1.1 Study aim and objectives
The aim of this trial is to pilot study procedures and assess feasibility prior to undertaking a full-scale clinical trial on DST to control body weight in obese patients with NAFLD. The primary objective is to assess the efficacy of DST in weight loss compared to placebo in obese patients with NAFLD. The secondary objective is to determine the effectiveness of DST in metabolic disease-related parameters, such as waist and hip circumferences, body composition, blood analysis, transient elastography analysis, NAFLD-related physical discomfort and obesity-related quality of life. As an exploratory outcome, changes in the gut microbial community will be analysed to evaluate the effect of DST on the gut microbiota.
This study is a randomised, double-blind, parallel-group, stratified (by gender), placebo-controlled pilot clinical trial. It will be conducted at Dunsan Korean Medicine Hospital, Daejeon University, Republic of Korea. A total of 60 participants will be recruited by local advertisements and the hospital bulletin board. All participants will be asked to sign the latest version of the informed consent form approved by the Institutional Review Board (IRB). When subjects voluntarily decide to participate, they will undergo screening, and specialists in Korean Medicine will confirm their eligibility based on the inclusion and exclusion criteria. If a subject has been qualified to participate, they will be invited for a baseline assessment within 2 weeks. At the second visit, subjects will be randomly allocated to either the DST group or placebo group with a 1:1 allocation ratio. Participants will take either DST or the placebo drug for 12 weeks. Participants will visit once monthly for evaluation while taking the test drugs. A follow-up assessment will be performed 4 weeks after the final drug administration. The study flow chart is presented in Figure 1 and a detailed schedule is given in Figure 2.
(1) Adults over 19 years to under 65 years
(2) Obese subjects with body mass index ≥25 kg/m2
(3) Subjects with non-alcoholic fatty liver findings on transient elastography: CAP (Controlled Attenuation Parameter) score over 268 dB/m
(4) Those who have voluntarily signed written consent approved by IRB, after sufficient explanation of this study
(1) Subjects who have had weight changes more than 10% of their previous weight within the last 6 months
(2) Subjects who are already on diet or heavy exercise for weight control purposes
(3) Subjects who had undergone surgical procedures for weight control
(4) Patients with endocrine disease that may affect body weight, such as hypothyroidism, Cushing's syndrome, or those with TSH concentration less than 0.1 uU/ml or more than 10.0 uU/ml
(5) Patients with evidence of chronic hepatitis, such as hepatitis B, hepatitis C, autoimmune hepatitis, and metabolic hepatitis (HBsAg or HCV Ab positive in screening tests)
(6) Subjects with alcohol intake over 210 grams per week for men and over 140 grams for women during the last year
(7) Liver Stiffness Measurement (LSM) of 12.5 kPa or higher on transient elastography
(8) Subjects who have received liver transplants
(9) Subjects who use insulin injections to control blood glucose concentration
(10) Subjects taking thiazolidinediones and high-dose vitamin E that may affect non-alcoholic fatty liver disease
(11) Those who had taken drugs that can be used to treat obesity, antidepressants, contraceptives, oral steroids, amphetamines, phenodiazines, ciproheptadins, female hormones or thyroid hormones within 3 months of screening (Short-term use of prednisone or equivalent corticosteroids less than 10mg/day within 7 days, which may not affect body weight will be allowed [17])
(12) Those whose AST or ALT exceeds 5 times the normal upper limit (200 IU/L)
(13) Kidney dialysis patients or those with creatinine concentration more than twice the normal upper limit
(14) Those who are in seriously unstable medical conditions such as cardiovascular disease, respiratory disease, gastrointestinal disease, hepatobiliary disease, metabolic disease, endocrine disease, renal disease or problems in urinary reproductive system and nervous system
(15) Those who have had diagnosed or have been treated for malignant tumors within the last 5 years
(16) Women who are pregnant or lactating, or subjects who do not agree to use effective methods of contraception during the clinical trial (Oral contraceptives are not allowed during the clinical trial)
(17) Subjects with genetic problems, such as galactose intolerance, Lapp lactase deficiency, or glucose-galactose malabsorption
(18) Subjects with a history of alcohol abuse or drug abuse within the past year
(19) Subjects who had taken other clinical trial drugs within 3 months
(20) Subjects who are thought to be inappropriate for this study
This study is a pilot clinical trial to explore the feasibility of DST in obese subjects with NAFLD. No clinical trial has evaluated the efficacy of DST on weight loss and/or NAFLD. Therefore, we calculated sample size based on an opinion from an expert in medical statistics and clinical trials similar to this study, which had used herbal supplements for weight loss [18, 19]. According to the previous studies, we assumed the mean and standard deviation of body weight would be 2.2 kg and 2.7, respectively. Thus, the sample size was 30 subjects in each group with a significance level of 5%, power of 80% and a drop-out rate of 20%. A total of 60 subjects will be recruited and divided into two groups of 30 subjects each.
