European Journal of Pharmacology 928 (2022) 175083

Contents lists available at ScienceDirect

European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar

Investigation of drugs for the prevention of doxorubicin-induced cardiac
events using big data analysis
Shiori Nishiuchi a, b, 1, Kenta Yagi c, 1, Hiroumi Saito a, Yoshito Zamami a, d, **, Takahiro Niimura c,
Koji Miyata a, Yoshika Sakamoto a, Kimiko Fukunaga a, Shunsuke Ishida b, Hirofumi Hamano a, d,
Fuka Aizawa b, Mitsuhiro Goda a, b, *, Masayuki Chuma e, Yuki Izawa-Ishizawa f, Hideki Nawa g,
Hiroaki Yanagawa c, Yasunari Kanda h, Keisuke Ishizawa a, b, ca

Department of Clinical Pharmacology and Therapeutics, Graduate School of Biomedical Sciences, Tokushima University, Tokushima, Japan
Department of Pharmacy, Tokushima University Hospital, Tokushima, Japanc
Clinical Research Centre for Developmental Therapeutics, Tokushima University Hospital, Tokushima, Japand
Department of Pharmacy, Okayama University Hospital, Okayama, Japane
Department of Hospital Pharmacy and Pharmacology, Asahikawa Medical University, Asahikawa, Japanf
Department of Pharmacology, Graduate School of Biomedical Sciences, Tokushima University, Tokushima, Japang
Department of Pharmacy, Faculty of Pharmacy, Shujitsu University, Okayama, JapanhDivision of Pharmacology, National Institute of Health Sciences, Kanagawa, Japanb

A R T I C L E I N F O

A B S T R A C T

Keywords:
Drug–drug interaction
Cardiology
Data analysis
Chemotherapy
Doxorubicin

Aim: Doxorubicin, an anthracycline anti-tumour agent, is an essential chemotherapeutic drug; however, theadverse events associated with doxorubicin usage, including cardiotoxicity, prevent patients from continuing
treatment. Here, we used databases to explore existing approved drugs with potential preventative effects against
doxorubicin-induced cardiac events and examined their efficacy and mechanisms.
Methods: The Gene Expression Omnibus (GEO), Library of Integrated Network-based Cellular Signatures (LINCS),
and Food and Drug Administration Adverse Events Reporting System (FAERS) databases were used to extract
candidate prophylactic drugs. Mouse models of doxorubicin-induced cardiac events were generated by intra­
peritoneal administration of 20 mg/kg of doxorubicin on Day 1 and oral administration of prophylactic candi­date drugs for 6 consecutive days beginning the day before doxorubicin administration. On Day 6, mouse hearts
were extracted and examined for mRNA expression of apoptosis-related genes.
Results: GEO analysis showed that doxorubicin administration upregulated 490 genes and downregulated 862
genes, and LINCS data identified sirolimus, verapamil, minoxidil, prednisolone, guanabenz, and mosapride as
drugs capable of counteracting these genetic alterations. Examination of the effects of these drugs on cardiac
toxicity using FAERS identified sirolimus and mosapride as new prophylactic drug candidates. In model mice,
mosapride and sirolimus suppressed the Bax/Bcl-2 mRNA ratio, which is elevated in doxorubicin-induced car­diotoxicity. These drugs also suppressed the expression of inflammatory cytokines Il1b and Il6 and markers
associated with myocardial fibrosis, including Lgal3 and Timp1.
Conclusion: These findings suggest that doxorubicin-induced cardiac events are suppressed by the administrationof mosapride and sirolimus.

* Corresponding author. Department of Clinical Pharmacology and Therapeutics, Graduate School of Biomedical Sciences, Tokushima University, Tokushima,
Japan.
** Corresponding author. Department of Clinical Pharmacology and Therapeutics, Graduate School of Biomedical Sciences, Tokushima University, Tokushima,
Japan.
E-mail addresses: zamami@tokushima-u.ac.jp (Y. Zamami), mgoda@tokushima-u.ac.jp (M. Goda).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejphar.2022.175083
Received 8 November 2021; Received in revised form 29 May 2022; Accepted 30 May 2022
Available online 31 May 2022
0014-2999/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

S. Nishiuchi et al.

European Journal of Pharmacology 928 (2022) 175083

1. Introduction

drugs with potential preventative effects against doxorubicin-induced
cardiac events and examine their efficacies and associated mechanisms.

