Protection of Cardiovascular Function With Crocin in BrEast Cancer Patients Undergoing Radiotherapy and Chemotherapy
Phase: NA
Conditions: Breast Cancer
Sponsor: Qilu Hospital of Shandong University

Version Number：20200818
Version Date：2020/08/18
Protection of Cardiovascular Function with Crocin in
BrEast Cancer patients undergoing Radiotherapy
and Chemotherapy
（ProtECtion Study）
Project undertaker: Qilu Hospital of Shandong University
Researcher: Mei Zhang, Xiaoling Liu, Mengmeng Li, Yufeng Cheng,
Jianbo Wang, Xiuwen Wang, Lian Liu, Li Li, Yanguo Liu, Qifeng
Yang, Xiaoli Kong, Ming Lv, Xiaorong Yang,Wuyun Bao

Version Number：20200818
Version Date：2020/08/18
Research Protocol
BACKGROUND
Cardiovascular disease (CVD) and tumors lead to more than 2/3 of the world's
death population each year [1]. Because CVD and tumors often share common risk
factors, such as diabetes, they often coexist. At the same time, in the course of tumor
drug therapy, radiotherapy and related adjuvant therapy, different degrees of
cardiovascular function impairment can also occur [2]. In recent years, with the
continuous development of medical technology, great progress has been made in the
diagnosis and treatment of patients with malignant tumors, and the survival time has
been significantly prolonged. Subsequently, the potential cardiovascular toxicity of
tumor treatment and the resulting cardiovascular events have gradually become an
important health risk for tumor survivors, and the death caused by cardiovascular
events in tumor patients during the treatment process ranks first among all causes of
death [3].
Traditional chemotherapeutic drugs, newly emerging biological agents, and
cardiothoracic radiotherapy can all act on the cardiovascular system, causing
structural and functional damage to the corresponding organs. Traditional cytotoxic
drugs such as anthracyclines and their derivatives, antimetabolites, paclitaxels,
alkylating agents, platinums, and alkaloids can cause irreversible cardiovascular
damage with increasing cumulative doses [2]. Among them, doxorubicin and
5-fluorouracil (5-FU) are reported more. It has been confirmed that doxorubicin can
produce cardiotoxicity by inhibiting mitochondrial and sarcoplasmic reticulase
activities, causing myocardial cell necrosis or apoptosis, resulting in cardiac
insufficiency [4-6]; 5-FU can damage endothelial cell function, interfere with Smooth
muscle cell signal transduction triggers abnormal vasoactive responses that ultimately
lead to coronary spasm and myocardial ischemia. Biologics, including monoclonal
antibodies, tyrosine kinase inhibitors (TKIs), and endocrine agents have the same
potential for cardiovascular toxicity, especially vascular endothelial growth factor
(VEGF) inhibitors. VEGF inhibitors can increase the risk of heart failure, coronary

Version Number：20200818
Version Date：2020/08/18
heart disease, hypertension and thromboembolic diseases through mechanisms such as
endothelial damage, vasoconstriction and remodeling, inflammation and platelet
activation [7-9]. Different from cytotoxic drugs, cardiovascular injury caused by
biological agents can be partially or completely alleviated after timely intervention
(such as adding cardioprotective drugs, adjusting anti-tumor regimen, etc.) [10]. In
addition, the cardiovascular effects of radiotherapy are mainly due to radiation-related
injury. When the radiation acts on the tumor tissue, it will inevitably cause damage to
the normal tissue in and around the radiation field, resulting in radiotherapy-related
heart disease such as diffuse myocardial or pericardial fibrosis, pericarditis, coronary
artery disease and valvular disease. The incidence rate is as high as 10% to 30%
[11-13].
To the above mentioned problems, an emerging discipline--Cardio-Oncology,
came into being. Cardio-Oncology is a discipline that studies the development of
cardiovascular disease, risk assessment, diagnosis and treatment, and prognosis follow
-up in tumor patients, involves all aspects of tertiary prevention of cardiovascular
disease in cancer patients. Screening of malignant tumor patients with high
cardiovascular risk factors, and then intervening in their early stages, in order to
maximize the protection of patients' cardiac function, is undoubtedly important in the
secondary prevention of cardiovascular disease in cancer patients. Because of its
non-invasive, simple and real-time characteristics, echocardiography is widely used in
the follow-up of cardiac function in cancer patients. However, due to the special
pathophysiology of tumor patients, traditional conventional ultrasound indicators such
as two-dimensional left ventricular ejection fraction (2D LVEF), left ventricular
fractional shortening and tissue doppler imaging cannot sensitively diagnose early
cardiovascular damage [14]. In recent years, new ultrasound technology based on
speckle tracking imaging (STI) has shown superiority in the diagnosis, risk
stratification and prognosis evaluation of cardiovascular diseases related to tumor
therapy [15]. STI can automatically track the natural acoustic speckle in the
myocardial tissue , and calculate the motion trajectory between two points. Through
the subsequent processing of the recorded trajectory, it can provide information for