1.5 Randomisation and allocation concealment
Subjects will be randomly allocated to either the DST or placebo group in a 1:1 allocation ratio. The gender distribution for each group will be controlled by an independent statistician using the stratified block randomisation method. The statistician will use SAS version 9.4 software (SAS Institute, Cary, NC, USA) to generate a random allocation list and will send the list to the pharmaceutical company for packing. The allocation for each subject will be sealed in an opaque envelope and will not be disclosed until the clinical trial is completely over, unless serious adverse events happen.
A statistician who is not involved in the clinical trial will send the randomisation codes to the pharmaceutical company for packing to maintain blindness of the assessors and the subjects. The placebo drug was manufactured in a form similar to DST by the same pharmaceutical company. It was confirmed in advance that the taste and flavour were similar (Additional file 1). According to the random sequence, drugs will be packed in the same form and will be delivered to the hospital. During the trial period, the pharmacist will supply the drug sequentially according to the randomisation code. To evaluate whether the blinding is successful, a quick questionnaire will be completed at the end of the study.
Participants in both groups will take DST or the placebo drugs three times daily (3 g/packet at a time), for 12 weeks. The drugs will be distributed on a monthly basis. Both the DST and placebo drugs will be manufactured by Hanpoong Pharmaceutical (Wanju, Republic of Korea), according to Good Manufacturing Practice guidelines. Both drugs will be prepared in the form of dark brown granules. The constituents of DST are shown in Table 1. The DST granules contain DST soft extract, lactose hydrate and corn starch. The placebo drug contains lactose hydrate, corn starch, caramel colour and ginseng flavoured powder. To assess adherence to the intervention, the subjects will be told to bring the empty packet of the ingested test drug and the leftover. Subjects who have < 70% drug compliance at the end of the study will be dropped-out. The research team will periodically contact subjects by phone or text message to improve compliance and check for safety.
1.8 Permission for use of concomitant drugs
Prohibited drugs include agents that can affect the primary outcome of body weight, such as anti-obesity medications, antidepressants, amphetamines, oral contraceptives, hormone drugs, oral steroids, cyproheptadine and phenothiazine. Agents that can affect NAFLD, such as insulin, thiazolidinedione and high-dose vitamin E (>800 IU per day) are also prohibited. Lipid-lowering agents, anti-diabetic medication and hepatoprotective agents are not included as prohibited drugs but subjects should not take as much as possible unless they were taking it from screening visit. Drugs that can cause significant changes in the gut microbial community, such as antibiotics and lactic acid bacteria, are not prohibited, but subjects should avoid them as much as possible to minimise the effect on intestinal microorganisms.
The subjects will be told to maintain their usual exercise routine and not to change the amount of exercise or kind of exercise. The Korean version of the International Physical Activity Questionnaire Short Form will be administered to evaluate physical activity status [20]. Daily intake of a low-calorie 20–25 kcal/kg diet will be recommended to increase the weight loss effect. Food intake status will be investigated using 3-day dietary recall before and after the drug intervention. Can-Pro (a computer aided nutritional analysis program for professionals version 5.0, Korean Nutrition Society, Seoul, Korea) will be used for the dietary data analysis. Can-Pro is commonly used in Korea to assess nutrient intake because it has a database that reflects the Korean diet.
1.10.1 Anthropometric measurements
Body weight will be assessed at every monthly visit, including the screening visit. Waist circumference and hip circumference will be measured according to World Health Organisation’s instructions at every monthly visit [21]. Anthropometric measurements will be measured by the same person to minimise errors.
Body composition, such as fat percentage, fat mass and lean body mass, will be measured at every monthly visit using a body composition analyser (InBody 770; Biospace Ltd., Seoul, Korea). The body mass index will be calculated as weight in kilograms divided by height in meters squared.
2.10.3 Non-alcoholic Fatty Liver assessment
Transient elastography, also called a Fibroscan analysis (Fibroscan 530 Compact, Echosens, Paris, France) will be completed by a trained operator after at least 2 hours of fasting. The transient elastography analysis is a non-invasive test to assess liver steatosis and fibrosis. Recent studies have shown that the controlled attenuation parameter (CAP) value is a reliable marker to quantify hepatic steatosis [22-24]. Liver stiffness measurement (LSM) values will also be obtained.
2.10.4 NAFLD-related symptoms
Physical symptoms related to NAFLD will be assessed using a visual analogue scale (VAS). The VAS is one of the most widely used measures to evaluate subjective symptoms [25-27]. Participants will be asked to mark their degree of fatigue and abdominal discomfort on a 100-mm horizontal line, where 0 indicates “no fatigue/discomfort” and 100 indicates “the worst fatigue/discomfort”. Assessors will record the length of the point marked by the subject.
