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Received: 20 October 2022    Accepted: 31 January 2023

DOI: 10.1002/prp2.1064

ORIGINAL ARTICLE

Synergism of amlodipine and telmisartan or candesartan on

blood pressure reduction by using SynergyFinder 3.0 and
probability sum test in vivo

Tian Xia1,2  | Lu-­Lu Xu1,2  | Peng-­Yue Guo3 | Wan-­Ting Shi1,2 | Yan-­Qiong Cheng1,2 |

Ai-­Jun  Liu1,2

1
Department of Pharmacology, School
of Pharmacy, Naval Medical University, Abstract
Shanghai, China
This study was designed to evaluate the synergism of two couples of antihyperten-
2
Institute of Pharmacy, Yueyang Hospital
of Integrated Traditional Chinese and
sive drugs (amlodipine + telmisartan and amlodipine + candesartan) on blood pressure
Western Medicine, Shanghai University of reduction in vivo by both SynergyFinder 3.0 and probability sum test. Spontaneously
Traditional Chinese Medicine, Shanghai,
China
hypertensive rats were treated with intragastric administration of amlodipine (0.5, 1,
3
Department of Clinical Pharmacy, Naval 2, and 4 mg/kg), telmisartan (4, 8, and 16 mg/kg), candesartan (1, 2, and 4 mg/kg), nine
Medical University, Shanghai, China combinations for amlodipine and telmisartan, and nine combinations for amlodipine
Correspondence and candesartan. The control rats were treated by 0.5% carboxymethylcellulose so-
Ai-­Jun Liu and Yan-­Q iong Cheng, dium. Blood pressure was recorded continuously up to 6 h after administration. Both
Institute of Pharmacy, Yueyang Hospital
of Integrated Traditional Chinese and SynergyFinder 3.0 and the probability sum test were used to evaluate the synergistic
Western Medicine, Shanghai University action. The synergisms calculated by SynergyFinder 3.0 are consistent with the prob-
of Traditional Chinese Medicine, Shanghai
200083, China. ability sum test both in two different combinations. There is an obviously synergistic
Email: mrliuaijun@163.com; interaction between amlodipine and telmisartan or candesartan. The combinations of
mschengyanqiong@126.com
amlodipine and telmisartan (2 + 4 and 1 + 4 mg/kg) and amlodipine and candesartan
Funding information (0.5 + 4 and 2 + 1  mg/kg) might exert an optimum synergism against hypertension.
Fund of Non Drug Therapy Clinical
Research Center; Fund of Yueyang Compared with the probability sum test, SynergyFinder 3.0 is more stable and reliable
Hospital of Integrated Traditional Chinese to analyze the synergism.
and Western Medicine, Grant/Award
Number: 2021yygm01; National Key
Research and Development Program KEYWORDS
of China, Grant/Award Number: amlodipine, candesartan, hypertension, probability sum test, SynergyFinder 3.0, telmisartan
2019YFC1708504; National Natural
Science Foundation of China, Grant/
Award Number: 82273984; Traditional
Chinese Medicine-­Clinical Evaluation
Platform of Chinese Patent Medicine,
Grant/Award Number: A1-­U21-­205-­902

Abbreviations: BRAID, Bivariate Response to Additive Interacting Doses; CMC, carboxymethylcellulose sodium; DBP, diastolic blood pressure; MBP, mean blood pressure; MuSyC,
Multidimensional Synergy of Combinations; SBP, systolic blood pressure; SD, standard deviation; SHRs, spontaneously hypertensive rats.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2023 The Authors. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and
Experimental Therapeutics and John Wiley & Sons Ltd.

