Study design and recruitment
This clinical study is a single-center, randomized, double-blind and placebo-controlled trial comparing TXYF granule with placebo in patients with IBS-D, which will be performed lasting totally 22 weeks. At the initially run-in period (week -2-0), we will assess eligibility and seek informed consent. Then comes to the treatment period (week 0-12), during which eligible subjects will be randomly assigned to one of two groups (TXYF granule group and placebo group) in a 1:1 ratio and get blinded treatment of 12 weeks. Finally，we will have a follow-up (week 20) two months after the treatment. This study involves 6 site visits (week -2, week 0, week 4, week 8, week 12 and week 20) and 12 call visits (weekly call follow-up during the treatment period), eventually the primary outcome and the secondary outcome will be evaluated. We will obtain the intestinal mucosae from partial IBS-D patients (n=10 for each group) before and after treatment for assessment of mechanistic outcome. The process of the study and visit schedule are schematically in Figure 1 and Figure 2. This clinical trial protocol refers to The Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) 2013 Checklist detailed in Additional file 1.
All 120 IBS-D patients participating in this study will be recruited from the gastroenterology department or ward at the First Affiliated Hospital of Guangzhou University of Chinese Medicine. And we will additionally recruit 10 healthy controls who will undergo intestinal mucosal biopsy for investigation of transcriptome profiling. Written informed consent which contains additional provisions for collection and use of colon samples will be obtained from each participant before enrollment. The trial process conforms to the Declaration of Helsinki (2008) (24). The Clinical Trials and Biomedical Ethics Committee of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (No.ZYYECK YJ125) has approved our trail.
- Patients have to satisfy the diagnostic criteria of IBS-D defined by ROME IV(25).
- The score of IBS Symptom Severity Score (IBS-SSS) is more than 75 at baseline visit (26).
- Patients have to satisfy the diagnostic criteria of diarrhea syndrome with liver depression and spleen deficiency based on TCM syndrome differentiation(27).
- The age is between 18 and 70 years old.
- Patients must make an agreement to participate and sign a written informed consent.
- Patients have a history or current evidence of inflammatory bowel disease, gastrointestinal bleeding or abdominal surgery.
- Patients have a history or current evidence of other organ diseases which influence gastrointestinal motility, such as hyperthyroidism, diabetes and chronic renal impairment.
- Patients have a history or current evidence of serious damages of the heart, liver, kidney, blood system or immune system.
- Patients used therapeutic medications for IBS-D or other diseases which could affect the trial within two weeks before enrollment.
- Patients are currently pregnant and lactating.
- Patients have an allergy to trial medication.
- Patients have a history of neurological disease or mental illness.
- Patients have participated in other clinical studies.
TCM syndrome differentiation
TXYF is typically applied to IBS-D patients with liver depression and spleen deficiency according to the expert consensus on diagnosis and treatment of irritable bowel syndrome in traditional Chinese medicine (2017)(27). Patients who have two primary symptoms plus one secondary symptom, integrated with signs of the tongue and pulse will be differentiated as syndrome of liver depression and spleen deficiency and enrolled:
- Primary symptoms: (1) abdominal pain accompanied by diarrhea; (2) prone to irascible or irritable
- Secondary symptoms: (1) distending pain in hypochondrium; (2) reduced appetite; (3) presented with fatigue
- Auxiliary symptoms: light and enlarged tongue or tooth-marked tongue with white greasy coating; string pulse
Randomization and blinding
Randomization scheme that generating a list of 120-case randomization sequence utilizing the stratified block randomization method by computer will be implemented by an independent statistician, which guarantees that enrolled patients will be evenly allocated to TXYF granule group or placebo group. Then 10 participants of each group will be randomly selected for RNA sequencing and whole transcriptome analysis. Concealment of allocation code relies on an opaque envelope. This trial requires that all participants and researchers involved in drug distribution, outcome evaluation and data analysis are completely blinded to allocation. Occurrence of severe adverse events or other unpredictable events allows unblinding under the permission of the principal investigator.
Experimental medication preparation and quality control
TXYF granule and placebo, uniformly manufactured by Guangdong YiFang pharmaceutical co., LTD under the guidance of Good Manufacturing Practices (GMP), which have the identical appearance and nearly similar taste. TXYF granule is made up of Rhizoma Atractylodis Macrocephalae, Paeoniae Radix Alba, Citri Reticulatae Pericarpium and Saposhnikoviae Radix (see table 1). These four crude herbs are weighed in proportion of the prescription, subsequently washed and crushed, and the thickness of the slices is 0.3-1.5 cm. Rhizoma Atractylodis Macrocephalae and Paeoniae Radix Alba are fired while Citri Reticulatae Pericarpium and Saposhnikoviae Radix are raw for use. According to the optimized implementation scheme for decocting, extracting and concentrating of the crude herbs, the ointments are prepared with a relative density of 1.07 to 1.09 g/ml, and then the ointments are screened to get granulated ointments. Finally, after spray drying and crushing, the granules are packed in sealed opaque packages. Placebo consisting of maltodextrin, dextrin, experimental medication extract (<5%), kudingcha extract and pigment is also prepared granules. The specific content is shown in Table 2.
