A Randomized Controlled 24-week Trial to Evaluate the Efficacy and Safety of Bi Qi Capsule for the Treatment of Knee Osteoarthritis

Objective: To evaluate the efficacy and tolerability of Bi Qi capsule in knee osteoarthritis. Patients and methods: An open-label, 24-week, parallel randomized controlled trial conducted between October 2016 to October 2018. Patients with knee osteoarthritis were randomly assigned 1:1 to receive Bi Qi capsule (1.2g, twice a day) and Calcitriol capsule (0.25ug, once a day). Western Ontario and McMaster Universities Osteoarthritis (WOMAC) was the primary outcome, including pain, stiffness, and function subscale scores. Secondary outcome measures were visual analogue scale (VAS) score for the patients assessment of pain, bone mineral density (BMD), patient’s assessment of function using the Health Assessment Questionnaire (HAQ). Safety was assessed by recording adverse events (AEs). Results: 100 patients were included in the study. 15(15%) patients discontinued the study because of lack of efficacy, adverse events and loss of follow up. At the end of the treatment ,the decrease in WOMAC total score was -8.7(4.9) and -5.4(5.6) and improvement of WOMAC function was -4.88(3.6) and -2.70(3.86) in the Bi Qi group and the controlled group, respectively (p0.01). The improvement in BMD of left femoral neck was 0.05(0.2) and -0.01(0.04) , improvement in T score of left femoral neck was 0.15(0.5) and -0.05(0.3) in the Bi Qi group and the controlled group, respectively (p0.01). No significant difference was observed between treatment groups for changes in WOMAC pain, WOMAC stiffness, VAS, BMD of lumbar spine, T score of lumber spine and HAQ over 24 weeks. Two adverse events were reported in Bi Qi group: facial edema (n=1), nausea (n=1) and one adverse events was reported in controlled group: lip swelling (n=1). All these adverse events were mild and could be alleviated after withdrawing treatment. Conclusion: It can be concluded that Bi Qi capsule

from KOA.

Introduction
Osteoarthritis (OA) refers to a syndrome of joint pain accompanied by functional limitation and reduced quality of life [1]. The pathology of OA involving the cartilage degradation, bone remodeling, osteophyte formation and synovial inflammation leading to pain, stiffness, swelling and even loss of normal joint function [2] . OA is the most common form of arthritis, affecting an estimated 302 million people worldwide, the most commonly of which is knee osteoarthritis (KOA) [3] .There are currently no effective disease-modifying remedies available to treat KOA. Oral non-steroidal anti-inflammatory drugs (NSAIDs) are strongly recommended for patients with KOA but only short-term and intermittent use because potential adverse of gastrointestinal, cardiovascular system. Other oral pharmacological agents such as glucosamine, chondroitin sulfate are strongly recommended against in patients with KOA because discrepancies in efficacy [4] .Thus, complementary and integrative therapies are favored in treatment of KOA. In fact, a growing number of patients with chronic musculoskeletal pain report utilizing such therapy, specifically traditional Chinese medication (TCM) [5] .
Bi Qi capsule is a TCM preparation for treatment arthritis including RA and KOA. It is composed of many natural products as follows: The dried fruit of Strychnos nux-vomica (Ma qian zi), the dried body of Pheretima aspergillum (Di long), the root of Codonopsis pilosula (Dang shen), the sclerotia of Poria cocos (Fu ling), the rhizome of Atractylodes macrocephala (Bai zhu), the root and rhizome of Glycyrrhiza uralensis (Gan cao), the rhizome of Ligusticum wallichii (Chuan xiong), the root and rhizome of Salvia miltiorrhiza (Dan shen), the root and rhizoma of Panax notoginseng (San qi) and the root of Achyranthes bidentata (Niu xi) [6] . Bi Qi capsule is widely used in treating KOA in China because of its good clinical efficacy. For example, a six-week clinical study involving 221 participants compared the Bi Qi capsule to Teng Huang Jian Gu pill (another Chinese patent), it concluded that Bi Qi capsule was better in improving the Visual Analogue Scale (VAS) score and Hospital for Special Surgery Knee Score (HSS) score than the control group [7] . Another non-randomized controlled trial involving 86 participants of KOA reported the Bi Qi capsule was superior to diclofenac tablets in improving total effectiveness after 84 days (12 weeks) [8] . But it is still insufficient evidence available to demonstrate the efficacy of Bi Qi capsule of high quality with a randomized design. For this reason, we conducted a randomized controlled clinical trial primarily to evaluate the efficacy and safety of Bi Qi capsule in KOA.

