2.1 Objective
The aim of this study is to assess the therapeutic effect of JGT in the relief of DOMS symptoms. The primary outcome is the difference between the JGT and placebo groups in pain intensity, measured every 12 hours from 0 to 72 hours after exercise. Our secondary outcomes include the pain threshold in the calf muscles, calf circumference, and serum markers of muscle damage. Furthermore, exercise-induced shifts in serum and urinary metabolites will be analyzed using a metabolomics-based analysis.
2.2 Study Design
This will be a randomized, placebo-controlled, cross-over design, investigator-initiated clinical trial. The trial will be conducted at Pusan National University Korean Medicine Hospital. Thirty healthy male adults will be recruited by hospital bulletin boards and local advertisements. Subjects who voluntarily wish to participate in this study and provide written informed consent will undergo a screening assessment to determine their eligibility. Subjects will be hospitalized for 4 days. On the first day, the subjects will perform eccentric exercise for 1 hour to induce DOMS. After exercise, the subjects will be administered either JGT or placebo for 3 days. After a more than 1 week wash-out period, the subjects will repeat the same process with the other drug. The duration of the wash-out period was calculated based on the major component of JGT with the longest half-life. As glycycoumarin has the longest half-life (T1/2) of 14.9 hours (20), we decided that the wash-out period should be 6.21 days or more, which is 10 times the half-life of glycoumarin needed to minimize the carry-over effect of JGT. The overall study flow is shown in Fig. 1. The detailed schedule is given in Fig. 2 and Table 1.
2.3 Participants
2.3.1 Inclusion criteria
(1) Subjects who have experienced muscle soreness with a numeric rating score (NRS) > 5 within 24 to 30 hours after exercise
(2) Healthy male subjects from 19 to 35 years of age without any recognized disease
(3) Subjects with a body mass index (BMI) > 18.5 kg/m2 and < 30 kg/m2
(4) Subjects who have not exercised regularly for 2 consecutive weeks or more in the last 6 months or more
(5) Subjects who have voluntarily signed the written consent approved by institutional review board (IRB) after sufficient explanation prior to the study
2.3.2 Exclusion criteria
(1) Subjects who have had an open wound or inflammatory disease within the last 6 months
(2) Subjects with neurological or muscular disorders that may affect muscle strength
(3) Subjects with missing limbs
(4) Patients with a seriously unstable medical condition during a physical examination, such as cardiovascular disease, respiratory disease, gastrointestinal disease, hepatobiliary disease, metabolic disease, endocrine disease, renal disease, urinary disease, or problems with the nervous system or mental health
(5) Those who are taking steroids, analgesics, muscle relaxants, or other medications that the researchers decide to be inappropriate, such as antispasmodics, antidepressants, antidiarrheals, antibiotics, and thrombolytics
(6) Subjects with a history of alcohol abuse or drug abuse within the past year
(7) Subjects who have taken other clinical trial drugs for less than 3 months
(8) Subjects with genetic problems, such as galactose intolerance, Lapp lactase deficiency, or glucose-galactose malabsorption
(9) Those who are unwilling to or unable to follow the study guidelines
(10) Others whom the researcher decides to be inappropriate for this study
2.4 Randomization and allocation concealment
Subjects will be randomly allocated to either the JGT or the placebo group with equal probability. An independent 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 random allocation list will assign 30 subjects to group A or B. According to the cross-over design, both groups will take JGT and the placebo drug. Subjects in group A will take JGT and those in group B will take the placebo drug during their first hospitalization.
2.5 Blinding
The statistician will send the randomization code to the pharmaceutical company for packing to maintain blindness on both the assessors and the subjects. JGT and the placebo drug will be manufactured by the same pharmaceutical company. The placebo drug will be manufactured in a form similar to the JGT. It was confirmed in advance that the taste and flavor will be similar. According to the random sequence, the drugs will be packed in the same form and will delivered to the hospital. The pharmacist will supply the drug sequentially during the trial period, according to the randomization code. The blinding will be maintained until all 30 subjects have completed the study and the database is locked. To evaluate whether the blinding was successful, a quick questionnaire will be conducted at the end of each hospitalization asking “which test drug do you think you were allocated to, JGT or placebo drug? And why do you think so?”
2.6 Exercise protocol for inducing muscle soreness
A standardized treadmill exercise protocol will be performed on the first day of hospitalization to induce DOMS. Previous studies have shown that downhill running protocols induce stress in the gastrocnemius muscles, as it is an unfamiliar eccentric exercise (29-31). The subjects will step backwards on a moving treadmill inclined at 13°. Starting with the right leg, the exercise will persist for 1 hour at a speed of 2.2 km/hour. The subjects will carry a 5–10 kg weight belt to ensure adequate stimulation to the muscles.