Doxorubicin, an anthracycline anti-tumour drug, exerts its effect byinserting into the DNA base pairs of tumour cells and inhibiting DNA
polymerase, RNA polymerase, and topoisomerase II reactions, thereby
suppressing DNA and RNA biosynthesis. The efficacy of doxorubicin has
been demonstrated in several cancer types, including lung, gastroin­
testinal, breast, and bladder cancers, as well as malignant lymphoma
and osteosarcoma (Damiani et al., 2016), resulting in its consideration
as an essential chemotherapeutic agent. Although doxorubicin exerts astrong anti-tumour effect, it is also associated with the development ofcardiotoxicity, which prevents patients from continuing treatment(Koleini and Kardami, 2017).
Doxorubicin cardiotoxicity includes acute and chronic disorders.
Chronic myocardial injury is known to occur in a cumulative, dosedependent manner (McGowan et al., 2017) and can lead to heart fail­ure, with a 3-year survival rate of approximately 50% in the absence oftreatment (Felker, 2000). Doxorubicin-induced chronic myocardial
injury is addressed in real-world clinical practice by dose restriction.
However, acute myocardial injury does not correlate with dosage and isreported to occur within a few days of doxorubicin administration,
resulting in transient ventricular hypofunction and other symptoms. No
prophylaxis for doxorubicin-induced acute myocardial injury has beendeveloped to date. The underlying mechanism of doxorubicin-induced
myocardial injury is probably associated with oxidative stress due tofree radical production, ferotosis, apoptosis owing to mitochondrial
damage in cardiomyocytes, and DNA damage due to topoisomerase II
inhibition (Mustafa et al., 2017; Songbo et al., 2019; Sheibani et al.,2020, 2021; Tadokoro et al., 2020). The effects of dexrazoxane on
doxorubicin-induced cardiotoxicity and doxorubicin-specific anti-­
tumour effects are controversial, according to various studies(Reichardt
et al., 2018; Lipshultz et al., 2014; Ghasemi et al., 2021; Chow et al.,2022). In clinical trials conducted in the United States, an increased riskof acute myeloid leukemia and myelodysplastic syndrome was reported
in paediatric patients and adolescents treated with dexrazoxane toprevent cardiomyopathy caused by long-term administration ofanthracyclines (Tebbi et al., 2007; Salzer et al., 2010; Schwartz et al.,2009; Swain et al., 1997). The Committee for Medicinal Products for
Human Use (CHMP) reviewed this report and concluded in 2011 that theuse of dexrazoxane to prevent cardiomyopathy in patients younger than
18 years of age was not warranted. It was recommended that the use ofdexrazoxane be contraindicated (EMA/491205/2011).
Recently, a new strategy was proposed for drug discovery called drug
repositioning, where existing approved drugs are investigated foralternate pharmacological effects, thereby exploiting their potential totreat other diseases (Ashburn and Thor, 2004). The construction oflarge-scale medical information databases providing information on
altered gene expression and side effects has made it possible to suc­cessfully repurpose drugs.
The Food and Drug Administration (FDA) Adverse Events Reporting
System (FAERS) is the largest database containing reports of sponta­
neous adverse events from medical professionals and companies in the
United States and abroad (Nagashima et al., 2016). The Gene Expression
Omnibus (GEO), maintained by the National Centre for Biotechnology
Information (NCBI), contains gene expression information that includes
microarray data of >3 million samples (Edgar et al., 2002). The Libraryof Integrated Network-based Cellular Signatures (LINCS) program is adrug-discovery tool that simulates changes in the expression of ~20,000
genes in response to chemical compounds in human cell lines and has
been used to study the development of novel therapeutic agents (Keenan
et al., 2018; Duan et al., 2016). These databases have been employed inmany studies, including those performed by our research team, toidentify drugs capable of preventing adverse events associated withhigh-risk drugs (Zamami et al., 2019a; Izawa-Ishizawa et al., 2019;
Okada et al., 2019).
In this study, we used these databases to explore existing approved