Version Number：20200818
Version Date：2020/08/18
evaluating the systolic and diastolic function of the whole and individual segments of
the myocardium. STI tracks myocardial motion well even with very small changes in
the position of the myocardium. STI has no Doppler angle dependence, can integrate
myocardial motion in longitudinal, radial, and circumferential directions, and has high
specificity and sensitivity in evaluating cardiac function [16]. STI-related parameters
mainly include: global longitudinal strain (GLS)/strain rate, global circumferential
strain, global radial strain, global area strain (3D-GAS), left ventricular torsion, left
ventricular rotation/ Untwisting speed, systolic dissynchrony index (SDI) and right
ventricular free wall systolic peak strain (RVFWS) and so on [17]. Among them, left
ventricular global longitudinal strain (GLS) has been proved to be a more sensitive
indicator of myocardial contractile function than LVEF. Studies have reported that
GLS decline in the early stage of chemotherapy has a strong predictive value for
patients with subsequent LVEF <50%. There is no literature report on the magnitude
of the effect of cardioprotective therapy on GLS in cancer patients treated with
chemoradiotherapy. A real-world study by our group used STI technology to monitor
the changes of cardiac function in breast cancer patients during chemotherapy. The
preliminary results showed that GLS in breast cancer patients was significantly lower
than the baseline level after 4 cycles of chemotherapy.
The prevention of cardiovascular events has become one of the bottlenecks in
improving the prognosis of cancer patients. However, because most classic clinical
studies in the cardiovascular field exclude cancer patients, the evidence-based
cardiology for first-line doctors is very limited. It is urgent to explore and develop
methods to effectively protect cardiovascular function. Crocus, also known as saffron,
is the dried stigma of the perennial Iridaceae plant, saffron, and is a precious Chinese
medicine. Crocin is a water-soluble carotene compound of saffron. It is a series of
ester glycosides composed of saffron acid and different sugars. It is one of the main
active components of saffron. It has various effects such as anti-oxidative stress,
anti-platelet aggregation, anti-thrombosis, and anti-myocardial ischemia [18]. Studies
have shown that crocin has good curative effect on cardiovascular diseases such as
hypertension,
hyperlipidemia
and
atherosclerosis,
and
also
has
anti-cancer,

Version Number：20200818
Version Date：2020/08/18
anti-inflammatory, anti-oxidative, hepatoprotective, choleretic and anti- diabetes
properties etc [19]. A number of basic and small-sample clinical studies have shown
that
crocin
can
protect
the
myocardium
from
ischemia-hypoxia
injury
by
anti-oxidative stress and reducing the activity of creatine kinase. On the other hand,
basic
research
has
also
found
that
crocin
can
significantly
improve
doxorubicin-induced cardiac injury and structural changes without affecting the in
vitro anticancer activity of doxorubicin [20]. Crocin can reduce the level of
malondialdehyde (MDA) and increase the level of glutathione in the heart tissue of
rats taking chemotherapy drugs, and by reducing the ratio of Bax/Bcl-2, the release of
cytochrome C and activation of caspase-3 attenuates cardiomyocyte apoptosis,
thereby ameliorating cardiac histopathological damage [21]. These studies suggest
that crocin may help prevent or improve the myocardial injury induced by
radiotherapy and chemotherapy in cancer patients and protect cardiovascular function.
The main component of saffron total glucoside tablets is crocin, which has been
approved to be used in patients with angina pectoris in China. At present, the
protective effect of crocin on cardiovascular mainly focuses on basic research and
small sample clinical research, and serological indicators are the main observation
indicators. There is still a lack of early application of saffron in patients with cardiac
function and vascular function damage. Therefore, the protective effect of crocin on
the cardiovascular function of tumor patients needs further research.
OBJECTIVES:
This study was to observe the effect of crocin on cardiovascular function in patients
with early breast cancer undergoing radiotherapy and chemotherapy.
RESEARCH DESIGN
This is a randomized, double-blind, parallel-controlled, single-center clinical
study.
Participant:
The patients are mainly from breast cancer patients who are hospitalized during

Version Number：20200818
Version Date：2020/08/18
the study period, planned to undergo radiotherapy and chemotherapy, and have high
risk factors for cardiovascular disease.
Inclusion criteria:
1)Age 25-80 years old, female.
2) Patients diagnosed with breast cancer by histopathology.
3) Patients who plan to receive adjuvant radiotherapy/chemotherapy or combined
adjuvant trastuzumab or pertuzumab targeted therapy.
4)Patients who completed at least 6 cycles of treatment after enrollment.
Exclusion criteria:
1)pregnant or breastfeeding women.
2)Patients with poor echocardiographic image quality.
3)Persistent atrial fibrillation and severe arrhythmia affect the collection and
analysis of ultrasound data.
4)Patients who are participating in other clinical studies.
Dosing regimen
1)Crocin group: The
chemotherapy/radiotherapy
protocols are
made by
oncologists adopted for patients depending on specific conditions, take saffron total
glucosides tablets (provided by REYOUNG Pharmaceutical Co., Ltd.) for 8 days
during each chemotherapy (started on the 1st day before chemotherapy), 4
tablets/time, 3 times a day.
2) Placebo group: Undergoing chemotherapy/radiotherapy protocols as planned,
take placebo piece during (the same appearance of saffron total glucosides tablets,
production
unit:REYOUNG
incorporated
company)
for
8
days
during
each
chemotherapy (started on the 1st day before chemotherapy), 4 tablets/time, 3 times a
day.
Criteria for Subject Discontinuation from Study
1) Allergic reactions that are clearly related to the study drug.
2) In the event of abnormal bleeding, other adverse symptoms or signs, and
abnormal examination results clearly related to the study drug, the investigator judges
that the study must be terminated.

Version Number：20200818
Version Date：2020/08/18
3) Women who become pregnant during the study period.
4) Continued research should be terminated at the request of the patient.
Criteria for withdrawing from the study (dropout)
1) The patient was lost to follow-up.
2) Poor patient compliance.
3) Blind release or emergency unblinding.
4) There is an adverse event related to the drug.
Criteria for full suspension of the test
1) The researchers found serious safety problems.
2) The curative effect is too poor, and there is no need to continue the test.
3) There are major mistakes in the research plan.
4) Unsustainable research funding.
5) The administrative department cancels the test.
A full suspension of the trial can be temporary or permanent. When the test is
terminated, all test records shall be retained for future reference
RESEARCH PROPOSAL
Estimation of Sample Size
Use PASS 11.0.7 software, bilateral inspections, α = 0.05, β = 0.2 (grasping
degree = 80%), and the number of allocations of the test group and the control group
should be allocated by 1: 1. It is expected that the LVEF of the crocin group will
decrease by no more than 0.17% compared with the baseline at the end of the 6-month
observation, and the average LVEF of the placebo control group will decrease by
3.28% compared with the baseline, with a standard deviation of 5.38%. The number
of cases should be 48 pairs. Considering the dropout rate of 20%, a total of 120
patients were randomly selected for this study.
Preliminary results from our real-world study showed that breast cancer patients
(n=79) had a GLS of -22.6 ± 1.77 before chemotherapy, -16.96 ± 3.65 after 6 cycles
of chemotherapy, and -15.94 ± 4.03 after 8 cycles of chemotherapy. Since there is no
literature report on the magnitude of the effect of cardioprotective therapy on GLS in