Study design
This was a single-site, two parallel-group, randomized controlled clinical trial of 24-weeks duration to assess the efficacy and safety of Bi Qi Capsule in patients with knee osteoarthritis in the Guangdong Provincial Hospital of Chinese Medicine from October 2016 to October 2018. The severity of KOA ranged from grade 1 to grade 3 by Kellgren-Lawrence system [9] . The study was approved by the institutional ethical review board.
The manuscript was in accordance with the populated CONDORT checklist (see Additional file 1) and flow diagram (see Additional file 2).

Setting and participants
Inclusion Criteria All participants were recruited from Guangdong Provincial Hospital of Chinese Medicine. To be eligible for this trial, patients had to meet the following criteria (1): 18-65 years of age;(2) diagnosed with KOA according to American College of Rheumatology criteria [10] with radiographic confirmation at screening by the radiologist (Kellgren-Lawrence score 4).
Exclusion Criteria Patients were excluded if they (1) had evidence of specific joint safety conditions (e.g., rapidly progressive KOA, osteonecrosis, bone tumor, bone tuberculosis); (2) combined with other diseases that could affect the efficacy assessment (e.g., rheumatoid arthritis, spondyloarthritis, gout); (3) had a history of trauma or surgery on knee; and pregnancy or breastfeeding; (4) had other conditions which made them ineligible for the study treatment. All patients provided written informed consent prior to study enrollment.

Study treatment
A computer-generated randomized code was used to assign patients with KOA in a 1:1 ratio to receive oral Bi Qi Capsule (1.2g, twice a day) (group A); Calcitriol capsule (0.25ug,once a day) (group B). Celecoxib (0.2g, once or twice a day) was permitted to take orally for up to 7 days for the patients who had a VAS for pain over 4 (cm) but not within 48 hours of a study visit. Study visits were scheduled for week 4, week 12 and week 24, during which safety and efficacy assessments were performed.

Outcomes and measurements
The primary efficacy end points were the change from baseline to week 24 in Western Ontario and McMaster Universities Osteoarthritis (WOMAC), including pain, stiffness, and function subscale scores [11] . Secondary outcome measures were VAS score for the patients assessment of pain, bone mineral density (BMD),patient's assessment of function using the Health Assessment Questionnaire (HAQ).The hematology (full blood count), biochemistry indices (alanine aminotransferase, aspartate aminotransferase, creatinine, urea nitrogen), routine urine test and electrocardiogram were recorded at baseline and after 4,12,24 weeks of treatment.

Statistical analysis
A full analysis set (FAS) was used to evaluate the baseline data, and a per-protocol (PP) analysis was performed to assess the efficacy and safety of the two treatments. The ITT analysis included all participants who received treatment for at least 4 weeks. The PP analysis included the participants who finished 24 week observation without violation of treatment. Continuous data are presented as mean (SD). Categorical data are presented as numbers (n) or proportions (%). Differences between groups were analyzed for significance using a one-way analysis of variance F test or a non-parametric test (continuous data) and χ 2 test (categorical data). The WOMAC, VAS and HAQ scores were analyzed using repeated measures Analysis of Variance (ANOVA) with post hoc between multiple comparisons. All analysis was computed using SPSS statistics V.17.0.

