Study protocol for a multicentre, randomised controlled trial of the external use of mirabilite to prevent pancreatitis in children after endoscopic retrograde cholangiopancreatography.

Acute pancreatitis is the most common complication of endoscopic retrograde cholangiopancreatography (ERCP) in children. To date, there is no suitable medicine for post-ERCP pancreatitis prophylaxis in children and few study has prospectively evaluated an intervention to prevent post-ERCP pancreatitis in children. Mirabilite, a well-known traditional Chinese medicine(TCM) has good therapeutic effects on acute pancreatitis and no side effect for children by basic research and clinical studies. Our protocol is designed to assess the ecacy and safety of the external use of mirabilite to prevent post-ERCP pancreatitis in children.

Methods/design: 520 patients planned for diagnostic and therapeutic ERCP will be enrolled according to the eligibility criteria . The patients will be randomly divided into two equal groups (Mirabilite and control, the external use of mirabilite in a bag on the projected abdominal area over the pancreas within 30 min before ERCP).
The primary end point is incidence of post-ERCP pancreatitis. Secondary end points include abdominal pain scores, the levels of in ammatory markers [tissue necrosis factor (TNF)-α, IL-6, and IL-8] and intestinal barrier function markers (diamine oxidase, D-lactic acid, and endotoxin). Additionally, the side effects of topical mirabilite is investigated.

Conclusion
This trial would be the rst experiment to determine mirabilite to prevent post-ERCP pancreatitis in children. Mirabilite maybe provide potential clinical bene ts and a new avenue with tremendous potential for the future preventing of post-ERCP pancreatitis.

Background
Endoscopic retrograde cholangiopancreatography (ERCP) is an advanced endoscopic technique that is well established for diagnosis and treatment in biliary and pancreatic disorders in children 1,2 . Post-ERCP pancreatitis is the most common adverse event following ERCP occurring in approximately 6-10.9% of children varying between case series dependent on multiple factors, including case mix, operator characteristics, underlying disease factors and variations in post-ERCP management [3][4][5] . Most episodes of post-ERCP pancreatitis are mild but a small proportion of patients develop severe pancreatitis, leading to prolongation of hospital stay, a long stay in the Intensive Care Unit that can increase the morbidity and mortality rates. The occurrence of post-ERCP pancreatitis limits the extensive application of ERCP in children.
The pathophysiology of post-ERCP pancreatitis is not entirely clear with several factors such as chemical, mechanical or microbiological factors that result from papillary instrumentation and/or hydrostatic injury from the over lling of the pancreatic duct with contrast material 6,7 . The in uence of these factors leads to activation of trypsinogen, oxidative stress, ischemia, at last inducing tissue injury. Activating in ammatory reaction chain and transcription of pro-in ammatory cytokines play an important role in tissue injury. It can induce chemo-attraction of mono-macrophages which increases the pro-in ammatory cascade and increase tissue necrosis.
To date, only non-steroidal anti-in ammatory drugs (NSAIDs) have been shown as effective in the prevention of post-ERCP pancreatitis in adult 8,9 . Questions remain regarding the dose of rectal indomethacin widely used in the adult population is not utilized in children and the method of rectal administration is not acceptable for children. Furthermore, hardly any reports have researched prophylaxis medicine for post-ERCP pancreatitis in children. Finding an ideal, effective, less-invasive and safe prevention for children are desired.
Mirabilite, a white granular mineral medicine primarily hydrous sodium sulfate (Na2SO4 10H2O) has been a well-known traditional Chinese medicine (TCM) for treating acute pancreatitis. Under the guidance of TCM pharmaceutical theory, acute pancreatitis is categorized as epigastric pain, splenic precordial pain, splenopyretic disease and knotted chest disease. The principle of treatment in mirabilite is to clear away the heat-evil (heat as a pathogenic factor that causes heat pattern/syndrome) and expelling super cial evils, supplementing qi (vital energy) and nourishing yin (body uid), activating blood circulation to dissipate blood stasis and inner communication and purgation. Many studies have shown that mirabilite plays an important role in the treatment of acute pancreatitis and other in ammatory diseases in adult in china. 10 15,16 . The likely mechanisms may operate through reducing ROS generation and regulating the nitric oxide pathway in rat with acute pancreatitis. Although there is no studies about mirabilite treating for acute pancreatitis in children, external use of mirabilite has been proved to be effective and safe for treating acute suppurative appendicitis, pneumonia, tonsillitis of children in china 17,18 .