2. Material and methods
2.1. Doxorubicin-induced changes in gene expression
The microarray dataset associated with doxorubicin-induced car­diotoxicity was obtained from the NCBI GEO database (http://www.
ncbi.nlm.nih.gov/geo/; accession no. GSE23598). The dataset
comprised mouse heart mRNA samples collected 4 days after adminis­
tration of 15 mg/kg doxorubicin or an equivalent amount of saline. Two
pre-processing methods were used to process the microarray data. The
robust multi-array average (RMA) method is a quantile normalisation
method that sorts the data of each sample by signal intensity and cor­
rects it to the same value if it is of the same rank in each sample. The
MAS5 method is a global normalisation protocol that corrects eachsample to ensure that all signal averages are the same.
The statistical analysis programming language R (https://www.r-pro
ject.org/) along with its packages “affy” and “RobLoxBioC” was used foreach process. We identified a group of genes with significantly alteredexpression ratios in the doxorubicin-treated group relative to the salinetreated group.
2.2. Extraction of drugs to counteract changes in gene expression
For the identified gene groups, we extracted drugs that counteract
the doxorubicin-induced changes in gene expression using the LINCS
search engine L1000CDS2. A group of genes upregulated by doxorubicin
was entered into L1000CDS2 as “up genes”, and a downregulated group
was entered as “down genes”. We then searched for compounds thatcountered the altered gene expression and selected the top 50 FDAapproved drugs as prophylactic candidate drugs.
2.3. Targeting prophylactic candidate drugs
The FAERS data for 7,738,415 spontaneous adverse events from Q1
2007 through Q1 2017 were downloaded from the FDA website (https://www.fda.gov/Drugs/GuidanceCompliance -RegulatoryInformation/
Surveillance/AdverseDrugEffects/ucm082193.htm). Duplicate data
were excluded according to the methodology recommended by the FDA,
and the remaining 6,994,117 events were used for analysis. In the
FAERS database, the names of drugs were standardised to genericnames, as this database allows drugs to be registered under any name,including trade names and abbreviations.
Adverse events were designated according to the 93 extracted terms
from the “10019280/Heart failures/Heart failure” group, which is basedon the Medical Dictionary for Regulatory Activities/J (v.21.0; Interna­
tional Glossary of Pharmaceutical Terms of International Council for
Harmonization of Technical Requirements for Pharmaceuticals for
Human Use) (Table S1).
The risk of the reported adverse events was evaluated by calculating
the 95% confidence interval (CI) and reporting odds ratios (RORs) ac­
cording to previously reported methods (Nagashima et al., 2016; Liet al., 2008). Doxorubicin-treated patients from the FAERS databasewere then classified into four groups: patients who (A) received thecandidate drug and reported heart failure as an adverse effect, (B)
received the candidate drug and did not report heart failure as anadverse effect, (C) did not receive the candidate drug and reported heart
failure as an adverse effect, and (D) did not receive the candidate drug
and did not report heart failure as an adverse effect. The number ofreports for each group was calculated based on these classifications,after which ROR and 95% CIs were calculated according to Eq. (1):
ROR = (A/B)/(C/D); 95% CI = exp [log(ROR) ± 1.96 square 1/A + 1/B + 1/
C + 1/D](1)
2

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European Journal of Pharmacology 928 (2022) 175083

where A, B, C, and D represent the number of reports in each group, andlog represents the natural logarithm. We defined drugs with a ROR > 1
as those that increased the risk of adverse effects, whereas drugs with a
ROR < 1 were considered to decrease the risk of adverse effects anddefined as prophylactic candidate drugs.

variance, followed by the Tukey test. Categorical variables werecompared using Fisher’s exact test. A p < 0.05 was consideredsignificant.