Version Number：20200818
Version Date：2020/08/18
cancer patients undergoing radiotherapy and chemotherapy, the sample content was
not calculated based on GLS.
Random grouping
Eligible patients were randomly divided into 1:1 group and divided into crocin
group and placebo control group. Both groups of subjects received crocin or placebo
on the basis of standard anti-tumor treatment. The randomization plan adopts the
method of stratified block randomization, and the statistician uses SAS14.0 software
to generate a random code table with a block of 4 and the treatment group
corresponding to the random number. The randomization table (blind bottom) was
sent to the project statistician by the random coders after the database was locked and
approved by the sponsor at the end of the study. The drugs were blinded according to
the serial number and random number. After the subjects are screened and qualified,
the researchers will distribute the drugs in order according to the drug number.
Research Process
After the selected patients signed the informed consent form, they were
randomly tested in the crocin group and the placebo group according to a randomized,
double-blind, placebo-controlled method. Dosage of crocin group or placebo: 3 times
a day, 4 tablets each time. The follow-up period was 6 months. Crocin group: take
saffron total glucosides tablets for 8 days during each radiotherapy and chemotherapy
(started on the 1st day before radiotherapy/chemotherapy), 4 tablets/time, 3 times a
day; Placebo group: placebo during radiotherapy and chemotherapy for 8 days, start
to take 1 day before radiotherapy/chemotherapy, 4 tablets/time, 3 times a day.
Follow-up was performed every 3 months after enrollment, and the follow-up period
was 6 months. After all patients were enrolled and followed up for 6 months, the
study was terminated and unblinded.
Chemotherapy regimens
The chemotherapy regimens are determined by the patient's condition.
(1) Patients with Her-2 positive breast cancer:
(i) A (E) C →PH/TH ± Pertuzumab regimen:
Doxorubicin (A) 50-60mg/m2 iv on Day 1 or Epirubicin (E) 80-100mg/m2 iv on

Version Number：20200818
Version Date：2020/08/18
Day 1, Cyclophosphamide (C) 600mg/m2 iv on Day 1, Day 21 is 1 cycle, a total of 4
cycles; the sequential A/B scheme is as follows:
A. Sequentially paclitaxel (P) 80 mg/m2 iv on day 1, once a week, for 12 weeks,
and trastuzumab (H) at the first dose of 4 mg/kg, then 2 mg/kg, once a week, for a
total of 12 weeks; it can be combined with Pertuzumab for the first dose of 840 mg,
followed by 420 mg each time, 21 days as a cycle, a total of four cycles.
B. Sequentially docetaxel (T) 100mg/m2 iv on the first day, 21 days as a cycle, a
total of 4 cycles. At the same time, the first dose of trastuzumab (H) is 8mg/kg,
followed by 6mg/kg, 21 days as a cycle, a total of 4 cycles, can be combined with the
first dose of Pertuzumab 840mg, and then 420mg each time, 21 days For 1 cycle, a
total of 4 cycles, used concurrently with trastuzumab.
(ii) TCbH±Pertuzumab regimen:
Docetaxel (T) 75 mg/m2 iv on day 1, carboplatin (Cb) AUC 6 iv on day 1, 21
days as a cycle, a total of 6 cycles; The first dose is 8mg/kg, followed by 6mg/kg, 21
days as a cycle, a total of 6 cycles, can be combined with the first dose of Pertuzumab
840mg, and then 420mg each time, and trastuzumab can be used at the same time.
A (E) C→PH/TH regimen and TCbH regimen both have a dose of 6 mg/kg of
trastuzumab (H) after chemotherapy, and can be combined with a dose of 420 mg of
pertuzumab, once every 3 weeks for full 1 year.
2) Patients with HER-2 negative breast cancer:
(i) A (E) C →P/T regimen;
Doxorubicin (A) 60mg/m2 iv on day 1 or epirubicin (E) 60-100mg/m2 iv on day
1, cyclophosphamide (C) 600mg/m2 iv on day 1, 1 on day 21 cycle, a total of 4
cycles.
Sequentially with paclitaxel (P) 80mg/m2 iv on the 1st day, once a week for a
total of 12 weeks; or docetaxel (T) 100mg/m2 iv on the 1st day, 21 days as a cycle, a
total of 4 cycle.
(ii) TC regimen:
Docetaxel (T) 75 mg/m2 iv on day 1, carboplatin (Cb) AUC 6 iv on day 1, 21
days as a cycle, a total of 6 cycles.

Version Number：20200818
Version Date：2020/08/18
The main test indicators in the selection and follow-up period
(1) The main observation and detection indicators at the time of selection
(i) Clinical conditions: chest tightness, chest pain, palpitation, fatigue and other
clinical symptoms and signs
(ii) ECG examination: use resting ECG and dynamic ECG to evaluate
arrhythmia and ST-T changes.
(iii) Laboratory tests: fasting for more than 6 hours, blood routine, urine routine,
fasting blood glucose, alanine aminotransferase, aspartate aminotransferase, blood
urea
nitrogen,
creatinine,
plasma
total
cholesterol,
triglyceride,
low-density
lipoprotein
cholesterol,
high-density
lipid
Protein
cholesterol,
high-sensitivity
C-reactive protein, prothrombin time, serum troponin, NT-proBNP.
(iv) Transthoracic echocardiography:
Two-dimensional dynamic images of left ventricular parasternal long axis, apical
four-chamber heart, apical two-chamber heart, apical long axis, left ventricular apex
level, papillary muscle level, and mitral valve level short-axis view; measure left
ventricular ejection fraction (LVEF ); Pulse wave Doppler was used to obtain the
diastolic mitral valve orifice blood flow spectrum, and tissue Doppler was used to
obtain the mitral valve annulus motion spectrum, and E/e' was calculated; Using
speckle tracking imaging (STI) ultrasound technology to measure global longitudinal
strain (GLS)/strain rate, global circumferential strain, global radial strain, global area
strain (3D-GAS), left ventricular torsion, left ventricular rotation/untwisting speed,
systolic dissynchrony index (SDI) and right ventricular free wall systolic peak strain
(RVFWS) and so on. See Annex 1 for details.
(2) The main observation and detection indicators of the first follow-up (3
months)
(i) Clinical situation: chest tightness, chest pain, palpitation, fatigue and other
clinical symptoms and signs
(ii) ECG examination: use resting ECG and dynamic ECG to evaluate
arrhythmia and ST-T changes.
(iii) Laboratory tests: fasting for more than 6 hours, blood routine, urine routine,

