2.7 Statistical analysis
The manuscript complies with BJP’s recommendations and requirements on experimental design and analysis [19]. The declared group size is the number of independent values, and statistical analysis was done using these independent values (i.e., not treating technical replicates as independent values). In terms of this study, data were presented as means ± S.E.M. Plots were produced using Graphpad Prism 9.0. Statistical analysis was performed using the SPSS software. For multiple groups comparison, the Levene’s test was used to test for equality of variances; if P>0.05, one-way ANOVA were performed; if P≤0.05, Kruskal-Wallis test were performed. For one-way ANOVA, if P≤0.05, the LSD post hoc test was performed. For the Kruskal-Wallis test, if P≤0.05, the LSD post hoc test was performed after the ranks were transformed into normal scores. Otherwise, no data normalization was performed. We conducted a full data analysis without excluding outliers. A P-value <0.05 was considered statistically significant.
3. Results
3.1 S086 lowered systolic blood pressure (SBP) in DSS rats
The SBP in DSS rats increased progressively over time in the vehicle high salt (8%) group, and was significantly higher compared to the sham low salt (0.3%) group at each measurement point between day 1 and day 28 after dosing (P<0.001). The SBP in the sham low salt (0.3%) group was approximately 150 mmHg, whereas the SBP in the vehicle high salt (8%) group ranged from 162.87 mmHg on day 1 to 199.15 mmHg on day 28. Notably, the peak SBP occurred between 9:00 PM and 2:00 AM while the valley SBP was observed between 1:00 PM and 6:00 PM. (Figure 2)
On day 1, there were no significant changes in SBP observed in each dosing group compared to the vehicle high salt (8%) group. However, on day 7, the LCZ696-68 mpk group, EXP3174-35 mpk group, and different doses of S086 (8, 23, 68 mpk) groups showed significant reductions in the 24-hour mean SBP compared to the vehicle high salt (8%) group. The reductions were 16.18 mmHg (P<0.001), 15.75 mmHg (P<0.01), 12.97 mmHg (P<0.05), 15.76 mmHg (P<0.01), and 22.56 mmHg (P<0.001), with corresponding reduction rates of 9.4%, 9.1%, 7.5%, 9.1%, and 13.1%, respectively. S086 demonstrated dose-dependent efficacy on SBP and had a better effect than the equimolar dose of LCZ696-68 mpk. The middle dose of S086-23 mpk showed similar efficacy compared to LCZ696-68 mpk. Additionally, S086 had a significantly better effect on SBP compared to the equimolar dose of EXP3174 (P<0.05) and Sacubitril (P<0.001) (Figure 2)
The efficacy of each dosing group on day 14, 21 and 28 showed similar results compare to day 7, and the efficacy increased over time compared to the vehicle high salt (8%) group. The reduction rate of SBP in LCZ696 group increased from 9.4% on day 7 to 16.5% on day 28, and from 13.1% to 19.5% for S086-68 mpk. The Sacubitril- 33 mpk group exhibited a significant effect on day 21 and 28, with the mean 24h SBP reduced by 16.91mmHg (P<0.01) and 14.86mmHg (P<0.05) compared to the vehicle high salt (8%) group.
The middle and high dose groups of S086 (23 and 68 mpk) could sustain SBP levels similar to the sham group for approximately 14 hours (11:00 AM to 8:00 PM and 6:00 AM to 11:00 AM) after 28 days of dosing. The other groups were unable to lower their SBP to the level of the sham group. (Figure 2)
3.2 S086 lowered diastolic blood pressure (DBP) in DSS rats
The DBP in DSS rats followed a similar trend to the SBP results. In the sham low-salt (0.3%) group, the DBP was approximately 100 mmHg, while in the vehicle high-salt (8%) group, it increased from 113.87 mmHg on day 1 to 146.31 mmHg on day 28 (Figure 3)
On day 7, 24-hour mean DBP decreased by 16.69 mmHg (P<0.001), 17.75 mmHg (P<0.001), 9.74 mmHg (P<0.05), 13.58 mmHg (P<0.05), 14.60 mmHg (P<0.05), and 21.02 mmHg (P<0.001) for the LCZ696-68 mpk group, EXP3174-35 mpk group, Sacubitril-33 mpk group, and different doses of S086 (8, 23, 68 mpk) groups. The reduction rates were 13.6%, 14.5%, 8.0%, 11.1%, 11.9%, and 17.2%, respectively. (Figure 3)
The middle and high dose groups of S086 (23 and 68 mpk) could sustain the DBP at or near the level of the sham group (some timepoints lower than the sham group) for about 14 hours (from 11:00 AM to 8:00 PM and from 6:00 AM to 11:00 AM) after 28 days of dosing. (Figure 3)
3.3S086 lowered mean arterial pressure (MAP) in DSS rats
As MAP is calculated from SBP and DBP, the effect of each treatment group on MAP was similar to its effect on SBP and DBP. The MAP in the sham low-salt (0.3%) group was approximately 120 mmHg. The vehicle high-salt (8%) group had significantly higher MAP than the sham low-salt (0.3%) group at all time points from day 1 to day 28 after dosing (P<0.001). Efficacy of S086 middle dose (23mpk) exhibited a non-inferiority compare to LCZ696-68 mpk. (Figure 4)