2.4. Animals

3.1. Identification of agents capable of preventing doxorubicin-induced
cardiotoxicity

3. Results

Twenty-four male C57BL/6J mice (aged 8 weeks) were purchasedfrom Nippon CLEA (Tokyo, Japan). The mice were housed and accli­
matised for 1 week before being placed at a constant temperature(23–24 ◦ C) with a 12-h light/dark cycle (light: 8 A.M. to 8 P.M.). Food andwater were provided ad libitum. All experimental procedures performedon the mice were in accordance with the guidelines of the Animal
Research Committee of Tokushima University Graduate School, and theprotocol was approved by the Institutional Review Board of Tokushima
University Graduate School for animal protection (permit no. T30-85).

First, we extracted genes showing altered expression following
doxorubicin administration. Following normalisation of data from the
GEO database (using the RMA method), doxorubicin treatment was
found to upregulate the expression of 1259 genes and downregulate thatof 1158 genes. Similarly, data normalised by the MAS5 method showedthat doxorubicin treatment upregulated 490 genes and downregulated
862 genes.
Next, the L1000CDS2 search engine identified six approved drugs
that counteracted doxorubicin-altered gene expression. Specifically,sirolimus was identified in the RMA-normalised gene groups, whereas
verapamil, minoxidil, prednisolone, guanabenz, and mosapride weredetected in the MAS5-normalised gene groups.
FAERS was subsequently used to examine the effects of these six
drugs on the prevention of doxorubicin-induced cardiotoxicity. From the
6,994,117 reports obtained following the exclusion of duplicate data,
36,329 patients using doxorubicin were included in the analysis. No
reports on the concomitant use of guanabenz with doxorubicin wereavailable. The RORs for sirolimus, mosapride, prednisolone, verapamil,and minoxidil were 0.32, 0.36, 0.65, 1.36, and 6.95, respectively(Table 1). However, the preventative effect exerted by prednisolone ismoderate (Aguilar et al., 2014). Therefore, sirolimus and mosapridewere identified as novel preventive drug candidates.

2.5. Doxorubicin-induced myocardial flame model mice and drug
administration
Mouse models of doxorubicin-induced cardiac disease were prepared
as previously described (Zhao et al., 2018a, 2018b; Gupta et al., 2018;
Yuan et al., 2018). Male C57BL/6J mice (8-weeks old) were randomised
into the following groups: vehicle (n = 8), doxorubicin-treated (n = 5),
mosapride (3 mg/kg) or sirolimus (2 mg/kg) only (n = 6/group), anddoxorubicin treatment plus mosapride (doxorubicin + mosapride) or
sirolimus (doxorubicin + sirolimus) (n = 5/group). The mice wereintraperitoneally administered a single dose of doxorubicin (20 mg/kg;
Funakoshi Co., Ltd., Tokyo, Japan) dissolved in saline. Vehicle-treatedmice were administered only saline. Mosapride (3 mg/kg; Wako,
Osaka, Japan) and sirolimus (2 mg/kg; Funakoshi Co., Ltd.) were dis­solved in 0.5% carboxymethyl cellulose, and each drug was orally
administered to mice once daily for 6 consecutive days beginning 1 day
before doxorubicin treatment. The method of administration and dosage
concentrations of mosapride and sirolimus were determined based on
previously reported methods used in animal experiments. After 5 days ofdoxorubicin administration, experimental mice were anaesthetised, andtheir hearts were collected. Anaesthesia was induced by inhalation of
isoflurane under a 4% diluted vaporiser setting (vaporised in oxygen, 1
L/min) and maintained with isoflurane under a 2% vaporiser setting.
The isoflurane was delivered via a small face mask.

3.2. Evaluation of cardiac effects elicited by doxorubicin and candidate
drug treatment
The contribution of candidate drugs to the protection of cardiac
function was examined in a mouse model of doxorubicin-induced car­diac events. The body weight of each group on Day 6 was 23.06 ± 0.49 g
for the vehicle group, 24.42 ± 0.79 g for the mosapride only group,
23.42 ± 0.22 g for the sirolimus only group, 17.60 ± 0.26 g for the
doxorubicin only group, 18.30 ± 0.50 g for the doxorubicin +mosapride group, and 18.70 ± 0.39 g for the doxorubicin + sirolimus
group. Body weights in the vehicle, mosapride only, and sirolimus only
groups were unchanged compared with those on Day 1. From the day

2.6. Quantitative reverse-transcription polymerase chain reaction (qRTPCR)

Table 1
Effect of concomitant medications on the rate of reported doxorubicin-induced
heart failure.