Pharmacol Res Perspect. 2023;11:e01064.  |

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1  |  I NTRO D U C TI O N
Drug combinations have become a standard therapy for many com-
plex diseases, including cancers and cardiovascular diseases.1,2 The
combination with different mechanism drugs might produce synergis-
tic interaction. It can enhance the treatment efficacy and avoid the ac-
quisition of monotherapy resistance. Good combination therapy also
reduces the dose of each drug. It is helpful to minimize the clinical and
metabolic side effects of each individual component in larger dosage.3
SynergyFinder, a web-­application, was put forward in 2017 and
has been widely used for the discovery of novel synergistic drug
combinations.4,5 SynergyFinder was designed to calculate the syn-
ergism between anti-­cancer medicines first. It also can be used in
many precision medicine areas, targeted drug combination discov-
ery.6 Recently, we reported that it can be used for anti-­platelet ag-
gregation in vitro.7 It is unclear whether this method can be used to
evaluate the synergism of antihypertensive drugs in vivo.
For the combination of different antihypertensive drugs, the proba-
bility sum test (q test) has been suggested to evaluate the synergism.8,9
This test derives from classic probability analysis, and several groups have
suggested that it may be useful for evaluating the synergism of combina-
8–­13
tions of two drugs in vivo. However, there are some disadvantages.
For example, about 20% data are discarded during the calculation. This
elimination might affect the accuracy of the synergistic conclusion.
In the present study, both SynergyFinder 3.0 (the latest upgrade)
and the probability sum test were performed to analyze the synergism
on blood pressure reduction in vivo. Two couples of antihypertensive
drugs (amlodipine + telmisartan and amlodipine + candesartan) were
used in spontaneously hypertensive rats (SHRs). We mainly focused
in the difference between the probability sum test and SynergyFinder
3.0 for the evaluation of synergism on blood pressure reduction.
2  |  M ATE R I A L S A N D M E TH O DS
2.1  |  Animals
Male SHRs (aged 3 months) were purchased from Beijing Vital River
Laboratory Animal Technology Co., Ltd. The rats were housed with
controlled temperature (23–­25°C) and lighting (8:00 AM to 8:00 PM
light, 8:00 PM to 8:00 AM dark) and with free access to food and tap
water. All the animals used in this experiment received humanitarian
concern. All animal experiments were approved by the Committee
on ethics of biomedicine of Naval Medical University and were con-
ducted according to the National Institutes of Health Guidelines for
the Care and Use of Laboratory Animals.
2.2  |  Blood pressure continuous recording in
conscious rats
The operation and measurement were conducted as described
previously in detail. 2,14 Briefly, rats were anesthetized by
20521707, 2023, 2, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1002/prp2.1064 by Cochrane Peru, Wiley Online Library on [09/04/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
inhalation of 5% isoflurane and maintained with 2% isoflurane.
The lower abdominal aorta was catheterized via the left femo-
ral artery with a polyethylene catheter full of heparin (100 U/
mL) for the recording of blood pressure. The catheters were tun-
neled subcutaneously, exposed through the interscapular skin
and fixed. Then the animals were treated by meloxicam (2 mg/
kg, subcutaneous injection) and penicillin (80 000 IU per animal,
intramuscular injection) to relieve pain and prevent infection for
2 days. After 2 days recovery (with free access to food and tap
water), rats were placed individually in cylindrical cages for blood
pressure recording. The aortic catheter was connected to a pres-
sure transducer via a swivel allowing rats to move freely. Beat-­to-­
beat blood pressure signals and heart period were digitized by the
MPA-­H BBS system (Alcott Biotech Co. Ltd) mounted in computer.
After approximately 3 h habituation, the blood pressure and heart
period signals were determined online. The data of blood pres-
sure from 12:00 to 13:00 are used as the baseline (before admin-
istration). Then, at 13:00, the drug was given via the catheter of
gastric fistula. The time reaching the maximum reduction in the
blood pressure of each group was about half hour. Systolic blood
pressure (SBP), diastolic blood pressure (DBP), and mean blood
pressure (MBP) values from every heart beat were recorded from
0.5 h after administration up to 6 h.
Experiment 1 protocol: The effect of a single dose or different
combinations of amlodipine and telmisartan on blood pressure re-
duction in SHRs.
Male SHRs were divided into 15 groups (n = 10 per group) and
administered by the following drugs: amlodipine (1, 2 and 4 mg/kg),
telmisartan (4, 8 and 16 mg/kg), and the combinations (1 + 4, 1 + 8,
1 + 16, 2 + 4, 2 + 8, 2 + 16, 4 + 4, 4 + 8 and 4 + 16 mg/kg).
The drugs were dissolved in the 0.5% carboxymethylcellu-
lose sodium (CMC) and were given into the stomach through a
catheter. The blood pressure was recorded according aforemen-
tioned method. The blood pressure values an hour before admin-
istration and mean values 6 h after administration were recorded
to analyze.
Experiment 2 protocol: The effect of a single dose or different
combinations of amlodipine and candesartan on blood pressure re-
duction in SHRs.
Male SHRs were divided into 13 groups (n  =  10 per group)
and administered by the following drugs: amlodipine (0.5 mg/kg),
candesartan (1, 2 and 4  mg/kg) and the combinations (0.5 + 1,
0.5 + 2, 0.5 + 4, 1 + 1, 1 + 2, 1 + 4, 2 + 1, 2 + 2 and 2 + 4  mg/kg).
The drugs were dissolved in the 0.5% CMC and were given into
the stomach through a catheter. The blood pressure was re-
corded according aforementioned method. Blood pressure data
of other 2 amlodipine alone groups (1 and 2  mg/kg) were got
from experiment 1.
Another 10 SHRs were treated by vehicle (0.5% CMC) with the
same volume as a control group. The blood pressure was recorded
accordingly. The blood pressure values 1 h before administra-
tion and mean values 6 h after administration were recorded to
analyze.