Version Number：20200818
Version Date：2020/08/18
fasting blood glucose, alanine aminotransferase, aspartate aminotransferase, blood
urea nitrogen, creatinine, high-sensitivity C-reactive protein, prothrombin time, serum
troponin, NT-proBNP.
(iv) Transthoracic echocardiography:
Two-dimensional dynamic images of left ventricular parasternal long axis, apical
four-chamber heart, apical two-chamber heart, apical long axis, left ventricular apex
level, papillary muscle level, and mitral valve level short-axis view; measure left
ventricular ejection fraction (LVEF )、E/e'；Using speckle tracking imaging (STI)
ultrasound technology to measure global longitudinal strain (GLS)/strain rate, global
circumferential strain, global radial strain, global area strain (3D-GAS), left
ventricular torsion, left ventricular rotation/untwisting speed, systolic dissynchrony
index (SDI) and right ventricular free wall systolic peak strain (RVFWS) and so on.
(3) The second follow-up (6 months, at the end of the study), the main
observation and measurement indicators
After
enrollment,
the
following
indicators
were
detected
before
each
chemotherapy cycle and every 3 months after the end of the chemotherapy cycle:
(i) Clinical situation: chest tightness, chest pain, palpitation, fatigue and other
clinical symptoms and signs，etc.
(ii) ECG examination: use resting ECG and Holter ECG to evaluate arrhythmia
and ST-T changes.
(iii) Laboratory tests: fasting for more than 6 hours, blood routine, urine routine,
fasting blood glucose, alanine aminotransferase, aspartate aminotransferase, blood
urea
nitrogen,
creatinine,
plasma
total
cholesterol,
triglyceride,
low-density
lipoprotein
cholesterol,
high-density
lipoprotein
cholesterol,
High-sensitivity
C-reactive protein, prothrombin time, serum troponin, NT-proBNP.
(iv) Transthoracic echocardiography:
Two-dimensional dynamic images of left ventricular parasternal long axis, apical
four-chamber heart, apical two-chamber heart, apical long axis, left ventricular apex
level,
papillary
muscle
level,
and
mitral
valve
level
short-axis
view
were
obtained;measure the LVEF, E/e’; Using STI ultrasound technology to measure global

Version Number：20200818
Version Date：2020/08/18
longitudinal strain (GLS)/strain rate, global circumferential strain, global radial strain,
global
area
strain
(3D-GAS),
left
ventricular
torsion,
left
ventricular
rotation/untwisting speed, systolic dissynchrony index (SDI) and right ventricular free
wall systolic peak strain (RVFWS) .etc.
DATA MONITORING
(1) Inform the participant that his/her personal study-related data will be used in
accordance with local data protection laws. The extent of disclosure must also be
explained to participants.
(2) All participant data related to the study will be recorded on the CRF.
Researchers are responsible for verifying the accuracy and correctness of data entry.
(3) The investigator must maintain accurate documentation supporting the CRF
information.
(4) Investigators must allow study-related monitoring and provide direct access
to source data files.
(5) Designate personnel to be responsible for data management for this study,
including data quality checks.
(6) Regulatory agency inspectors are responsible for contacting and visiting
investigators to inspect facilities and, upon request, to inspect various records of
clinical studies (eg, case report forms and other relevant data).
(7) Monitors are responsible for regularly validating case report forms
throughout the study to verify compliance with the protocol; completeness, accuracy,
and consistency of data; and compliance with local regulations for conducting clinical
studies.
(8) Educate patients to promptly report any symptoms and unusual signs. Safety
Assessment Physicians record the patient's symptoms and physical examination
results. Patients with serious adverse events can discontinue their assigned medication
and be closely monitored or receive emergency treatment until the adverse event
resolves.
EFFICACY ASSESSMENT

Version Number：20200818
Version Date：2020/08/18
Primary study endpoint:
1) Differences between groups in the difference in LVEF measured by
echocardiography at the end of the experiment compared to baseline.
2) Differences between groups in the difference in GLS measured by
echocardiography at the end of the experiment compared to baseline.
Secondary study endpoints
1) Differences in the incidence rates of serum troponin exceeding the upper limit
of normal value and NT-proBNP higher than the normal age reference value between
the two groups during follow-up.
2）The differences in the incidences of chest tightness, chest pain and palpitation
between the two groups.
3）Differences between the two groups in the incidences of arrhythmia and ST-T
changes displayed by dynamic electrocardiogram.
4 ）Differences between groups in the difference in the E/e', 2D-STI indexes,
3D-STI indexes measured by echocardiography at the end of the experiment
compared to baseline.
5) Differences between groups in the difference in the indexes of left ventricular
diastolic function measured by echocardiography at the end of the experiment
compared to baseline.
6) Differences between groups in the difference in the right ventricular function
monitoring indicators measured by echocardiography at the end of the experiment
compared to baseline.
SAFETY ASSESSMENT
Including clinical adverse reactions, laboratory indicators, etc.
ADVERSE EVENT OBSERVATION AND REPORTING
Definition of Adverse Events
Adverse events refer to any adverse medical events that occurred between the
time the patient signed the patient's informed consent and were enrolled in the trial