The collected cardiac apex tissue was placed in ISOGEN II (Nippon
Gene, Tokyo, Japan) and homogenised on ice, followed by extraction of
total RNA according to the manufacturer’s instructions. The extracted
RNA concentration was measured using a Nanodrop 1000 (Thermo
Fisher Scientific, Waltham, MA, USA), and cDNA solutions were pre­pared using a PrimeScript RT reagent kit (Takara Bio, Shiga, Japan).
Each reaction was performed using a TaKaRa PCR thermal cycler(Dice; Takara Bio). qRT-PCR was performed using Thunderbird SYBRqPCR mix (Toyobo, Osaka, Japan) and a StepOnePlus system (Applied
Biosystems, Waltham, MA, USA) with the following cycling conditions:
one cycle at 95 ◦ C, followed by 40 cycles of 15 s at 95 ◦ C and 35 s at
60 ◦ C. The expression level of each gene was determined using mouse
β-actin gene as the endogenous standard gene. All data were analysedusing CFX Manager software (Bio-Rad, Hercules, CA, USA).

Concomitantwith Drug B

Without
Drug B (%)

With
Drug B(%)

RORa

95% CIbp

Sirolimus

2092/
36221
(5.78)
2093/
36281(5.77)
1912/
31666(6.04)
2090/
36278(5.76)
2092/
36324(5.76)

2/108(1.85)

0.32

0.04–1.19

0.13

1/48
(2.08)

0.36

0.01–2.11

0.52

182/
4663
(3.90)
4/51(7.84)

0.65

0.55–0.76<0.0001

1.36

0.36–3.71

0.54

2/5 (40)

6.95

0.66–42.46

0.05

Mosapride
Prednisolone
Verapamil

2.7. Statistical analysis

Minoxidil

All experiments were independently repeated a minimum of threetimes, and the resulting data were expressed as the mean ± standard
error of the mean (SEM). Data were analysed using a two-way analysis ofab3

ROR: reporting odds ratio.
CI: confidence interval.

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European Journal of Pharmacology 928 (2022) 175083

after doxorubicin treatment (Day 2), body weights were reduced in the
doxorubicin only, doxorubicin + mosapride, and doxorubicin + siroli­
mus groups, with a significant reduction observed after 5 days ofdoxorubicin treatment. Although combining doxorubicin with theseprophylactic agents did not significantly inhibit weight loss, reduced
weight loss was observed compared with that in the doxorubicin only
group (Fig. 1).
3.3. Changes in apoptosis-related gene expression by doxorubicin andcandidate drug treatment
We then evaluated the effects of doxorubicin and candidate drug
administration on B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated Xprotein (Bax) expression in the mouse heart. The Bax/Bcl-2 mRNA
expression ratio was 1.00 ± 0.12 for the vehicle group, 0.75 ± 0.07 forthe mosapride only group, 0.96 ± 0.08 for the sirolimus only group,
2.87 ± 0.71 for the doxorubicin only group, 1.37 ± 0.22 for the doxo­
rubicin + mosapride group, and 1.53 ± 0.17 for the doxorubicin +sirolimus group. Although doxorubicin treatment significantly increased
the Bax/Bcl-2 mRNA ratio, a trend towards the reduction of the ratio was
observed in the doxorubicin + mosapride and doxorubicin + sirolimus
groups (Fig. 2).
We then evaluated changes in the expression of inflammatory
markers associated with cardiac fibrosis following doxorubicin andcandidate drug treatment. Quantification of the expression of inflam­
matory cytokines in cardiomyocytes revealed no increases ininterleukin-1b (Il1b) mRNA expression in the mosapride only and siro­
limus only groups compared with that in the vehicle group, although adecreasing trend in Il6 mRNA expression was observed in these groups
compared with that in the vehicle group. Additionally, we found that
doxorubicin treatment significantly increased Il1b and Il6 expression;
however, combined treatment with mosapride or sirolimus resulted in adecreasing trend of Il1b expression (Fig. 3A) and significant suppressionof Il6 expression (Fig. 3B).
Furthermore, we evaluated mRNA levels of Lgal3 (encoding galectin3) and tissue inhibitor of metalloproteinase (Timp)1 as biomarkers ofcardiac remodelling. We found no increases in Lgal3 or Timp1 mRNAlevels in the mosapride and sirolimus groups compared with those in thevehicle group, but the expression of both was significantly increased bydoxorubicin treatment. Moreover, their expression was significantly
suppressed by combined treatment with either mosapride or sirolimus
compared with that observed in the doxorubicin alone group (Fig. 4A