2.7 Intervention
After the treadmill exercise, the subjects will be administered either JGT or the placebo drug, three times daily before meals for 3 days (9 times in total). Both the JGT and placebo drug will be manufactured by Kyungjin Pharmaceutical Co., Ltd. (Gyeonggi-do, Republic of Korea), according to Good Manufacturing Practice guidelines. Both drugs will be prepared in the form of light brown granules. The constituents of each drug are shown in Table 2. After the first hospitalization, subjects will be re-admitted to the hospital after the wash-out period of more than 1 week. During the second hospitalization, subjects will be administered the other drug to the one taken before in accordance with the cross-over design. No over-the counter drugs or prescription-based medicines will be permitted throughout the clinical trial. If a subject already has taken a medication during the trial or the subject is suffering from a medical condition that needs medication, the subject will be dropped from the trial.
2.8 Outcome measures
2.8.1 Pain intensity
The primary outcome will be the visual analog scale (VAS) score to measure pain with motion. To assess the perception of DOMS after the treadmill exercise, four separate baseline pain intensity measures will be recorded: the numeric rating scale (NRS), a VAS with motion, a VAS at rest, and a VAS after walking downstairs. Pain intensity will be measured before the exercise, immediately after the exercise, and 1, 6, 12, 24, 36, 48, 60, and 72 hours after the exercise.
The VAS is one of the most widely used measures of pain intensity, determined on a 100-mm horizontal line, where 0 indicates “no pain” and 100 indicates “the worst imaginable pain” (32, 33). Assessors will record the length of the point marked by the subject. For the NRS pain score, the subjects will be asked to report overall pain intensity on a single 11-point numeric scale, with 0 indicating “no pain” and 10 representing “the worst imaginable pain” (33). Although studies have shown that both VAS and NRS have similar sensitivity in pain assessments, VAS is believed to have a higher degree of precision (34, 35). On the other hand, NRS has the advantage that it can be used effectively across all type of disorders because it is easy and simple. Taking advantage of each methods, VAS will be used to evaluate the detailed pain intensity with motion and NRS will be used to evaluate the overall pain intensity.
To assess time-weighted overall pain intensity, the sum of the pain intensity differences (SPID), which is an outcome measure that summarizes the treatment response over a period of time, will be calculated using the NRS scores that were evaluated before exercise and at 24, 48, and 72 hours after exercise (36). The SPID72 value will be obtained according to the following calculation: (1) PID (pain intensity difference) = NRSt – NRSbaseline (2) SPIDt = ∑PIDt * (time in hours elapsed since the previous observation). A higher SPID72 value indicates larger differences in pain intensity over the 72 hours after exercise.
2.8.2 Calf circumference
The circumference around both legs will be measured at the level of acupuncture point BL57, horizontally on the ground to obtain a constant reference point. BL57 is in the middle of the calf between the two heads of the gastrocnemius muscle. After marking the acupuncture point with a pen for repeatability, the same measurer will use the same tape to repeat the measurements. The average value will be calculated after measuring three times consecutively. Calf circumferences will be measured before the exercise, immediately after the exercise, and 1, 6, 12, 24, 36, 48, 60, and 72 hours after the exercise.
2.8.3 Pain threshold
Pain threshold will be measured using a digital algometer (model FPX25, Wagner Instruments, Greenwich, CT, USA). The point of measurement will be acupuncture point BL57 in both calves, at which the rubber tip will be pressed perpendicularly into the muscle with a consistently increasing force of 0.25 kg/cm2/sec (37). The values will be obtained when the subject indicates the onset of pain. Each leg will be measured three times after brief resetting times. Average values will be obtained for further analysis. Pain threshold will be measured immediately after measuring calf circumference.
2.8.4 Muscle damage and inflammation
Venous blood samples will be collected to assess muscle damage and inflammation. Plasma CK activity will be used to assess muscle damage. C-reactive protein (CRP) and interleukin-6 (IL-6) concentrations will be measured to evaluate the inflammation induced by eccentric exercise. Whole blood samples will be obtained and centrifuged to isolate the serum. The samples will be stored at −80℃ and analyzed at the end of the study. As plasma concentrations of the markers will differ depending on the reaction time, each marker will be collected at the appropriate time (38, 39). Blood samples for CK activity will be collected at six different time points: before exercise, and 12, 24, 48, 60, and 72 hours after the exercise. Blood samples for the inflammation markers will be collected at six different time points, including before the exercise, immediately after the exercise, and 1, 6, 12, and 24 hours after the exercise.