XIA et al.
2.3  |  SynergyFinder 3.0
An excel was set up. There are several lines, including “Pair Index”,
“Drug 1”, “Drug 2”, “dose 1”, “dose 2”, “Response” and “Dose Unit”.
“Pair Index” represents how many pairs of drugs. “Drug 1” and
“Drug 2” are the name of drugs in a dose-­response, while “dose 1”
and “dose 2” are the doses of the drugs in combination. “Response”
means the effect of drug combinations at the doses input by “dose
1” or “dose 2”. It is the reduction percentage of blood pressure. The
range of the “Response” is expected from 0 to 100. The “Dose Unit”
means the unit of drugs' dose.
The inhibition ratio of blood pressure reduction was used for
SynergyFinder 3.0 system. Inhibition ratio = (blood pressure after
administration − blood pressure before administration)  × 3.33/blood
pressure before administration × 100%. Unlike tumor cells, blood
pressure cannot be reduced or inhibited by 100%. About 30% reduc-
tion in blood pressure is most appropriate in clinical.15 So we defined
a 30% reduction in blood pressure as 100% inhibition. Dose-­effect
curves for each drug on blood pressure were drawn and the half max-
imal inhibitory dose were calculated by using Prism software, version
8 (GraphPad). Choosing the percentage reduction as the phenotypic
response, the blood pressure reduction of each drug combination was
analyzed using SynergyFinder 3.0 (https://syner​gyfin​der.fimm.fi).5
Briefly, the mean percentage of blood pressure reduction of a
pair of antihypertensive drugs, and their combinations at different
doses were input respectively. According to the software recom-
mendation, highest single agent (HSA) reference model was selected
in this study. This model states the expected combination effect is
the maximum of the single drug responses at corresponding concen-
trations. Finally, the comprehensive synergy score and synergy maps
of drug combination were obtained automatically by the software
using HSA model. The synergy score was used to evaluate the effi-
cacy of the combination. According to the user guide in the website,
synergy score was evaluated as follows: scores <−10: the interaction
between two drugs was likely to be antagonistic; from −10 to 10: the
interaction between two drugs was likely to be additive; and > 10:
the interaction between two drugs was likely to be synergistic.7
The synergy scoring can be visualized as either a two-­dimensional
or a three-­dimensional interaction surface over the dose matrix. The
depth of the color reveals the degree of percentage of blood pres-
sure reduction in the two-­dimensional image and the height of the
3D drug interaction landscape is standardizing as the percentage of
blood pressure reduction to show the comparison of the degrees of
interaction among drug combinations.
2.4  |  Probability sum test
The probability sum test was used to evaluate the synergism of the
10–­12
combination according to the references. Compared with the
control group, the experimental groups with a decrease in blood
pressure >15 mmHg were defined as responders. Similarly, rats with
a decrease in blood pressure <12 mmHg (80% of 15 mmHg) were
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      3 of 12
defined as non-­responders and blood pressure data ranging from 12
to 15 mmHg were discarded. We calculate the q value as follows:
q = PA+B/(PA + PB − PA × PB). A and B represent drug A and drug B. P
is the percentage of responders in each group. PA+B is the respond-
ers' real percentage and (PA + PB − PA × PB) is the expected response
ratio. (PA + PB) is the sum of the probabilities when drug A and drug B
are used alone. PA × PB is the probability of ratios responding to both
drugs when they were used alone. We considered that the combi-
nation was synergistic when q was >1.15 and the combination was
antagonistic when q was <0.85. When q was between 0.85 and 1.15,
the combination was additive.
2.5  |  Statistical analysis
The investigators were blinded to the animal groups when they
assessed the blood pressure and analyzed the synergistic score
using SynergyFinder 3.0 or the probability sum test. The animals
were randomly assigned by using the random permutations table.
Continuous variables were expressed as mean ± standard deviation
(SD). Student's t-­test (two-­t ailed paired t-­test) was used for compari-
sons between before and after drug administration. p < .05 was con-
sidered statistically significant.
3  |  R E S U LT S
3.1  |  Effects of amlodipine, telmisartan alone, and
their combination on SBP, DBP, and MBP in SHRs
The effects of a single dose of amlodipine and telmisartan alone or in
their combinations on SBP, DBP, and MBP are shown in Figure 1. In
amlodipine alone treatment groups, compared to before administra-
tion, SBP values in 1, 2, and 4 mg/kg amlodipine groups were signifi-
cantly reduced by approximately 8, 12, and 18 mmHg, respectively
(Student's t-­test, p < .05). DBP values were significantly reduced by
5, 8, and 14 mmHg, respectively. MBP values were reduced by 6,
10, and 16 mmHg, respectively. In telmisartan treatment groups (4,
8, and 16 mg/kg), SBP values showed significant reduction by ap-
proximately 6, 10, and 16 mmHg, respectively. DBP was reduced by
16, 10, and 16 mmHg, and MBP was reduced by 6, 10, and 16 mmHg,
respectively. Blood pressure values were also significantly reduced
by the different combinations (Student's t-­test, p < .01).
In combination groups with amlodipine 1 mg/kg, adding telmis-
artan (4, 8, and 16 mg/kg) significantly reduced SBP by 15, 12, and
14 mmHg, respectively. In combination groups with amlodipine 2 mg/
kg, adding telmisartan (4, 8, and 16 mg/kg) significantly reduced SBP
by 21, 14, and 17 mmHg, respectively. In combination groups with
amlodipine 4 mg/kg, adding telmisartan (4, 8, and 16 mg/kg) signifi-
cantly reduced SBP by 17, 19, and 21 mmHg, respectively. The similar
results were also got from DBP and MBP.
No significant change in blood pressure was observed in control
rats compared with the blood pressure before administration.
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4 of 12       XIA et al.

