatment in a mouse model of MERS-CoV infection
The two best compounds (6j and 6h) in the series were examined for their in vivo efficacy using 10-week-old male hDPP4-KI mice infected with MERSMA-CoV (30). In the first study, animals were divided into three groups (n = 5 to 6) and were lightly anesthetized with ketamine/xylazine and infected with 50 µl of 750 PFU MERSMA-CoV via intranasal inoculation. Compound 6j or 6h were formulated in 10% ethanol and 90% PEG400 and given to mice from 1 to 10 dpi at 50 mg/kg per day (once per day) via intraperitoneal administration. The control mice received vehicle. Animals were weighed daily and monitored for 15 days. Animals were euthanized when an animal lost 30% of initial weight or at 15 dpi.
In the next study, treatment with compound 6j was delayed up to 3 dpi to determine the impact of delayed treatment on mouse survival. Animals were divided into five groups (n = 5), and compound 6j (50 mg/kg per day, once per day) was administered to mice starting at 1, 2, or 3 days after virus challenge (1, 2, or 3 dpi, respectively) until 10 dpi. Mice were monitored for weight loss and survival as described above for 15 days after virus challenge. As controls, vehicle (10% ethanol and 90% PEG400) was administered equivalently to the experimental compound, or animals received no treatment (untreated). The third study was conducted to assess the effects of therapeutic treatment of compound 6j in the lungs. For lung harvest and virus titration, animals were divided into three groups (n = 4) of mice, and compound 6j (50 mg/kg per day, once per day) or vehicle was administered to mice starting at 1 dpi until euthanasia. Animals were euthanized at 3 or 5 dpi, and lungs were removed aseptically, disassociated with a manual homogenizer in 1× PBS, briefly centrifuged, and supernatants removed. Samples were titered on Vero81 cells as reported elsewhere (49). For lung histopathology analyses, animals were divided into two groups (n = 5), and compound 6j (50 mg/kg per day, once per day) or vehicle was administered to mice starting at 1 dpi for 5 days. Mice were euthanized at 6 dpi, lungs were fixed with 10% formalin, and hematoxylin and eosin–stained tissues were examined by a veterinary pathologist using the postexamination method of masking (50). Briefly, tissues were scored in an ordinal manner for edema and hyaline membrane formation using the following scale: 0, none; 1, rare (<5 alveoli); 2, <33% of lung fields; 3, 34 to 66% lung fields; and 4, >66% lung fields (30).
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