Version Number：20200818
Version Date：2020/08/18
and the last follow-up visit in this clinical trial, regardless of whether the event had a
causal relationship with the above-mentioned drugs.
Note: Adverse reactions may be any discomfort and unintentional signs
(including abnormal laboratory test results), symptoms, or illnesses (new or
worsening) associated with the drug.
Events that meet the definition of an adverse event include:
(i) Exacerbation of existing chronic or intermittent disease, including increased
frequency and/or intensity.
(ii) Newly detected or diagnosed disease after taking the trial drug, even though
it may have existed before the trial started.
(iii) Signs, symptoms or clinical sequelae of suspected interaction.
(iv)
Signs,
symptoms
or
clinical
sequelae
of
suspected
overdose
of
investigational drug or concomitant drug (overdose itself is not an adverse
event/serious adverse event).
(v) "Lack of efficacy" or "not achieving expected pharmacological effects" by
themselves are not reported as adverse events or serious adverse events. However,
signs, symptoms and/or clinical sequelae due to lack of efficacy will also be reported
as adverse events or serious adverse events if they meet the definition of an adverse
event or serious adverse event.
Events that do not meet the definition of an adverse event include:
(i) Medical or surgical procedures (eg, endoscopy, appendectomy); events
leading to surgery are adverse events.
(ii) Situations where adverse medical events would not occur (social security
and/or hospital admissions).
(iii) No worsening of pre-existing disease or conditions present or detected at the
start of the trial, only expected day-to-day fluctuations.
(iv) The disease/disorder under study, or the expected progression, signs or
symptoms of the disease under study. Unless the situation is more serious than
expected.
Definition of Serious Adverse Events

Version Number：20200818
Version Date：2020/08/18
A "serious adverse event" is an adverse event that can occur at any dose and will:
(i) Causing death.
(ii) Life-threatening.
Note: "Life-threatening" means that the subject is in danger of dying when the
event occurs, not that death could theoretically result if the event were more severe.
(iii) Requiring hospitalization or prolonged hospitalization.
Note: Generally, hospitalization refers to a subject's stay in a hospital or
emergency room (usually at least overnight) for observation and/or treatment that
cannot be accomplished in a doctor's office or outpatient clinic. Complications that
occurred during hospitalization were considered adverse events. An event was
classified as a serious adverse event if the complication prolonged hospital stay or met
the criteria for any other serious adverse event. Treat as a serious adverse event when
it cannot be determined whether "hospitalization" occurred or was necessary.
Selective hospitalization with no worsening of pre-existing disease compared to
baseline was not considered an adverse event.
(iv) Causing disability, or affect the ability to work or live.
Note: "Disability" refers to a material that impairs an individual's ability to lead a
normal life. Discomforts of insignificant clinical significance, such as common
headache, nausea, vomiting, diarrhea, influenza, and accidental trauma (eg, sprained
ankle), which may interfere with quality of life but are not substantially disruptive, are
excluded.
(v) Causing congenital deformities.
In other cases, medical or scientific judgment should be used to determine the
appropriateness of adverse event reporting. For example, some important medical
events may not be immediately life-threatening, lead to death or hospitalization, but
may harm the subject or require medical or surgical intervention to avoid the
occurrence of the serious adverse events listed above. These events should also be
considered serious adverse events, such as invasive or malignant cancer, allergic
bronchospasm requiring intensive monitoring in the emergency room or at home,
blood cachexia, or convulsions that do not result in hospitalization, drug dependence,

Version Number：20200818
Version Date：2020/08/18
or drug abuse.
Medical side effects
Definition: Refers to the harmful reactions of qualified drugs that are not related
to the purpose of drug use under normal usage and dosage.
Causal judgment indicators for adverse reaction judgment:
Item 1: Whether there is a reasonable relationship between the time when the
drug was started and the time when the suspicion appeared.
Item 2: Whether the suspected adverse reaction conforms to the known adverse
reaction type of the drug.
Item 3: Whether the suspected adverse reaction can be explained by the patient's
pathological condition, concomitant medication, concomitant therapy, or previous
therapy.
Item 4: Whether the suspected adverse reaction is alleviated or disappeared after
drug discontinuation or dose reduction.
Item 5: Whether the same reaction reappears after the suspected drug is used
again.
Criteria for causal judgment: according to the order of the above five judgment
indicators.
Possible associations between adverse events, study drug, and concomitant
medications were assessed by the investigator according to the table below.
Adverse reaction cause and effect judgment form
critical
result
Judgment index
Item1
Item 2
Item 3
Item 4
Item 5
Definitely
related
＋
＋
－
＋
＋
Might be
related
＋
＋
－
＋
？
May be
＋
＋
±
±
？

Version Number：20200818
Version Date：2020/08/18
related
suspicious
＋
－
±
±
？
Impossible
＋
－
＋
－
－
Description: + affirmative, - negative, ± difficult to affirm or deny, ? The
situation is unknown.
1) According to the above table, determine the relationship between the adverse
events and drugs to the following five grades
(i) definitely related, (ii) likely related, (iii) likely related, (iv) doubtful, (v)
unlikely to be related.
2) The incidence of adverse reactions was calculated using the total number of
1+2+3+4 cases as the numerator, and all the selected cases for evaluation of adverse
reactions as the denominator.
Records of adverse event
For adverse events that occurred during the trial, the type, degree, time of
appearance, duration, treatment measures, and treatment process should be recorded
in the case report form, and on the basis of comprehensive consideration of
comorbidities and concomitant medication, the evaluation of their relationship with
the
experiment
should
be
made.
Dependence
of
medication
and
detailed
documentation by physician.
If adverse events are found, the observing physician can decide whether to
discontinue the observation according to the condition. Cases of discontinuation due
to adverse events should be followed up and the results recorded in detail. If abnormal
safety test indicators (blood, urine, stool routine, electrocardiogram, liver function,
renal function) occur during and after treatment, the adverse event table should be
filled in in time, and re-examination should be conducted at an appropriate time, and
the subject should be consulted the incidence, treatment, etc. are comprehensively
analyzed to determine whether it is related to the test drug.
Management of serious adverse events