Fig. 2. Expression of apoptosis-related genes in the heart tissue of mousemodels of doxorubicin-induced cardiotoxicity. Ratio of Bax/Bcl-2 mRNAlevels. mRNA levels were adjusted to those in the vehicle group, which were set
to 1. Error bars represent the mean ± standard error of the mean (SEM; n =5–8). *p < 0.05 vs. vehicle; #p < 0.05 vs. the doxorubicin-treated group.

and B).
4. Discussion
In this study, we searched for candidate drugs that reduced
doxorubicin-induced cardiotoxicity by analysing three medical infor­
mation databases (GEO, LINCS, and FAERS) and determining the effi­cacy and mechanism of action of these candidates.
Doxorubicin-specific adverse effects, including cardiotoxicity, causes
significant clinical challenges by interfering with the continuation oftreatment (Koleini and Kardami, 2017). Using GEO, we found that
doxorubicin alters the expression of genes associated with inflammatory
responses and apoptosis, which are involved in the mechanism of car­diotoxicity (Yu et al., 2020; Tacar et al., 2013). Therefore, we used
LINCS to search for drugs capable of suppressing changes in the gene
expression induced by doxorubicin, which resulted in the identificationof six drugs (verapamil, minoxidil, prednisolone, guanabenz, mosapride,and sirolimus).
FAERS was then used to compare the frequency of doxorubicininduced cardiac events in patients administered with doxorubicin,
with and without the use of candidate drugs. Prednisolone, mosapride,and sirolimus achieved an ROR < 1, suggesting that they reduced thedevelopment of doxorubicin-induced heart disease (Table 1). Prednis­
olone, a steroid, should not be administered indiscriminately for an
extended period, although a study has shown its efficacy against car­diotoxicity (Aguilar et al., 2014). Therefore, we chose mosapride andsirolimus as candidate drugs for further analysis. Although the multiple
databases helped identify drugs capable of preventing the developmentof adverse reactions and predict mechanisms associated with drug
combinations, this strategy does not allow the elucidation of the true
effects of drug combinations. Therefore, we investigated the associatedmechanisms using a mouse model of doxorubicin-induced cardiac
events. Since epidemiological studies and clinical reports suggest thatmyocardial damage is generally more common in males than in females(Kytö et al., 2013; Zamami et al., 2019b), this study was conducted onmale mice. Since FAERS does not provide information on the number ofdoses, to evaluate the effects of cumulative toxicity is challenging.
Therefore, when trying to obtain results linked to FAERS in animal ex­periments, it is appropriate to consider a single-dose acute toxicity
model. Specifically, using a model of acute cardiotoxicity induced by asingle 15 mg/kg dose of doxorubicin (Yuan et al., 2018; Jiao et al.,2022), we examined the effects of doxorubicin and prophylactic drug

Fig. 1. Changes in body weight in the mouse model of doxorubicininduced cardiotoxicity. Changes in body weight of 8-week-old male C57BL/
6J wild-type mice treated with doxorubicin alone or in combination withmosapride or sirolimus for 6 days. Error bars represent the mean ± standard
error of the mean (SEM; n = 5–8). **p < 0.01 vs. vehicle.
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European Journal of Pharmacology 928 (2022) 175083

Fig. 3. Inflammatory cytokine gene expression in the heart tissue of mouse models of doxorubicin-induced cardiotoxicity. (A) Il1b and (B) Il6 mRNA levels.
mRNA levels were adjusted to those of the vehicle group, which were set to 1. Error bars represent the mean ± standard error of the mean (SEM; n = 5–8). *p < 0.05;**p < 0.01 vs. vehicle; #p < 0.05; ##p < 0.01 vs. the doxorubicin-treated group.

