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)