Research Hypothesis
Basing on mirabilite can treat acute pancreatitis, we hypothesis that mirabilite is effective in preventing post-ERCP pancreatitis. Furthermore, previous clinical evidences have shown that mirabilite is a safe and bene cial treatment option for children with in ammatory diseases.

Trial design
In this study, mirabilite is applied for preventing Post-ERCP pancreatitis in children and the efficacy and safety of the external use of mirabilite is assessed by a the medical staff and patient blinded, multicentre, controlled, observe, randomised with a 1:1 allocation trial.

Methods/design
Study design In this research, clinical trial will be performed in Shanghai Children's Medical Center and Shanghai Shuguang Hospital in China. A brief owchart of the entire study is shown in Fig.1, and the schedule of events is provided in Fig. 2. pro-in ammatory reaction.

Study populations
Patients who planned to undergo ERCP in the participated hospitals will be invited to the study. A screening session and physical examination prior to inclusion will be conducted by a medical doctor according to the following criteria.
An investigator who is masked to treatment allocation records the patient demographics, post-ERCP adverse events and follow-up data and the procedure-related parameters including cannulation methods, numbers of cannulation attempts, and inadvertent pancreatic duct cannulation, pancreatography, and prophylactic placement of pancreatic duct stent.
2. Planned for diagnostic or therapeutic ERCP.
4. Informed consent obtaining from the guardians of all patients, and assent obtaining from patients greater than 10 years of age.
Exclusion criteria 1. Organic gastrointestinal disease such as upper digestive tract stenosis or obstruction.
2. Pancreatitis or use of pancreatic enzyme medication within 7 days.
4. Dermatological disorders such as fresh abdominal wounds, skin lesions, or angioma. Allergy to contrast agents or mirabilite.

Intervention
Surgical details All pediatric patients undergo a comprehensive review and specialist consultations before ERCP. This process aims to ensure an objective and comprehensive analysis, as well as the appropriateness of ERCP, and to rule out contraindications to endoscopy. Patients are asked to undergo routine preoperative laboratory testing (complete blood count, coagulation, blood amylase and lipase concentrations, and hepatic function markers), upper abdominal ultrasonography, computed tomography (CT), magnetic resonance cholangiopancreatography (MRCP), and iodine allergy testing.
Each patient is required to fast for 12 hours before surgery. Duodenoscopy is performed using the JF240V devices (Olympus Corp, Tokyo, Japan). ERCP is conducted by an experienced digestive endoscopy specialist who had performed >30000 ERCPs. The following procedures are completed under radiographic guidance: endoscopic sphincterotomy, balloon dilation, stone extraction, and stenting and naso-pancreatic or naso-bilary drainage tube placement.
Standard post-ERCP treatment Standard treatment will be administered for post-ERCP pancreatitis of both groups, including fasting, pancreatic enzyme control, and uid and electrolyte balance maintenance. Complications such as infection, bleeding, or perforation within 1 month after discharge will be treated accordingly.
Intervention Mirabilite bag is designed with a rectangular shape and in two sizes based on the projected area of the pancreas of the pediatric patients. Children aged ≤6 years receive bags with dimensions of 17 mm × 14 mm, and those aged >6 years received bags with dimensions of 24 mm × 14 mm. Two layers of medical gauze are sewn into rectangular bags, and with four 8-cm attachment bands are sewn to the two longer sides. The bags are used for the topical application of mirabilite to the abdomen and are attached to the patient's back using the attachment bands.

Randomisation and masking
In this study, patients will be randomly assigned into two treatment groups by 1:1 ratio using a block randomization stratified by centres. The randomization is performed before ERCP (about 7h before ERCP, usually on the morning of the procedure).
Mirabilite will be administered in the procedure room before or after ERCP by one investigator in each site who do not participate in data collection and analysis. These investigators and patients will be instructed not to disclose if or when mirabilite is used. Endoscopists and assistances who participated in ERCP procedures will be masked to group allocation. Investigators who collected demographic or procedurerelated data or participated in the assessment of post-ERCP complications will be also masked from group allocation.

Assessments and measurements
Abdominal pain scores (visual analogue scale[VAS] scores) 19 will be recorded for all patients at 24h after surgery. On postoperative days 1, 2 and 3, TNF-α, IL-6, IL-8, diamine oxidase, blood D-lac, and endotoxin of serum will be measured. TNF-α, IL-6, IL-8 will be determined using the ELISA method (sandwich ELISA format). Diamine oxidase will be determined by spectrophotometry (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). D-lac will be determined using modi ed enzymatic spectrophotometry (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The quantitative determination of endotoxin will be performed with a limulus kit (Sigma, St Louis, MO, USA).
According to the criteria diagnosis of post-ERCP pancreatitis, serum amylase levels and upper abdominal ultrasonography will be measured 24 h after ERCP.