F I G U R E 1  Effects of a single dose of amlodipine (1, 2 and 4 mg/kg) and telmisartan (4, 8 and 16 mg/kg) alone and their combination
on the SBP, DBP and MBP in SHRs. Open bars mean before administration; solid bars mean after administration. Values are expressed as
mean ± SD. *p < .05, **p < .01 versus before administration.

3.2  |  Effects of amlodipine, candesartan
alone, and their combination on SBP, DBP, and MBP
in SHRs
The effects of amlodipine and candesartan alone or in their
combinations on SBP, DBP, and MBP are shown in Figure 2. SBP
values in 0.5  mg/kg amlodipine group were reduced by 3 mmHg
(p  =  .08). DBP and MBP values were both reduced by 4 mmHg
(Student's t-­test, p < .05). In candesartan treatment groups (1, 2,
and 4 mg/kg), SBP values showed reduction by approximately 4,
4, and 8 mmHg, respectively. DBP was significantly reduced by 7,
10, and 11 mmHg, and MBP was significantly reduced by 5, 7, and
10 mmHg, respectively. Blood pressure values were significantly
reduced by the different combinations (Student's t-­test, p < .05)
except for one combination group (amlodipine 0.5 mg/kg + cande-
sartan 2 mg/kg).
In combination groups with amlodipine 0.5 mg/kg, adding can-
desartan (1, 2, and 4 mg/kg) reduced SBP by 8, 3, and 9 mmHg, re-
spectively. In combination groups with amlodipine 1 mg/kg, adding
candesartan (1, 2, and 4 mg/kg) significantly reduced SBP by 7, 14,
and 11 mmHg, respectively. In combination groups with amlodip-
ine 2  mg/kg, adding candesartan (1, 2, and 4  mg/kg) significantly
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XIA et al.       5 of 12

F I G U R E 2  Effects of a single dose of amlodipine (0.5, 1 and 2 mg/kg) and candesartan (1, 2 and 4 mg/kg) alone and their combination
on the SBP, DBP and MBP in SHRs. Open bars mean before administration; solid bars mean after administration. Values are expressed as
mean ± SD. *p < .05, **p < .01 versus before administration.