The quality inspection department of Guangdong YiFang pharmaceutical company was responsible for the quality control of experimental medication based on pharmacopoeia and national drug quality standards. Thin layer chromatography and high-performance liquid chromatography-mass spectrometry were respectively utilized for qualitative and quantitative testing. Among them, the content of paeoniflorin in extract of Paeoniae Radix Alba, the content of hesperidin in Citri Reticulatae Pericarpium extract and the total content of Prim-O-Glucnylcimifugin and 4′-O-β-glucosyl-5-O-Methylvismmiside in Saposhnikoviae Radix extract were 8.97%, 0.84% and 0.7% respectively. Microbial limit of each herb extract was qualified.
TXYF granule or placebo will be orally administered one packet (strength: 8.1g) after dissolved twice a day (one hour after the meal) for 12 weeks. Over the course of the study, participants are required to discontinue therapeutic medications associated with IBS-D. Simultaneously, probiotic foods, functional foods and dietary supplements are also prohibited. Participants are obligated to fill in study diary daily involved medication administration record, concomitant treatments and dietary questionnaire which avail to check compliance and statistically rectify dietary bias. Onset of exacerbated IBS-D symptoms allows rescue medications for less than one week, which need to be documented in the electronic Case Report Form (eCRF).
Colonoscopy and biopsies
Participants who didn’t have a colonoscopy one year prior to enrollment will undergo colonoscopy and sigmoid mucosal biopsy (week 0) for assessment of microscopic colitis, from which 10 participants of each group will be randomly selected to undergo mechanistic assessment and 2 additional samples of sigmoid mucosa will be obtained. After 12 weeks of treatment, these 20 participants will undergo a second colonoscopy and two sigmoid colon biopsies (week 12). Besides, 10 healthy subjects will be recruited to undergo colonoscopy and two sigmoid colon biopsies. The obtained samples will be promptly snap-frozen in liquid nitrogen and then stored at -80℃ for the next-generation high-throughput RNA sequencing (RNA-seq) and quantitative Real-time PCR (qRT-PCR). Samples of intestinal mucosa will be destroyed after use.
RNA isolation, library preparation and sequencing
According to the manufacturer’s instructions, total RNA will be isolated from sigmoid colon samples using TRIzol (Life Technologies). RNA purity, concentration and integrity by using NanoPhotometer spectrophotometer (IMPLEN, CA, USA), Qubit RNA Assay Kit and RNA Nano 6000 Assay Kit respectively will be measured. 2-3ug total RNA per sample as input material is needed for cDNA library construction and subsequent PCR amplification and library quality will be evaluated lastly on the Agilent Bioanalyzer 2100 system. RNA-seq will be conducted on an Illumina Hiseq 2000 platform.
RNA-seq data processing and bioinformatics analysis
Raw data of fastq format through quality control including removing a series of hybrid reads and low-quality reads will be filtered as clean data. Clean data then will be mapped to the human reference genome employing HISAT2 v2.0.4 and the aligned reads of per sample will be assembled by StringTie (v1.3.3). Both ncRNAs and coding transcripts will be normalized and expressed as fragments per kilo-base of exon per million fragments mapped (FPKM). Differential Expression will be analyzed by using Cuffdiff. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes will be implemented by GOseq R package and KOBAS software respectively. To predict the targets of ncRNAs, databases including TargetScan(www.targetscan.org), starbase(http://starbase.sysu.edu.cn/), deepbase2.0(http://deepbase.sysu.edu.cn/) and circInteractome (https://circinteractome.nia.nih.gov) will be used. Visual networks of ncRNAs and mRNAs will be constructed by Cytoscape software.
The primary outcome of our trial is the response rate of IBS-Symptom Severity Score (IBS-SSS). Responder is defined as a 50% or more reduction in IBS symptoms compared to baseline (28). IBS-SSS is a hitherto unique symptom severity questionnaire of IBS, which evaluates the condition of abdominal pain, abdominal distension and defecation satisfaction. This definition of a responder that reports of a 50% or more reduction in IBS symptoms is less disturbed by initial severity of IBS. IBS-SSS will be assessed at baseline, week 4, week 8, week 12 and week 20 while response rate of IBS-SSS will be assessed at week 4, week 8, week12 and week 20.
Participants will receive 12 call visits (week 1-12) and the following question will be asked: “In the past week, have you had adequate relief of your irritable bowel syndrome pain and discomfort?”. Participants only need to answer "yes" or "no". Responder is defined as a "yes" answer to the aforementioned question for at least 6 of the 12 weeks. AR will be accessed at week 12.