Fig. 4. mRNA levels of cardiac fibrosis-related markers in the heart tissue of moue models of doxorubicin-induced cardiotoxicity. (A) Lgal3 and (B) Timp1
levels. mRNA levels were adjusted to those of the vehicle group, which were set to 1. Error bars represent the mean ± standard error of the mean (SEM; n = 5–8). *p< 0.05; **p < 0.01 vs. vehicle; #p < 0.05; ##p < 0.01 vs. the doxorubicin-treated group.

candidates, focusing on body weight changes, apoptosis and ferotosis indoxorubicin-treated mice. Previous reports have shown that apoptosis
involving Bax/Bcl and ferotosis involving Gpx4 (glutathione peroxidase4) are involved in doxorubicin-induced cardiotoxicity as a pathway ofcell death. The combination of prophylactic drug candidates withdoxorubicin treatment tended to reduce weight loss compared withdoxorubicin treatment alone (Fig. 1). In addition, the expression of
apoptosis- and ferotosis-related genes was investigated: Bax promotes
apoptosis in the mitochondrial apoptosis pathway by increasing thepermeability of the mitochondrial outer membrane, whereas Bcl-2 in­hibits apoptosis by preventing the influx of apoptosis-inducing proteins
into the cytoplasm (Aguilar et al., 2014; Adams and Cory, 1998; Oltvai
et al., 1993). Gpx4 is also known to be involved in ferotosis (Tadokoro
et al., 2020). We found that doxorubicin treatment significantly
increased the Bax/Bcl-2 mRNA expression ratio in cardiomyocytes ofdoxorubicin-induced heart failure mice, but this effect was suppressedby the co-administration of mosapride or sirolimus (Fig. 2). On the other
hand, no difference in Gpx4 mRNA was observed with prophylactic
treatment (data not shown). Thus, we suggest that mosapride and siro­
limus may inhibit doxorubicin-induced myocardial apoptosis andreduce doxorubicin-induced cardiotoxicity.
Next, we evaluated inflammatory cytokines and markers associatedwith cardiac fibrosis. Inflammatory cytokines are implicated in thedevelopment of myocardial infarction, with high serum levels of IL-1b
and IL-6 that are associated with poor prognosis(Tsutamoto et al.,
1998; Zhu et al., 2010, 2011). Additionally, other studies have shown
the involvement of the inflammatory response in doxorubicin-induced
cardiotoxicity (Sheibani et al., 2020, 2021). In the present study, we
found that doxorubicin-induced elevations in Il6 expression weresignificantly suppressed by combination treatment of doxorubicin withmosapride or sirolimus; a trend towards suppression of Il1b, a proin­
flammatory cytokine that promotes cardiac remodelling (Bujak et al.,2008), expression was also observed by combination treatment (Fig. 3).