Version Number：20200818
Version Date：2020/08/18
Any serious adverse events during the trial (including events requiring
hospitalization,
prolonged
hospitalization,
disability,
affecting
work
ability,
endangering life or death, causing congenital malformations, etc.) In addition to
taking urgent measures, the subject must also immediately report to the Ethics
Committee of the National Drug Clinical Trial Institution of Qilu Hospita of
Shandong University, the main research unit, and REYOUNG Pharmaceutical Co.,
Ltd. The report of serious adverse events should include : patient's name, random
number, length of study participation, start and stop dates of serious adverse events,
maximum intensity of serious adverse events, possible relationship between serious
adverse events and study drug, due to serious adverse events. Whether the adverse
event required a change in study drug, the treatment given to the patient due to the
serious adverse event, concomitant medications in the event of a serious adverse event,
and the outcome of the serious adverse event.
DATA MANAGEMENT AND STATISTICALANALYSIS
Data management
(1) Establishment of database: The data administrator establishes the EXCEL
database according to the research protocol and CRF, and sets up logical verification
according to the data verification plan (DVP). After the test is passed, it will be
released for use.
(2) Data entry: The data entry personnel conduct independent double entry and
double verification. For inconsistent results, check and correct each item against the
CRF until the results are completely consistent.
(3) Data questions and answers: After the data entry is completed, the data
administrator conducts question screening according to the manual verification plan
of the DVP, opens the database access rights to the researchers, and answers the
questions remotely. The data administrator replies to the query, and if necessary, can
re-issue the query until the data is "clean".
(4) Database lock: After the principal investigator, sponsor, statistical analyst and
data administrator jointly sign the "database lock record", the data administrator locks

Version Number：20200818
Version Date：2020/08/18
the database.
(5) Database submission: The data administrator submits the database to the
statistician.
Statistical analysis dataset
(1) Full Analysis Set (FAS): A collection of all randomized patients who received
at least one study drug.
(2) Per-Protocol Set (PPS): is a data set generated by subjects who are fully
compliant with the trial protocol, including the treatment received, the availability of
measurements of the primary endpoint, and the absence of major deviations from the
trial protocol. PPS analysis was used for the primary efficacy measure.
(3) Safety Data Set (SS): Actual data on at least one treatment with recorded
safety indicators after treatment. The incidence of adverse reactions was taken as the
denominator of the number of SS cases.
Statistical methods
Subject Distribution Analysis
(1) The number of subjects enrolled and who completed the trial was listed, and
three data sets for analysis (FAS, PPS, SS) were identified.
(2) The reasons for not entering PPS were classified and analyzed, and the
number of subjects in different categories was calculated.
(3) List the detailed list of population groups, including the reasons for not being
included in PPS/FAS/SS.
(4) Draw the subject distribution flow chart.
Demographic data and baseline analysis
Descriptive Statistics Demographics and other baseline characteristic values:
(1) Calculate the number of cases, mean, standard deviation, 95% CI, minimum
and maximum value of continuous variables.
(2) Count and grade data to calculate frequency and composition ratio.
(3) Inferential statistical results (P values) are listed as descriptive results.
Analysis of the compliance and consolidation of medication
(1) Calculating the percentage of the subjects with the compliance of the

Version Number：20200818
Version Date：2020/08/18
medication in the range of 80%-120%, and the difference between the comparison of
the comparison of the χ2 test or the Fisher accurate probability method.
(2) Drug exposure, use T test comparison between group differences.
(3) Calculate the percentage of the subjects with consolidated medication, and
the difference between the comparison of the comparison of the χ2 test or the Fisher
accurate probability probability method.
Analysis of efficacy
Main efficacy index analysis
At the end of treatment, the differences between the LVEF and GLS measured by
ultrasound compared with baseline were compared between groups using the t test.
PPS and FAS analyses were performed simultaneously.
Secondary efficacy index analysis
(1) During the follow-up period, the incidence rates of serum troponin
(cTnI)>30ng/L and NT-proBNP higher than the normal age reference value were
compared between groups by χ2 test or Fisher's exact test. The actual data in FAS
were used for analysis.
(2) The frequency and duration of chest tightness, chest pain and palpitation
were compared between groups using t test. The actual data in FAS were used for
analysis.
(3) Holter electrocardiogram showed the degree of arrhythmia and ST-T changes,
and t-test was used to compare the differences between groups. The actual data in
FAS were used for analysis.
(4) Compared with baseline, ultrasound-measured global radial strain, systolic
dyssynchrony index (SDI), and t-tests were used to compare differences between
groups. The actual data in FAS were used for analysis.
Security analysis
(1) Calculate the incidence of adverse events/reactions, serious adverse
events/reactions, and adverse events/reactions leading to dropout.
(2) A detailed list of cases of various adverse events/reactions, serious adverse
events/reactions, adverse events/reactions leading to dropout.