- Irritable Bowel Syndrome-Quality of Life Questionnaire (IBS-QOL)
IBS-QOL reflects the union of the physical and mental well-being covering eight dimensions, which will be assessed at baseline, week 4, week 8, week 12 and week 20.
- Long-term efficacy assessment
Participants will be followed up for long-term effectiveness 2 months after treatment period(week 20). IBS-SSS response rate and IBS-QOL will be evaluated.
We will analyze the whole transcriptome profiling of intestinal mucosae from TXYF granule group (n=10) and placebo group (n=10) at baseline and week 12 and healthy subjects (n=10) at baseline. The whole set of transcriptional deviations including mRNA, miRNA, lncRNA, circRNA and functional networks of ncRNAs and mRNAs in IBS-D will be disclosed, which makes us to seek for the underlying molecular mechanisms and find out specific diagnostic biomarker. Meanwhile, therapeutic targets and pathways about TXYF in IBS-D will be clarified. On an Illumina Hiseq 2000 platform, the preparation of whole-set transcriptome libraries and deep sequencing will be executed by Novogene Bioinformatics Technology Cooperation (Beijing, China).
Participant will receive relevant laboratory test at baseline and week 12, including liver and kidney function (ALT, AST, Scr, BUN), blood routine test, urine routine test and fecal occult blood test. The other test includes electrocardiograph examination.
Adverse events (AEs) defined as any unpredictable and unexpected nocuous effects resulting from TXYF granule or placebo granule throughout the trial will be recorded in detail by a research assistant, including symptom, sign, severity, start date, duration, laboratory result, intervention and outcome at every visit. Once severe adverse events (SAEs) occur, it must be immediately reported to the principal investigator, the ethics committee of the hospital, and the Guangdong Food and Drug Administration within 24 h and subject safety should be the priority. If necessary, principal investigator has the right to terminate this trial.
Sample size calculation
The sample size calculation of this superiority trial depends on our previous pilot study and literature . It can be speculated that the response rates of TXYF granule and placebo will reach 70% and 40% respectively. We calculate by the software Gpower3.1 under the setting of 80% power and 5% type I error that 42 cases of each group are needed. With a drop-out rate of 15%, accumulated 100 cases are needed and we expect to recruit 120 IBS-D patients.
Data management and monitor
Data management relies on a convenient follow-up system maintained by Empower Electronic Data Collection (EDC) (Solutions, shanghai, China) and the company is also responsible for converting paper study diary and any other paper source documents (dietary questionnaire) to electronic version. The collected data from participants will be kept absolutely secret and the source data will be just accessible to the data administrator and statistician. All the paper source documentation about this trial will be properly preserved five years after the end of the trial.
Our group made up of a principal investigator, 3-5 clinicians, 2 research assistants, a data administrator, a drug manager and a statistician will receive rigorous training and perform this trial in accordance with the Standard Operating Procedures (SOP) and Good Clinical Practice (GCP). This trial is unconditionally subject to the supervision and monitor of the Science and Technology Department of the First Affiliated Hospital of Guangzhou University of Chinese Medicine. This independent department is responsible for the annual audit of our trial.
Clinical data analysis
Based on the principle of intention-to-treat (ITT), all randomly assigned participants will be included in the statistical analysis. For participants who prematurely terminate this trial, outcomes of last visit will be used as the final outcomes. Statistical analyses will be completed by SAS (version 9.4, SAS Institute, Cary, NC). Continuous variables will be presented in the form of means and standard deviations while the categorical variables will be presented in the form of counts and percentages. The primary outcome (week 4, week 8, week 12 and week 20) and the secondary outcome AR (week 12) will be assessed by using χ 2 test or Fisher’s exact test. Another secondary outcome IBS-QOL (baseline, week 4, week 8, week 12 and week 20) will be assessed by using T-test or Mann-Whitney U-test accompanied with Wilcoxon test. A Two-sided P<0.05 indicates statistical significance. For missing data, sensitivity analysis will be conducted and the optimal approach to the imputation of missing data will be proposed.
Mechanistic data analysis
The differential expression analysis about mRNA, miRNA, lncRNA and circRNA for all pairwise comparisons: IBS-D patients versus healthy controls, TXYF granule (pre-treatment) versus placebo (pre-treatment), TXYF granule (post-treatment) versus placebo (post-treatment), TXYF granule (pre-treatment) versus TXYF granule (post-treatment) and placebo (post-treatment) versus placebo (post-treatment), will be performed by Cuffdiff. An adjusted P <0.05 (Student’s t-test accompanied with Benjamini-Hochberg FDR adjustment) will be used as the cut-off for significantly differentially expressed genes. Pearson correlation test will be used for target genes prediction.