2.8.5 Safety assessment
Blood pressure and heart rate will be measured every day using an automated device. In addition, blood tests will be conducted before and after the drug intervention during each hospitalization. The blood tests will include aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, gamma-glutamyl transpeptidase, protein, albumin, blood urea nitrogen, creatinine, red blood cells, hematocrit, hemoglobin, white blood cells, and lipids.
2.9 Exploratory outcomes
2.9.1 Metabolomics analysis
During the hospitalization, plasma and urine samples will be obtained three times (before the exercise, 1 hour after the exercise, and 72 hours after the exercise) to examine the drug-induced and exercise-induced changes in metabolites. Urinary samples and plasma will be aliquoted into empty vials and maintained at −80℃ until further analysis. When the trial has been completed, all samples will be analyzed simultaneously. Non-targeted analysis by mass spectrometry will be used to understand the metabolic changes after exercise and the drug intervention, which is called “sportomics” (11-13). Briefly, the supernatants will be obtained by adding acetonitrile or methanol and the solution will be injected into an ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS, Synapt G2Si; Waters, Milford, MA, USA) system. Information from the mass data, including retention time, ion intensity, and m/z will be extracted. After alignment and normalization, the multivariate data matrix will be exported into SIMCA-P (Umetrics, Umea, Sweden) for a multivariate statistical analysis. The ions contributing to separation of the JGT and placebo groups will be further investigated by searching the METLIN and human metabolomics databases.
2.9.2 Other outcomes
The degree of pain induced by eccentric exercise can vary depending on the usual momentum and body composition. To determine the effects of usual physical activity and body composition on DOMS symptoms, the Korean version of the International Physical Activity Questionnaire Short Form (IPAQ-SF) and a body composition analysis will be performed on the first day of hospitalization. The IPAQ-SF consists of seven questions asking about the amount of time spent per week for activities (walking and moderate and vigorous activities) and sitting (11, 12). Total weekly physical activity for each activity category will be estimated by a metabolic equivalent energy expenditure (11). Body composition parameters, such as body fat percentage, fat mass, and muscle mass will be obtained with the InBody 720 instrument (Biospace, Seoul, Republic of Korea).
2.10 Sample size
As no clinical trial has evaluated the efficacy of JGT on DOMS, the sample size calculation was impossible. A study estimating the sample size for a pilot study proposed 15 subjects per treatment arm as an optimal sample size for the standardized effect size with 90% power and two-sided 5% significance (8).
2.11 Statistical analysis
The statistical analysis will be conducted by an independent statistician using SAS version 9.4 (SAS Institute). The significance level will be set at 0.05 in a two-tailed test. All continuous data will be presented as means and 95% confidence intervals (CIs). Categorical data will be reported as frequencies and percentages. 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 (FAS) criteria, including subjects who had the primary outcomes assessed once or more except for the baseline measures and who took the test drug at least once. Subjects who do not meet the eligibility criteria, those who did not take the test drug throughout the study, and those who have never been evaluated after the screening visit will be excluded from the FAS dataset. The per-protocol set will be analyzed, which will only include subjects who have completed the study as a supplementary analysis.
The baseline characteristics of the study subjects will be presented using a descriptive analysis for each group. The primary outcome will be the differences between the VAS score with motion measured at various intervals from 0 to 72 hours after exercise. A mixed-effect model repeated measures (MMRM) model will be used to assess differences along with the time effect. The dependent variable will be the change in the VAS score (pain with motion) measured 0 to 72 hours after exercise. The model will include treatment and protocol-specified visits as fixed effects, and subjects as a random factor. Dunnett’s test will be used for multiple comparisons. The results of the secondary effective analysis will be analyzed using the same method as the primary outcome, in the case of continuous variables with time variation. Additionally, 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 analyzed using the chi-square or Fisher’s exact test. Missing values by maximum likelihood will be considered when the MMRM model is used. The last observation carried forward method will be adopted for missing values for Student’s paired t-test, the Wilcoxon signed-rank test, or repeated measures analysis of variance.
The incidence of serious adverse events (SAEs) and adverse events (AEs) related to the treatment will be analyzed for the safety assessment using the independent t-test or Wilcoxon’s rank-sum test. The percentage of subjects who experienced one or more side effects during the study will be analyzed using Pearson’s chi-square test or Fisher’s exact test.
2.12 Data management and monitoring strategies
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 electronic case report forms (eCRF) using the Medidata RAVE data management system (Medidata Solutions Inc., New York, NY, USA). 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 performed in accordance with the protocol.
2.13 Adverse events
During the hospitalization, subjects will be asked if they have any physical discomfort other than DOMS symptoms. Occurrence of any AEs will be recorded on the eCRF after assessing severity and causality. If a SAE occurs, it will be reported to the IRB as soon as possible. When suspected unexpected serious adverse reactions occur, they will also be reported to the Korea Food and Drug Administration according to the relevant regulations.