reduced SBP by 20, 13, and 20 mmHg, respectively. The significant
reduction was also got from DBP and MBP.
3.3  |  The synergistic effects of amlodipine and
telmisartan by SynergyFinder 3.0
The mean dose-­response of SBP, DBP, and MBP in rats administrated with
amlodipine and telmisartan or their combinations are shown in Table S1–­
S3. The comprehensive synergy score and synergy maps of amlodipine
and telmisartan combination are shown in Figures  3–­5. The reduction
ratio of blood pressure is positive relative to the dose of amlodipine or
telmisartan (Figures 3–­5A,B). For SBP, DBP, and MBP, the comprehensive
synergy scores of amlodipine combined with telmisartan are 10.10, 10.30,
and 10.24, respectively, which indicates that amlodipine and telmisartan
exhibit synergistic effect in these doses for blood pressure reduction.
When the combined dose is 2 mg/kg amlodipine +4 mg/kg telmisartan,
the synergy scores are 19.94, 16.70, and 17.93 for SBP, DBP, and MBP,
respectively. The scores also reach a high level when the dose is 1 mg/
kg amlodipine and 4 mg/kg telmisartan(15.96, 21.63, and 18.69 for SBP,
DBP, and MBP, respectively). The data indicate that these two combina-
tions (2 + 4 and 1 + 4 mg/kg) might exert an optimum synergistic effect.
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6 of 12       XIA et al.

F I G U R E 3  The drug interaction test

for amlodipine and telmisartan on SBP by
SynergyFinder 3.0. (A) the curve of the
dose-­inhibition response for amlodipine
& telmisartan output. X-­axis reflects
the concentration of amlodipine &
telmisartan, Y-­axis reflects the inhibition
ratio on SBP. (B) dose-­response matrix
(inhibition) for amlodipine & telmisartan.
The inhibition ratio is positively relative to
the degree of red. (C) the drug interaction
landscapes based on the HSA model on
SBP. The synergy score is showed by red
(>0) and green (<0). Left is plane figure
and right is solid figure.

F I G U R E 4  The drug interaction test

for amlodipine and telmisartan on DBP by
SynergyFinder 3.0. (A) the curve of the
dose-­inhibition response for amlodipine
& telmisartan output. X-­axis reflects
the concentration of amlodipine &
telmisartan, Y-­axis reflects the inhibition
ratio on DBP. (B) dose-­response matrix
(inhibition) for amlodipine & telmisartan.
The inhibition ratio is positively relative to
the degree of red. (C) the drug interaction
landscapes based on the HSA model on
DBP. The synergy score is showed by red
(>0) and green (<0). Left is plane figure
and right is solid figure.
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XIA et al.       7 of 12

F I G U R E 5  The drug interaction test

for amlodipine and telmisartan on MBP
by SynergyFinder 3.0. (A) the curve of the
dose-­inhibition response for amlodipine
& telmisartan output. X-­axis reflects
the concentration of amlodipine &
telmisartan, Y-­axis reflects the inhibition
ratio on MBP. (B) dose-­response matrix
(inhibition) for amlodipine & telmisartan.
The inhibition ratio is positively relative to
the degree of red. (C) the drug interaction
landscapes based on the HSA model on
MBP. The synergy score is showed by red
(>0) and green (<0). Left is plane figure
and right is solid figure.

