This study was conducted as a prospective and contrastive intervention trial with a follow-up period of 24 weeks. According to the efficacy, dose and safety of diacerein in the treatment of hand and knee OA reported in literatures 12-14, although patients were followed up 24 weeks, 12 weeks was the primary end point and 24 weeks was the secondary end point. Questionnaire and physical examinations were conducted once every 12 weeks. The primary efficacy parameters were assessed at primary and secondary end points, and the secondary efficacy parameters were assessed at baseline, 12weeks and 24 weeks.
Subjects in this trial were grouped according to their own health situation, medication history and contraindications of drug use. No placebo was included because the constraint of Administration Village Committee. Subjects were assigned to receive chondroitin sulfate or diacerein for 24 weeks.
The selections of test sites
Heilongjiang Province is a historically severe KBD endemic area and located in northwestern China. In accordance with principles of matching natural and social factors, such as geologic environments, economic situations and educational standards, towns were included if they met the following eligibility criteria: (1) the monitoring data of KBD in recent 30 years were complete; (2) historically severe KBD endemic areas; (3) the X-ray detective rate of KBD in children was < 3% in recent 3 years; (4) the number of long-term residents above 45 years old were more than 50. Ultimately, five towns were chosen as our test sites, they were Fanrong Town, Fulu Town, Longanqiao Town, Shaowen Town of Fuyu County and Lianghe Town of Shangzhi City.
A simple questionnaire was performed and data on demographic variables (age and sex) for all residents who were living in above-mentioned towns were gained firstly. Next, radiologic and clinical examinations of both hands and/or feet were performed for villagers aged 45-70 years in order to screen adult KBD patients. Adult KBD patients were diagnosed and those in degree I and II were selected in according to the Diagnosis of Kashin-Beck Disease (WS/T 207-2010 15).
After then, standard questionnaire and physical examinations were conducted for all selected KBD patients in degree I and II. Standard questionnaire mainly included health status, previous medication history, WOMAC pain and stiffness subscales or Assessment for Therapeutic Efficacy on Kashin-Beck Disease scale (WS/T 79-201116). Physical examinations included measurements of patients’ height, weight, liver and renal function, etc. The heights and weights of patients were measured using height and weight scales. Body mass index (BMI) was calculated as the weight in kilograms divided by the square of the individual's height in meters. According to World Health Organization (WHO) criteria, the BMIs were categorized into four grades: underweight (BMI<18.50 kg/m2), normal (18.5≤BMI<25 kg/m2), overweight (25≤BMI<30 kg/m2) and obese (BMI≥30.00 kg/m2).
KBD patients aged 45-70 years in degree I and II would be enrolled in this trial if they had lived in our test sites since they were children (6 years old). Individuals with other joint diseases, such as joint inflammation, metabolic bone diseases, neoplasia, osteoporosis, etc., or coexisting conditions such as a history of diabetes mellitus, stroke, emaciation, long-term use of drugs and cardiovascular, gastrointestinal, kidney, liver, respiratory diseases, or used drugs for OA/KBD in past three months and those who declined to participate in the study were excluded. Finally, 308 patients entered the study cohort.
Group setting and interventions
A total of 308 KBD patients were divided into two different groups (154 in group A and 154 in group B). On the basis of the literatures 12-14 and drug instructions, subjects in group A and group B were assigned to receive chondroitin sulfate tablet (300mg once, three times daily) and diacerein capsule (50mg once, twice daily), respectively. Drinking was not allowed during medical treatment. Patients should stop taking drugs immediately as soon as suspicious side effects appeared.
The collection of blood sample
At 0, 12 and 24 weeks, the morning fasting blood samples of subjects (not less than 3 mL/person) were collected in 5-mL tubes with or without 10% ethylene diamine tetraacetic acid, then left at room temperature for 1-2 h. After that, the blood samples were centrifuged at 3000 rpm for 10 min to separate plasma or serum. The plasma and serum were dispensed into 120 μL aliquots into microcentrifuge tubes that were stored at -80 °C until assay.
The subjects were followed up every 4 weeks by doctors from township hospital. The main jobs of each follow-up were to record amount of drug use and occurrence of AEs or SAEs. Meanwhile, drugs of next phase were distributed to subjects with good compliance. The compliance of subjects was mainly assessed by following up their medication use, including whether they taken drugs according to the instructions, the amount of drugs left over from this cycle and whether they taken other drugs at the same time.