Primary outcome
The primary outcome is the incidence of post-ERCP pancreatitis at 24h after surgery. The post-ERCP pancreatitis (PEP) is de ned if a children after ERCP met 2 of the 3 following criteria: classical abdominal pain, a plasma amylase or lipase concentration exceeding 3 times the normal upper limit at 24 hours postoperatively, and radiographic (B-type ultrasonography or CT) suggestive of pancreatitis.
Secondary outcomes: Several secondary outcome measures are also recorded. Abdominal pain is measured 24 h after ERCP using a visual analogue scale (VAS), as follows: 0 points, no tenderness, no pain; 1-3 points, mild but tolerable discomfort and pain; 4-6 points, sleep quality affected by tolerable discomfort and pain; and 7-10 points, strong discomfort and intolerable pain that severely affects sleep quality. The intestinal barrier function assessed by diamine oxidase, D-lactic and endotoxin and proin ammatory cytokine assessed by concentrations of TNF-α, IL-6, and IL-8 are recorded. Inpatient days, the rate of mortality and transfer to the ICU because of severe PEP are also recorded.Safety endpoints Patients are monitored for adverse reactions to mirabilite, including skin damage and diarrhea. The following adverse reactions to ERCP are also monitored: post-surgical intestinal perforation, bleeding, bile duct infection, and other procedure-related complications requiring an extension of the hospital stay. Patients are followed up for 1 month postoperatively. Detailed definitions for other adverse events are provided in Table 1 20 .

Sample size estimation and statistical Analysis
In our previous pilot study, the incidence of post-ERCP pancreatitis in control group was 26% and the incidence in mirabilite group was 16%. The sample size was 260 subjects per group with a of 0.05 and statistical power of 0.8.
We will report our results according to the CONSORT 2010 Statement. The data of all patients who underwent randomization will be analyzed. The qualitative variables will be presented as absolute numbers and proportions. Quantitative variables will be presented as mean ±SD or medians and IQRs as appropriate. The primary endpoint, the incidence of PEP will be shown in absolute frequency and ORs with 95CI, and compared by χ² tests. The secondary endpoints between the two groups will be compared by Mann Whitney U test or t-test as appropriate. All tests are two-sided, and a p value of less than 0.05 is considered statistically significant. Subgroup analysis will be performed according to the patient demographics (age, sex, centre, et al) and the procedure-related parameters. Missing values will be imputed with multiple imputation under the assumption of missing at random. All statistical analyses will be performed with R (version 3.5.1).

Discussion
To our knowledge, our study is the rst to evaluated prophylactic medications for pediatric post-ERCP pancreatitis inmulti-centre randomized controlled trial.
Mirabilite can reduce in ammation, promote the recovery of large intestinal function and the excretion of intestinal toxins, thus reducing damage to the intestinal mucosa. Mirabilite can also reduce symptoms such as abdominal pain, and may also decrease the risk of complications. Therefore, many clinical studies in China have evaluated the use of mirabilite for the treatment of acute pancreatitis. The use of topical mirabilite for post-ERCP pancreatitis prophylaxis may be better tolerated in pediatric patients by its external use nature and according toits safety pro le in treatment of acute pancreatitis compared to rectal, oral, or intravenous applications.
In this study, we aim to assess the e cacy of mirabilite for post-ERCP pancreatitis prophylaxis by comparing the incidence of this condition between the intervention and control groups. Comparisons of the concentrations of in ammatory markers and the translocation of amine dioxide, diamine oxidase, and endotoxin between the two groups will enable a further demonstration of the probable mechanism of action of mirabilite via improved intestinal function consequent to in ammation suppression. This study will also enable an observation of the side effects of mirabilite. We expect that mirabilite will reduce the incidence of post-ERCP pancreatitis and suppress pain, reduce in ammation, and improve intestinal function in patients after ERCP. We expect that mirabilite will be con rmed as an effective and safe treatment option for post-ERCP pancreatitis prophylaxis in pediatric patients.
The approximate date when recruitment will be completed: Oct 22th, 2021. Figure 1 Study design ow chart Figure 2 Treatment schedule and outcome measures.