3.4  |  The synergistic effects of amlodipine and
candesartan by SynergyFinder 3.0
The mean dose-­response of SBP, DBP, and MBP in rats adminis-
trated with amlodipine and candesartan or their combinations are
shown in Table S4–­S 6. The comprehensive synergy score and syn-
ergy maps of amlodipine and candesartan combination are shown
in Figures  6–­8 . The reduction ratio of blood pressure is positive
relative to the dose of amlodipine or candesartan (Figures  6–­
8A,B). For SBP, DBP, and MBP, the comprehensive synergy scores
of amlodipine combined with candesartan are 11.74, 20.91, and
17.30, respectively, which indicates that amlodipine and telmisar-
tan exhibit synergistic effect in these doses for blood pressure
reduction. When the combined dose is 0.5 mg/kg amlodipine and
4 mg/kg candesartan, the synergy scores are 19.06, 35.55, and
28.11 for SBP, DBP, and MBP, respectively. These data indicate
that the combination (0.5 + 4 mg/kg) might exert an optimum syn-
ergistic effect.
3.5  |  Synergistic effects of amlodipine and
telmisartan by probability sum test
Table  1 presents the results of the probability sum test of SBP,
DBP, and MBP in data from SHRs treated with amlodipine, tel-
misartan, and their combinations. For SBP, the q values only in
two combination groups (amlodipine + telmisartan, 1 + 8 and
1 + 16 mg/kg) are <1.15 (1.05 and 0.90). These data indicate that
all the other combination groups might have a synergistic effect
on SBP. For DBP, the q values only in one group (1 + 8 mg/kg) are
<1.15 (0.97). All q values of MBP are larger than 1.15. In the com-
bination group (2 + 4 mg/kg), the q values (6 for SBP and 6.25 for
MBP) are larger than any other groups. These data indicate that
this combination (2 + 4 mg/kg) might exert an optimum synergistic
effect.
3.6  |  Synergistic effects of amlodipine and
candesartan by probability sum test
Table 2 shows the probability sum test of SBP, DBP, and MBP in data
from SHRs treated with amlodipine and candesartan. For SBP, the
q values only in two combination groups (amlodipine + candesartan,
1 + 1 and 1 + 2 mg/kg) are <1.15 (1.13 and 1.11). All the other q val-
ues of combination groups are larger than 1.15. These groups might
have a synergistic effect on SBP. For DBP and MBP, the q values
only in one group (0.5 + 2 mg/kg) are <1.15 (0.50 and 1.00 for DBP
and MBP, respectively). All the other q values are larger than 1.15.
For SBP and MBP, the q values in two combination groups (2 + 1 and
0.5 + 4 mg/kg) are larger than any other group. All these data indi-
cates that these two combinations (2 + 1 and 0.5 + 4  mg/kg) might
exert an optimum synergistic effect.
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20521707, 2023, 2, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1002/prp2.1064 by Cochrane Peru, Wiley Online Library on [09/04/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
8 of 12       XIA et al.

F I G U R E 6  The drug interaction test

for amlodipine and candesartan on SBP
by SynergyFinder 3.0. (A) the curve of the
dose-­inhibition response for amlodipine
& candesartan output. X-­axis reflects
the concentration of amlodipine &
candesartan, Y-­axis reflects the inhibition
ratio on SBP. (B) dose-­response matrix
(inhibition) for amlodipine & candesartan.
The inhibition ratio is positively relative to
the degree of red. (C) The drug interaction
landscapes based on the HSA model on
SBP. The synergy score is showed by red
(>0) and green (<0). Left is plane figure
and right is solid figure.

F I G U R E 7  The drug interaction test

for amlodipine and candesartan on DBP
by SynergyFinder 3.0. (A) the curve of the
dose-­inhibition response for amlodipine
& candesartan output. X-­axis reflects
the concentration of amlodipine &
candesartan, Y-­axis reflects the inhibition
ratio on DBP. (B) dose-­response matrix
(inhibition) for amlodipine & candesartan.
The inhibition ratio is positively relative to
the degree of red. (C) the drug interaction
landscapes based on the HSA model on
DBP. The synergy score is showed by red
(>0) and green (<0). Left is plane figure
and right is solid figure.
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XIA et al.       9 of 12

F I G U R E 8  The drug interaction test

for amlodipine and candesartan on MBP
by SynergyFinder 3.0. (A) the curve of the
dose-­inhibition response for amlodipine
& candesartan output. X-­axis reflects
the concentration of amlodipine &
candesartan, Y-­axis reflects the inhibition
ratio on MBP. (B) dose-­response matrix
(inhibition) for amlodipine & candesartan.
The inhibition ratio is positively relative to
the degree of red. (C) the drug interaction
landscapes based on the HSA model on
MBP. The synergy score is showed by red
(>0) and green (<0). Left is plane figure
and right is solid figure.

TA B L E 1  The probability sum test in rats administrated with amlodipine and telmisartan or their combinations.

Amlodipine (mg/kg) Telmisartan (mg/kg) PSBP PDBP PMBP q SBP q DBP q MBP

0 4 0% 0% 0%
0 8 22.2% 33.3% 25.0%
0 16 42.9% 37.5% 42.8%
1 0 22.2% 11.1% 0%
2 0 12.5% 0% 10%
4 0 60.0% 57.1% 66.7%
1 4 66.7% 57.1% 57.1% 3.00 5.15 /
1 8 28.6% 42.9% 37.5% 1.05 0.97 1.50
1 16 50.0% 66.7% 71.4% 0.90 1.50 1.67
2 4 75.0% 44.4% 62.5% 6.00 / 6.25
2 8 71.4% 71.4% 66.7% 2.24 2.15 2.05
2 16 100% 100% 100% 2.00 2.67 2.06
4 4 77.8% 87.5% 87.5% 1.30 1.53 1.31
4 8 100% 100% 100% 1.45 1.40 1.33
4 16 100% 100% 100% 1.30 1.37 1.24

Note: P SBP, PDBP, and PMBP were the percentages of the effective decrease (≥15 mmHg) in SBP, DBP and MBP respectively by amlodipine, telmisartan
and their combinations. The values of q > 1.15 represents synergistic; q < 0.85 represents antagonistic.