In this trial, Assessment for Therapeutic Efficacy on Kashin-Beck Disease (WS/T 79-2011) 16 was applied to evaluate therapeutic effect of drugs, and the proportion of patients with effective therapeutic effect and overall improvement rate were calculated and set as primary efficacy parameters. Firstly, joint dysfunctions including joint rest pain (0 to 2 points), joint kinesthetic pain (0 to 2 points), morning stiffness (0 to 2 points), maximum walking distance (0 to 2 points) and lower limb mobility (0 to 2 points) before and after treatment were evaluated. Then, the recovery rates of joint functions after interventions were calculated using above joint dysfunctions scores. The therapeutic effects of drugs were judged to be significant, effective or invalid when the recovery rate of joint functions ≥70%, 30%-70% and <30%, respectively. Afterwards, the proportion of patients with effective therapeutic effect was calculated and compared. Finally, the proportion of patients with both significant and effective therapeutic effects (i.e. overall improvement rate) after interventions in different groups was gained and compared.
The WOMAC scale is a validated questionnaire addressing severity of joint pain (five questions), stiffness (two questions) and limitation of physical function (seventeen questions) of OA, researchers can use the whole system or choose parts of it. In this trial, the mean changes in WOMAC pain and stiffness subscales were chosen as the secondary efficacy parameters.
Safety evaluations in this trial included occurrences of AEs, SAEs at each visit and changes of liver and renal functions at 0, 12 and 24 weeks. AEs were mainly listed in the package inserts of drugs, including diarrhea, stomach upset, nausea, dry mouth and dizziness. SAEs were defined as any fatal or life-threatening clinical experience or disabling event, or requiring long-term hospitalization, whether or not it is judged to be related to treatments.
After multiple quality control corrections, eight liver function indexes including albumin (ALB, 34-48 g/L), alkaline phosphatas (ALP, 30-90 U/L), alanine amino- transferase (ALT, 0-40 U/L), aspartate aminotransferase (AST, 0-40U/L), direct bilirubin (DBIL, 1-6.8 umol/L), γ-glutamyl transpeptadase (GGT, 7-50 U/L), total bilirubin (TBIL, 5-21 umol/L), total protein (TP, 65-85 g/L) and four renal function indexes including urea (UREA, 1.8-7.5 mmol/L), creatinine (CREA, 35-79 umol/L), micro albumin (mALB, <20mg/L), N-acetyl-β-D-glucosidase (NAG, 0.3- 14.6 U/L) were measured by use of automatic biochemical analyzer. Reagents for all indexes were purchased from Ningbo Meikang Biotechnology Co., Ltd., Ningbo, China.
Our study was carried out by a well-trained research staff along with doctors, and they were trained before the trial. The training module contents included purpose of this trial, study procedures and how to implement the questionnaire, etc. The instruments used in trial were standardized by local technical supervision bureau. Additionally, to unify various instrument operation methods and formulate standard operation procedures so that the team could complete all diagnosis and follow-up tasks according to unified standard.
Three exports or technologists read same X-ray image and provide their own evaluations based on the Diagnosis of Kashin-Beck Disease (WS/T207-2010) 15 to our team. The diagnosis of KBD patients was finalized by our team according to views of these experts.
A health education program was implemented to our subjects. The purpose and significance of treatment were clarified three times so that subjects could actively cooperate with treatment. Patients changed groups without permission and terminated trial unauthorized were recorded in order to control the rate of loss of follow-up strictly. The corresponding data of subjects withdrew from the trial should be handled statistically.
Research ethics and patient consent
The trial was approved by the Human and Ethics Committee for Medical Research of Center for Endemic Disease Control, Chinese Center for Disease Control and Prevention, Harbin Medical University and the Administration Village Committee in accordance with the Helsinki Declaration. The trial was registered complementally on 31/10/2020, and the registration number in the Chinese Clinical Trial Registry is ChiCTR2000039600 (http://www.chictr.org.cn). The purpose, significance, intended diagnostic and therapeutic methods, dosage and duration of medication and possible AEs or SAEs of this trial were informed to all subjects when they were enrollment in, after then, written informed consents were obtained from them.
Data were analyzed by using SPSS software (version 22.0 SPSS Inc., Chicago IL). Basic characters, BMI, X-ray diagnosis of patients, primary efficacy parameters and other categorical variables were analyzed with Chi-square test. Data of WOMAC pain and stiffness scores was expressed as mean ± standard deviation and analyzed by use of Two-way Repeated Measures Analysis of Variance. Intent-to-treat (ITT) analysis was applied in this study. All tests were two-tailed, and P<0.05 was considered statistically significant.