Version Number：20200818
Version Date：2020/08/18
(3) List the cross-tabulation of laboratory examinations and electrocardiogram
examinations before and after medication.
(4) Descriptive statistics of laboratory examinations, electrocardiograms, and
changes in vital signs from baseline and measured values.
(5) A detailed list of abnormal values of laboratory tests, electrocardiograms, and
physical examinations is listed.
Statistics software
(1) SAS software (version 9.4) was used for analysis.
(2) All statistical tests are two-sided, and a P value less than or equal to 0.05 will
be considered to be statistically significant for the differences tested.
(3) Detailed statistical methods will be provided in the statistical analysis plan.
RESEARCH PLAN
(1) Case entry stage: from March 2021 to December 2022—complete the entry
of each group of cases.
(2) Follow-up time: 6 months. For follow-up time points, please refer to the
attached trial procedure. All subjects were followed up by June 2023.
(3) Data analysis: June 2023 to September 2023—Data collection and analysis,
writing papers .
REFERENCES
1. Mozaffarian D, Benjamin EJ, Go AS, et al. Heart disease and stroke statistics--2015
update: a report from the American Heart Association. Circulation, 2015, 131:
e29-e322.
2.Zamorano JL, Lancellotti P, Rodriguez MD, et al. 2016 ESC Position Paper on
cancer treatments and cardiovascular toxicity developed under the auspices of the
ESC Committee for Practice Guidelines: The Task Force for cancer treatments and
cardiovascular toxicity of the European Society of Cardiology (ESC)[ J]. Eur Heart J,
2016, 37(36):2768-2801.
3. Tarantini L, Massimo GM, Di Lenarda A, et al. ANMCO/AIOM/AICO consensus
document on clinical and management pathways of cardio-oncology: executive

Version Number：20200818
Version Date：2020/08/18
summary. Eur Heart J, 2017, 19(Suppl D):D370-D379.
4. Arai M, Tomaru K, Takizawa T, et al. Sarcoplasmic reticulum genes are selectively
down-regulated in cardiomyopathy produced by doxorubicin in rabbits[ J]. J Mol Cell
Cardiol, 1998, 30(2): 243-254.
5. Papadopoulou LC, Theophilidis G, Thomopoulos GN, et al. Structural and
functional impairment of mitochondria in adriamycin-induced cardiomyopathy in
mice: suppression of cytochrome c oxidase II gene expression[ J]. Biochem
Pharmacol, 1999, 57(5): 481-489.
6. Shi J, Guo Y, Cheng L, et al. Early change in left atrial function in patients treated
with anthracyclines assessed by real-time threedimensional echocardiography[ J]. Sci
Rep, 2016, 6: 25512.
7. Chen XL, Lei YH, Liu CF, et al. Angiogenesis inhibitor bevacizumab increases the
risk of ischemic heart disease associated with chemotherapy: a meta-analysis[ J].
PLoS One, 2013, 8(6): e66721.
8. Schutz FA, Je Y, Azzi GR, et al. Bevacizumab increases the risk of arterial ischemia:
a large study in cancer patients with a focus on different subgroup outcomes[ J]. Ann
Oncol, 2011, 22(6): 1404-1412.
9. Ranpura V, Hapani S, Chuang J, et al. Risk of cardiac ischemia and arterial
thromboembolic events with the angiogenesis inhibitor bevacizumab in cancer
patients: a meta-analysis of randomized controlled trials[ J]. Acta Oncol, 2010, 49(3):
287-297.
10. Suter TM, Ewer MS. Cancer drugs and the heart: importance and management[ J].
Eur Heart J, 2013, 34(15): 1102-1111.
11．Carver JR, Shapiro CL, Ng A, et al. American Society of Clinical Oncology
clinical evidence review on the ongoing care of adult cancer survivors: cardiac and
pulmonary late effects[ J]. J Clin Oncol, 2007, 25(25): 3991-4008.
12. Malanca M, Cimadevilla C, Brochet E, et al. Radiotherapy-induced mitral stenosis:
a three-dimensional perspective[ J]. J Am Soc Echocardiogr, 2010, 23(1): 101-108.
13. Darby SC, Cutter DJ, Boerma M, et al. Radiation-related heart disease: current
knowledge and future prospects[ J]. Int J Radiat Oncol Biol Phys, 2010, 76(3):

Version Number：20200818
Version Date：2020/08/18
656-665.
14. Plana JC，Galderisi M，Barac A，et al．Expert consensus for multimodality imaging
evaluation of adult patients during and after cancer therapy: a report from the
American
Society
of
Echocardiography
and
the
European
Association
of
Cardiovascular Imaging［J］．Eur Heart J Cardiovasc Imaging，2014，15( 10) :
1063-1093．
15. Amundsen BH，Helle-Valle T，Edvardsen T，et al．Noninvasive myocardial strain
measurement
by
speckle
tracking
echocardiography:
validation
against
sonomicrometry and tagged magnetic resonance imaging［J］．J Am Coll Cardiol，2006，
47( 4) : 789-793．
16. ZHAO R ，CHENG LL. Utility and latest developments of echocardiographic
speckle tracking imaging in cardio-oncology [J]. Adv Cardiovasc Dis, 2018,39 (2):
138-142.
17. Huang SJ，Orde S．From speckle tracking echocardiography to torsion，research
tool today，clinical practice tomorrow［J］．Curr Opin Crit Care，2013，9( 3) :
250-257．
18. Chu X，Liu T，Han X，et al. Research on chemical constituents of crocus sativus
and their cardiovascular pharmacological effects [J]. Guangzhou Chemical Industry,
2016, 44(18): 30-32.
19. Han Y，Xie GY，Li R，et al.
Research progress on pharmacological activities of
crocin [J]. Drugs & Clinic, 2017,32 (9):1806-1814.
20. Razmaraii N, Babaei H, Mohajjel Nayebi A, et al. Crocin treatment prevents
doxorubicin-induced cardiotoxicity in rats [J]. Life Sci, 2016, 157: 145-151.
21. Razavi B M, Hosseinzadeh H, Movassaghi A R, et al. Protective effect of crocin
on diazinon induced cardiotoxicity in rats in subchronic exposure [J]. Chem Biol
Interact, 2013, 203(3): 547-555.