4  |   D I S C U S S I O N telmisartan or candesartan are also quite different. They might have

synergistic effect on blood pressure reduction.
Amlodipine is a calcium channel blocker (CCB).16 Telmisartan It's difficult to determine the synergistic interaction in prac-
and candesartan belong to angiotensin II type 1 (AT (1)) receptor tice for antihypertensive drugs, especially in vivo. For anti-­c ancer
blocker (ARB).17 The action mechanisms between amlodipine and drugs, there are some new methods to evaluate the synergism of
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10 of 12       XIA et al.

TA B L E 2  The probability sum test in rats administrated with amlodipine and candesartan or their combinations.

Amlodipine Candesartan
(mg/kg) (mg/kg) PSBP PDBP PMBP q SBP q DBP q MBP

0 1 0% 0% 0%
0 2 10.0% 22.2% 11.1%
0 4 12.5% 37.5% 14.3%
0.5 0 0% 0% 0%
1 0 22.2% 11.1% 0%
2 0 12.5% 0% 10.0%
0.5 1 50.0% 60.0% 60.0% / / /
0.5 2 0% 11.1% 11.1% / 0.50 1.00
0.5 4 77.8% 100% 100% 6.22 2.67 7.00
1 1 25.0% 37.5% 33.3% 1.13 3.38 /
1 2 33.3% 44.4% 40.0% 1.11 1.44 3.60
1 4 37.5% 70.0% 62.5% 1.17 1.58 4.38
2 1 87.5% 100% 88.9% 7.00 / 8.89
2 2 44.4% 62.5% 55.6% 2.09 2.81 2.78
2 4 80.0% 80.0% 80.0% 3.41 2.13 3.50

Note: PSBP, PDBP, and PMBP were the percentages of the effective decrease (≥15 mmHg) in SBP, DBP and MBP respectively by amlodipine, candesartan
and their combinations. The values of q > 1.15 represents synergistic; q < 0.85 represents antagonistic.

drugs combination.18–­21 For example, the Combenefit is a new
software tool. It uses classical Synergy models and enables the
visualization and analysis of drug combination effects in cancer
cells model.18 DeepSynergy is an accurate tool for synergy anal-
ysis. DeepSynergy learns to distinguish different cancer cell lines
and find certain drugs combinations. The results will be output
19
in visual two-­dimensional graph. Multidimensional Synergy of
Combinations (MuSyC) is a formalism based on a generalized, multi-­
dimensional Hill-­e quation. It will decouple synergistic potency and
efficacy. MuSyC is also mainly used in calculating the synergy be-
tween anti-­c ancer medicines. The results can be presented in co-
ordinate system. 20 The Bivariate Response to Additive Interacting
Doses (BRAID) model is designed to maintain a balance between
versatility and simplicity for anti-­c ancer medicines. Through eight
parameter model in cancer cells, BRAID quantifies the synergy and
makes it visual. 21 More evidence is needed to clarify whether these
methods can be used for in vivo research in other diseases, includ-
ing hypertension.
SynergyFinder 3.0 might be an answer. SynergyFinder was de-
signed to calculate the synergism between anti-­cancer medicines
firstly. It has been widely used for the discovery of novel synergis-
4,5
tic drug combinations and updated to version 3.0. It's designed
to enable unbiased identification of synergistic combinations from
high-­throughput experimental data. It shows the highest synergy
among all the combinations and searches further development and
testing toward safe and effective treatment options. It also can be
used in many precision medicine areas, targeted drug combination
discovery.6 Recently, we reported it can be used for anti-­platelet ag-
gregation in vitro.7
In this study, we selected two different couples of antihyper-
tensive drugs (amlodipine plus telmisartan or amlodipine plus can-
desartan) as an example, and calculated the synergistic effect on
blood pressure reduction by SynergyFinder 3.0 in vivo. The inhibi-
tion ratio of blood pressure reduction was used for SynergyFinder
3.0 system. For hypertensive patients, a 30% reduction in blood
pressure is most appropriate in clinical. So we defined a 30% re-
duction in blood pressure as 100% inhibition. The results output
by SynergyFinder 3.0 are more credible. The results are consis-
tent with the probability sum test both in the combination of am-
lodipine and telmisartan or amlodipine and candesartan (effective
reduction of blood pressure defined as ≥15 mmHg). In the combi-
nation groups of amlodipine and telmisartan, the q values in two
doses groups (2 + 4 and 1 + 4  mg/kg) are larger than any other
group. The synergy scores by SynergyFinder 3.0 are also highest
in these two groups. The two combinations groups of amlodip-
ine and candesartan (0.5 + 4 and 2 + 1 mg/kg) also get the similar
results.
The probability sum test also has been widely used to ana-
lyze the synergism of the combinations of two antihypertensive
drugs. 8–­13 The synergistic or antagonistic effect is mainly defined
by the q value (PA+B/(PA + PB − PA × PB)). However, there are some
disadvantages for this method compared with SynergyFinder 3.0.
First, the definition of responders for blood pressure reduction.
We usually define a decrease in blood pressure >15 or 20 mmHg
as responders. 2,10 P is the percentage of responders in each
group. When the reduction percentage is equal to zero, it will
have a great impact on the q values, which might cause a big dif-
ference between different responders. Second, the elimination