2.10.5 Korean version of obesity-specific quality of life (KOQOL) questionnaire
The KOQOL questionnaire was developed to evaluate quality of life in obese patients by reflecting the cultural and linguistic expression of Koreans [28]. The reliability and validity of the KOQOL has been verified (Cronbach's α: 0.6–0.8) [28]. The KOQOL consists of 15 questions and six domains: psychological health, physical health, work-related quality of life, routine life, sexual life and diet distress. The total score is 60 and the higher the score, the lower the quality of life.
2.10.6 Obesity pattern identification questionnaire
The obesity pattern identification questionnaire was developed by the Korean Institution of Oriental Medicine to categorise obese patients into different constitutions, which is the fundamental basis of Korean traditional medicine[29, 30]. Obese subjects can be classified into six patterns: spleen deficiency, yang deficiency, food accumulation, liver depression, phlegm fluid and blood stasis. The questionnaire will be completed at the screening visit. The obesity pattern of each subject can be used in a subgroup analysis to evaluate whether treatment effects differ according to the obesity pattern.
Blood testing will be done at the screening visit, after 12 weeks of drug intervention and at the follow-up visit (week 16). The blood tests will include aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, direct bilirubin, gamma-glutamyl transpeptidase, blood urea nitrogen, creatinine, protein, albumin, uric acid, white blood cells, red blood cells, erythrocyte sedimentation rate, haemoglobin, haematocrit and platelets. It will also include glucose, insulin, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and high sensitivity C-reactive protein. Haemoglobin A1c will be examined at the screening and after 12 weeks of drug intervention. Thyroid-stimulating hormone, HbsAg, anti-HCV and urine human chorionic gonadotropin (only women of childbearing age) will be evaluated at the screening visit to determine eligibility.
Blood pressure, heart rate and body temperature will be measured at every visit using an automated device. Participants will be asked if they have any physical discomfort. The frequency of adverse events will be presented for the safety assessment. Any adverse events will be recorded on the electronic case report form (eCRF) after assessing severity and causality. If a severe adverse event occurs, it will be reported to the IRB as soon as possible.
2.10.9 Gut microbiome analysis
The subjects will be instructed to collect faecal samples within 24 hours before visits on weeks 0 and 12, and freeze them until the visit. A stool collecting kit and instructions will be provided to collect the faecal samples, which will be stored at − 80°C for further analysis. The microbiome community will be evaluated by 16s rRNA sequencing analysis. The relative abundance of the bacterial taxa in each sample will be obtained, and significant differences in the bacterial taxa between the DST group and placebo group will be evaluated.
The primary outcome of this study will be the mean change in body weight from baseline to the end of the 12-week intervention. Secondary outcomes will include body composition, anthropometric measurements. A transient elastography and physical symptoms questionnaire will be completed at every monthly visit. As this is a pilot trial, the recruitment rate and completion rate will be calculated to determine feasibility of the study design.
The Korea Institute of Oriental Medicine will be responsible for quality control throughout the study. An independent clinical research associate will regularly monitor the overall process to determine if the trial is being performed in accordance with the protocol, standard operating procedures and the guidelines for good clinical practice. Monitoring will ensure that the investigators have been trained properly and that written consent has been obtained by the appropriate procedures. Monitoring will also include source data verification, and a check for maintenance and completeness of essential documents and the eCRF.
2.13 Data collection and management
All source documents, including informed consent forms, questionnaires and worksheets will be collected in compliance with standard operating procedures. The data will be collected on an electronic data capture system through eCRFs using the Medidata RAVE data management system (Medidata Solutions Inc., New York, NY, USA). The principle investigator will ensure anonymization of the subjects and the confidentially of all information collected during the course of the trial. The final dataset of the clinical trial will be accessible to all authors. The principle investigator will coordinate dissemination of data. The results will be submitted for publication in peer-reviewed journals.
2.14 Statistical analysis
The statistical analysis will be conducted by an independent statistician using SAS version 9.4. The significance level will be set to 5% in a two-tailed test. The multiple imputation method will be adopted for missing data. The intention-to-treat (ITT) principle will be applied for the primary analysis. The ITT principle will include subjects who meet the full analysis set criteria, including subjects who had the primary outcome assessed once or more except for the baseline measures and who took the test drug at least once. The per-protocol set will be analysed in a supplementary analysis, which will only include subjects who have completed the study as the supplementary analysis.
The independent t-test or Wilcoxon rank-sum test will be used for continuous variables, and the chi-square test or Fisher’s exact test will be used for categorical variables. Analysis of covariance will be performed for the primary outcome and the continuous data of the secondary outcomes. The model will include baseline values and gender as covariates and treatment as a fixed effect. Student’s t-test or Wilcoxon’s signed-rank test will be used to compare before and after the treatment within each group. Categorical data will be analysed using the chi-square or Fisher’s exact test. Additionally, repeated measures-analysis of variance will be used to compare differences in trends per visit.
The incidence of serious adverse events and adverse events related to the treatment will be analysed. The percentage of subjects who experienced one or more side effects during the study will be analysed and presented using a descriptive analysis for each group.