Trial Population
Patients were recruited from the Outpatient Department of Rheumatology in Guangdong Provincial Hospital of Chinese Medicine over a 2-year period from October 2016 to October 2018. One hundred and ten patients were examined by the rheumatologists for KOA and were assessed for eligibility to enter the study according to the inclusion and exclusion criteria. One hundred participants met the inclusion criteria, of which 50, 50 were randomized into the treatment group (group A, Bi Qi capsule 1.2g twice a day) and  Questionnaire (HAQ) were not significant different between group A and B (Table 1).   There was no significant difference between group A and group B for the BMD and T score of lumbar spine (p> 0.05). The mean VAS and HAQ scores in both groups were comparable at the baseline visit. Although there was also a tendency of both outcome measures in the two groups, no significant difference was found between the group A and group B at each visit and no significant difference was observed for change scores of VAS and HAQ between the two groups (p 0.05) ( Table 3,4).

Safety Assessment
There were no significant abnormal changes in hematology, hepatic and renal functions at the end of treatment (Table 5). Two adverse events were reported in group A: facial edema (n=1), nausea (n=1) and one adverse events was reported in group B: lip swelling (n=1). All these adverse events were mild and could be alleviated after withdrawing treatment.

Discussion
KOA is a chronic and low-grade inflammation, involving mainly innate immune mechanisms. It is well know that inflammatory mediators associated with OA include cytokines, chemokine, growth factors, prostaglandins, nitric oxide, and neuropeptides [12] .
Cyclooxygenase inhibitors (e.g., NSAIDS) have been the most commonly recommended medicine in the guideline of KOA but only with time limited use due to its adverse effects [2] .Bi Qi capsule was widely used in the treatment of KOA for its efficacy of antiinflammatory, detumescence, analgesia and improvement of arterial blood flow [13][14][15] .
Previous studies had explained the mechanism of Bi Qi capsule, which could play a protective role in articular cartilage damage and promoted cartilage repair by regulating serum interleukin-1 (IL-1), matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels [16] . By regulating the expression of Janus Kinase-3 and other inflammatory cytokines [17] . Joint inflammations, synovial hyperplasia and cartilage destruction can be reduced by up-regulating the express of osteoprotein (OPG), down-regulating the expression of receptor activator of nuclear factor κB ligand (RANKL) and ratio of RANKL/OPG [18] .The dried fruit of Strychnos nux-vomica is the most important component of Bi Qi capsule. Brucine, a major alkaloid monomer from the dried fruit of Strychnos nux-vomica, relieves pain, reduces inflammation, regulates cytokine expression and inhibits the proliferation of synovial fibroblast [19] . It has been reported that the injection of total alkaloids of nux vomica cuts down the nitric oxide (NO) and improves the level of superoxide dismutase (SOD) in synovial fluid of KOA on rabbit model. Thus, the repair effect of total brucine on OA cartilage injury may reduce the generation of free radicals by inhibiting lipid peroxidation and increasing the expression of SOD to promote the scavenging of free radicals. It leads to a reduction in free radicals that inhibits chondrocyte apoptosis [20][21][22][23] . In addition, it has been demonstrated that cryptotanshinone, brucine and strychnine are the major anti-inflammatory components in the aqueous extract of Bi Qi capsule [24] . Cryptotanshinone not only inhibits the secretion of NO but also suppresses the secretion of interleukin (IL)-6 in lipopolysaccharide (LPS)induced RAW 264.7 (leukemia cells in mouse macrophage) cells [24] . Taken
Ethics approval and consent to participants: This trial was approved by ethical review board of Guangdong Hospital of Traditional Chinese Medicine (B2016-073-01) and was consent to participants.

Consent for publication:
This trial was consent for publication.

Data Availability
These data are owned by Guangdong Provincial Hospital of Chinese Medicine. Access to these data will be considered by the author upon request. She can be reached at americaxx@gzucm.edu.cn  Change from baseline to Week 24 in WOMAC total score, WOMAC pain, WOMAC stiffness, WOMAC function at each visit