Version Number：20200818
Version Date：2020/08/18

Version Number：20200818
Version Date：2020/08/18
Annex 1.
Standard Operating Procedures for Ultrasound
Examination
Personnel and equipment requirements
In order to ensure the quality of image acquisition and the standardization of
measurement data, two experienced sonographers from the central laboratory-Qilu
Hospital of Shandong University first conducted a demonstration of standard image
acquisition according to the research protocol. In the screening stage, the researchers
of each research unit need to submit the sample images to the central laboratory for
review, which will be evaluated and fed back by the sonographer. All participants in
the collection and analysis have no stake in this research. Each operator needs to have
corresponding qualifications and training, and the ultrasound machine that collects
images has a clear imaging function, so as to better perform ultrasound examinations
and capture images.
Operation method
Standardized
cardiac
anatomical
parameters
and
hemodynamic
function
parameters
were
measured
using
internationally
recognized
standardized
echocardiographic cardiac views. All measured values are determined uniformly to 1
digit after the decimal point. Three cardiac cycle parameters were measured for each
parameter, and the mean value was taken.
Specific implementation methods and steps:
Preparation before inspection
Rest for 5 minutes before the test.
Connect synchronized ECG monitoring electrodes to determine cardiac cycle
phases. The end of the T wave of the ECG was used to define the end systole of the
ventricle, and the peak of the R wave of the QRS wave was used to define the end of
the ventricular end diastole.
Position
The left lateral decubitus position was used as the detection position for
echocardiographic parasternal and apical views.

Version Number：20200818
Version Date：2020/08/18
Breathing
In order to exclude the influence of respiration on the measured value, the
respiration should be controlled at the end of expiration and temporarily held before
the image acquisition (except when observing the inner diameter of the inferior vena
cava).
Inspection site
The following sound-transmitting windows are used for detection: the detection
area of the left sternum and the left apex of the heart. After the detection area is
determined, enough ultrasonic couplant is filled between the skin of the detection area
and the ultrasound probe to remove air and reduce gas interference.
Parameter setting of echocardiography equipment and technical requirements
before image acquisition
(1) It is recommended to use the fundamental wave image for cardiac structure
observation and measurement to avoid the distortion effect of tissue harmonic
imaging. Optimize the resolution of two-dimensional gray-scale ultrasound images:
try to use the smallest detection depth and the highest ultrasonic emission frequency;
use
standard
two-dimensional
cardiac
slices
and
appropriate
detection
sound-transmitting windows to ensure that the observation slices are displayed clearly
and completely.
(2) For two-dimensional echocardiographic grayscale images, the frame rate of
observed and recorded images should be greater than or equal to 30 frames/second;
for color tissue Doppler velocity images, the frame rate of observed and saved images
should be greater than or equal to 80 frames/second , in order to facilitate subsequent
functional parameter analysis.
(3) In order to avoid the artificial shortening effect of the long axis of the left
ventricle when observing the apex, the left lateral position should be used, the use of
an overly soft mattress should be avoided, and the position of the apex palpable by
palpation should be avoided. The section with the largest inner diameter of the long
axis is measured.
(4) In order to accurately determine the end-diastolic and end-systolic phases of

Version Number：20200818
Version Date：2020/08/18
the ventricle, both the mitral valve motion and the chamber diameter change should
be referenced, and excessive reliance on ECG to determine the phase should be
avoided.
(5) The tissue Doppler sampling frame was set to 1 mm, and color Doppler flow
imaging should be canceled during sampling to determine the spatial location of
cardiac anatomy. Adjust the filter appropriately to avoid spectral distortion caused by
too low or too high (too high filter results in low-speed frequency shift signal filtering,
too low filter results in too strong noise signal).
(6) Image acquisition and image storage requirements: use a high-end
cardiovascular color Doppler diagnostic instrument to acquire cardiac images, and
image storage requires 5 continuous cardiac cycle dynamic images, which are stored
in DICOM format. Each patient is a folder, including each image acquisition time and
operator .
(7) Image analysis and repeatability test: The image information is sent to the
central laboratory by the responsible personnel of each center by email for collection
and storage, and then two experienced sonographers apply professional multi-center
data analysis methods in accordance with the double-blind and random principles.
Professional software is used for data reading, and each data is measured three times
and averaged. The data with poor image quality will be re-examined by a team of
professionals, analyzed multiple times, and finally decided whether to use it or not. In
order to test the reproducibility of the measurement, this study randomly selected 50
subjects' images, and repeated the measurement of all key parameters. Inter-observer
variability is a comparison of the difference between two sonographers' measurements,
and intra-observer variability is a comparison of the difference between two
measurements of the same physician at different times. Differences were compared
using the Bland-Altman method to analyze and draw scatter plots, and to calculate
correlation coefficients.
(8) The specific operation diagram of each measurement parameter is as follows:

Version Number：20200818
Version Date：2020/08/18
Parasternal long-axis view of the left ventricle showing end-diastolic left
ventricular diameter measurement by two-dimensional echocardiography
Standard apical four-chamber view for left ventricular end-diastolic and end-systolic
volume and LVEF measurements using the biplanar Simpson method

Version Number：20200818
Version Date：2020/08/18
Standard apical four-chamber view-guided pulse wave tissue Doppler sampling
of mitral valve sidewall and septal annulus tissue motion e' velocity, and calculating
the average.
Standard apical four-chamber view-guided pulse-wave Doppler sampling Peak
E-peak velocity across the mitral valve during diastole
Data storage and analysis
All data are saved in DVD disc or mobile hard disk in DICOM format. Each
patient is a folder, including the time of each image acquisition and the operator. The
image information is sent to the central laboratory by the responsible personnel of
each center by email for summary and storage, and then analyzed by two experienced
sonographers. According to the double-blind and random principle, professional
software for multi-center data analysis was used for data reading, and each data was

Version Number：20200818
Version Date：2020/08/18
measured three times to obtain the average value. The data with poor image quality
will be re-examined by a team of professionals, analyzed multiple times, and finally
decided whether to use it or not.
Intra-observer variation and inter-observer variation observed
To test the reproducibility of the measurement, 50 subjects were randomly
selected in this study, and all key parameters were repeatedly measured. The
interobserver
variability
compared
the
difference
between
two
sonographers'
measurements, and the intraobserver variability compared the difference between the
same
physician's
two
measurements.
Differences
were
compared
using
the
Bland-Altman method to analyze and draw scatter plots, and the correlation
coefficient was calculated.