Experiment 1: Effect of administration of nafamostat into the nasal cavity
The 200 µM nafamostat solution was sequentially diluted with saline to prepare 50 µM, 12.5 µM, and 3.1 µM nafamostat solutions for intranasal inoculation. Each weanling mouse was administered 2 µL once per day for 1 week. One group (five mice in a cage) was administered saline as a control, and another one was administered 9.3mM NH4Cl solution, which was a pilot experiment for administering NH4Cl in the following experiment. The change of mean body weight of each group was shown in Figure 1. All the mice used in this experiment grew steadily, and the final mean body weight at day 14 was approximately 17 g in all groups. The mice showed favorable fur, and active movements. The mice administered NH4Cl showed a movement of frequently rubbing their nose immediately after the administration. Any other pain symptom due to the administration was not observed under the conditions of Experiment 1. This result suggests that the administration of 200 µM nafamostat into the nasal cavity is acceptable in the weanling mice.
Experiment 2: Effect of the administration of nafamostat 200 µM plus NH4Cl into the respiratory tract through the nasal cavity
Based on the results of Experiment 1, the 5-old-week adult mice were administered a mixture of 200 µM nafamostat and various concentration of NH4Cl, 20µL per day for 1 week in Experiment 2. The concentrations of NH4Cl in the mixtures were 74mM, 37mM, 18.5mM, and 9.3mM. As a control group, mice were administered normal saline.
Body weight change of individual mouse was expressed with per centage of the body weight at the biggening of Experiment 2 and illustrated in Figure 2. Some mice administered 200 µM nafamostat plus 74 mM, 37 mM, and 18.5 mM NH4Cl (refer to Figure 2-A, B, C) showed transiently decreased weight (by 1 g or less) by the 3rd day of administration. However, the mice had a steady weight gain on the 4th day of administration and showed the same weight gain as the control mice at the end of the observation period. The mice administered 200 µM nafamostat plus 9.3 mM NH4Cl and the control mice did not show decreased weight and gradually increased in weight (Figure 2-D, E).
In addition to the above groups, one group (three mice) was administered 2x20µL of 200µM nafamostat plus 74mM NH4Cl under anesthesia per day, and another group (three mice) 37µL of 200µM nafamostat alone under anesthesia without anesthesia per day. In the former group the mice resisted the intranasal administration and spat some of the inhaled doses. In the latter group the solution was observed to be running out of the nasal cavity several times, probably because the inoculum volume was too much. All the mice showed favorable fur and active movements and showed a steady weight gain similarly to the pattern of the control mice (data not shown).
Although all mice showed favorable fur and active movements, the mice administered 200 µM nafamostat plus 18.5mM NH4Cl (Group C) seemed to be docile with low physical activity. To examine their health status more precisely, sera were separated from blood drawn by cardiac puncture after the observation period, and a testing company was requested to perform biochemical tests. The amount of serum required for testing was obtained from four out of five mice in Groups A, C, and E, respectively (refer to the supplemental Table 1).
As shown in the Table 1, two out of four mice administered 200 µM nafamostat plus 18.5mM NH4Cl (Group C) showed higher liver function markers than the normal ranges. The mice in Group C were also administered 200 µM nafamostat at 2 µL for 1 week at the weanling age (refer to the supplemental Table 1) in Experiment 1. The high dose administration at that time and/or the twice administration of high dose of nafamostat throughout Experiment 1 and 2 were considered to have stressed the liver of the mice. Meanwhile, the laboratory data on the mice of Group A were all within the normal ranges similarly to those in the control mice of Group C.
These outcomes reveal that the protease inhibitor, nafamostat at 200 µM 20 µL/day for 1 week could be administered directly to the respiratory epithelium of adult mice without any adverse effects, and that 74 mM NH4Cl could also be administered in the same manner.