Similarly, we identified significant doxorubicin-induced upregulation of
Lgal3 and Timp1 expression as biomarkers of cardiac remodelling, withsubsequent significant suppression in their levels observed by combi­
nation treatment with mosapride or sirolimus (Fig. 4). These results
suggest that treatment with mosapride or sirolimus improves prognosis
after the onset of cardiac disease. In doxorubicin-induced cardiomyop­
athy, the inflammatory response causes fibrosis, which leads to cardiac
dysfunction (Tanaka et al., 2020), and it is suggested that the inflam­
matoryresponseandsuppressionoffibrosis
attenuate
doxorubicin-induced cardiomyopathy.
This study has certain limitations. First, the gene-expression data
recorded in LINCS is based on data from cell lines. However, we eval­uated the efficacy of LINCS data against the reported rate ofdoxorubicin-induced cardiac events using FAERS rather than deter­mining the candidate drugs by LINCS alone. In fact, the results of FAERS
analysis showed that verapamil and minoxidil (extracted from LINCS)
were ineffective against doxorubicin-induced cardiac events, resulting
in their exclusion as candidate drugs. Second, FAERS data report spon­taneous adverse events; however, all patients who have used doxoru­
bicin or concomitant medications are not included, because those whohave not experienced adverse events are not registered. Moreover, somepatients who experienced adverse events are not reported. Therefore,
the rate of doxorubicin-induced heart failure calculated by FAERS
analysis might differ from that observed in the clinic. Third, the effect ofany confounding factors was not applied to FAERS analysis. Although
the ROR was calculated based on the number of reports on the use ofconcomitant medications, we did not adjust for age, gender, or medical
history. A high percentage of patients with heart failure has high bloodpressure (Adams et al., 2005), with the average age of these patients at
70 years (Nieminen et al., 2006). We were unable to evaluate themedical history of patients, as this information is not provided in FAERS.
Owing to these limitations, the results of the FAERS review do not reflect
real-world patients. Nevertheless, considering the difficulty in collecting
5

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European Journal of Pharmacology 928 (2022) 175083

case reports of inconsistently occurring adverse effects, such as
doxorubicin-induced cardiac events, the results of FAERS analysis,
which incorporates a large amount of actual patient data, are effective ininvestigating the real-world clinical practice. Therefore, further evi­
dence, including multicentre and randomised clinical trials, is needed todetermine the possible effects of mosapride or sirolimus in preventing
cardiac events in patients receiving doxorubicin.
In the present study, a model of doxorubicin-induced acute car­diotoxicity was generated in male mice with a single dose of 15 mg/kg
doxorubicin (Yuan et al., 2018; Jiao et al., 2022). The current study
examined the prophylactic effects of mosapride and sirolimus on
doxorubicin-induced acute myocardial injury, but further studies areneeded to examine their effects under other conditions. Since sex dif­ferences in doxorubicin-induced cardiotoxicity have been reported, it
may be useful to perform studies using female mice. It may also be
worthwhile to examine the prophylactic effects of mosapride and siro­
limus using a model of chronic cardiotoxicity induced by multiple dosesof doxorubicin.
In conclusion, this study demonstrates that doxorubicin-induced
adverse cardiac events may be suppressed by the administration of thecandidate drugs mosapride and sirolimus.

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for signal detection on adverse drug reaction spontaneous reporting database ofFunding
This work was supported by a grant from the Japan Society for the
Promotion of Science Grants-in-Aid for Scientific Research [grant no.
18K06785], a Grant-in-Aid for Transformative Research Areas (B) [grantno. 20H05798], and the Japan Research Foundation for Clinical Phar­macology [grant no. 2018A10].
The funding sources had no involvement in the study design; in thecollection, analysis, and interpretation of data; in the writing of thereport; and in the decision to submit the article for publication.
CRediT authorship contribution statement
Shiori Nishiuchi: Methodology, Investigation, Writing – originaldraft. Kenta Yagi: Writing – original draft. Hiroumi Saito: Methodol­
ogy, Investigation, Software, Validation. Yoshito Zamami: Conceptu­
alization, Project administration. Takahiro Niimura: Methodology,
Software, Validation, Formal analysis. Koji Miyata: Software, Valida­tion. Yoshika Sakamoto: Methodology, Software, Validation. Kimiko
Fukunaga: Software, Validation. Shunsuke Ishida: Writing – review &editing. Hirofumi Hamano: Methodology, Software, Validation. Fuka
Aizawa: Methodology, Software, Validation. Mitsuhiro Goda:
Conceptualization, Formal analysis, Visualization, Project administra­tion. Masayuki Chuma: Writing – review & editing. Yuki Izawa-Ishi­
zawa: Writing – review & editing. Hideki Nawa: Writing – review &editing. Hiroaki Yanagawa: Writing – review & editing. Yasunari
Kanda: Writing – review & editing. Keisuke Ishizawa: Writing – review& editing, Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
We thank Editage (www.editage.com) for English language editing.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ejphar.2022.175083.
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European Journal of Pharmacology 928 (2022) 175083
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