XIA et al.
of some data might affect the accuracy of q values. In this ex-
periment, a reduction in blood pressure >15 mmHg is defined as
responders. A decrease <12 mmHg (80% of 15 mmHg) is defined
as non-­r esponders and data ranging from 12 to 15 mmHg are dis-
carded, which might affect the synergistic conclusion.
In SynergyFinder 3.0, all the data are input to the system and
are reflected in synergy pictures and scores. SynergyFinder 3.0
combines multiple synergy scoring models and obtains a novel con-
sensus synergy score. During the data processing, the false positive
results will be removed with an improved outlier detection function-
ality.5 Hence, individual extreme data will not have much influences
on synergy scores. Different with the probability sum test, the syn-
ergy scores of SBP, DBP, and MBP by SynergyFinder 3.0 are high
consistent.
In conclusion, SynergyFinder 3.0 is more stable and reliable to
analyze the synergism on blood pressure reduction in vivo than
the probability sum test. There is an obviously synergistic interac-
tion between amlodipine & telmisartan or candesartan. The com-
binations of amlodipine & telmisartan (2 + 4 and 1 + 4  mg/kg) and
amlodipine & candesartan (0.5 + 4 and 2 + 1 mg/kg) might exert an
optimum synergistic effect against hypertension.
AU T H O R C O N T R I B U T I O N S
Conceived the idea: Ai-­Jun Liu. Designed the experiments: Ai-­Jun Liu
and Yan-­Qiong Cheng. Performed the experiments: Tian Xia, Lu-­Lu
Xu, and Wan-­Ting Shi. Analyzed the data: Tian Xia, Lu-­Lu Xu, Yan-­
Qiong Cheng, and Ai-­Jun Liu. Wrote the paper: Ai-­Jun Liu and Tian
Xia.
AC K N OW L E D G M E N T S
This work was supported by the National Key Research and
Development Program of China (No. 2019YFC1708504), the
National Natural Science Foundation of China (No. 82273984),
the Fund of Yueyang Hospital of Integrated Traditional Chinese
and Western Medicine (2021yygm01), the Traditional Chinese
Medicine-­clinical evaluation platform of Chinese patent medicine
(A1-­U21-­205-­9 02) and the Fund of Non drug therapy clinical re-
search center.
C O N FL I C T O F I N T E R E S T S TAT E M E N T
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
DATA AVA I L A B I L I T Y S TAT E M E N T
Available per request.
E T H I C S S TAT E M E N T
All experiments were approved by the Committee on ethics of bio-
medicine of Naval Medical University and were conducted accord-
ing to the National Institutes of Health Guidelines for the Care and
Use of Laboratory Animals.
|
20521707, 2023, 2, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1002/prp2.1064 by Cochrane Peru, Wiley Online Library on [09/04/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
      11 of 12
ORCID
Tian Xia  https://orcid.org/0000-0001-9971-0122
Lu-­Lu Xu  https://orcid.org/0000-0001-7879-6908
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Pharmacol Res Perspect. 2023;11:e01064. doi:10.1002/
S U P P O R T I N G I N FO R M AT I O N
prp2.1064
Additional supporting information can be found online in the
Supporting Information section at the end of this article.