Comparison of combination therapy with methotrexate and sinomenine or leflunomide for active rheumatoid arthritis: A randomized controlled clinical trial

Accepted Manuscript

Comparison of combination therapy with methotrexate and sinomenine or leflunomide for active rheumatoid arthritis: a randomized controlled clinical trial Run-yue Huang , Hu-dan Pan , Jia-qi Wu , Hua Zhou , Zhan-guo Li , Ping Qiu , Ying-yan Zhou , Xiu-min Chen , Zhi-xin Xie , Yao Xiao , Qing-chun Huang , Liang Liu PII: DOI: Reference:

S0944-7113(18)30624-X https://doi.org/10.1016/j.phymed.2018.12.030 PHYMED 52807

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Phytomedicine

Received date: Revised date: Accepted date:

20 October 2018 18 December 2018 23 December 2018

Please cite this article as: Run-yue Huang , Hu-dan Pan , Jia-qi Wu , Hua Zhou , Zhan-guo Li , Ping Qiu , Ying-yan Zhou , Xiu-min Chen , Zhi-xin Xie , Yao Xiao , Qing-chun Huang , Liang Liu , Comparison of combination therapy with methotrexate and sinomenine or leflunomide for active rheumatoid arthritis: a randomized controlled clinical trial, Phytomedicine (2018), doi: https://doi.org/10.1016/j.phymed.2018.12.030

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Comparison of combination therapy with methotrexate and sinomenine or

leflunomide for active rheumatoid arthritis: a randomized controlled clinical trial

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Run-yue Huang , Hu-dan Pan , Jia-qi Wu , Hua Zhou , Zhan-guo Li , Ping Qiu , Ying-yan Zhou , Xiu-min d

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Guangdong Provincial Academy of Chinese Medical Sciences/Second Affiliated Hospital of Guangzhou

University of Chinese Medicine, Guangzhou, China. b

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Chen , Zhi-xin Xie , Yao Xiao , Qing-chun Huang , Liang Liu

State Key Laboratory of Quality Research in Chinese Medicine/Macau Institute for Applied Research in

Medicine and Health, Macau University of Science and Technology, Macau, China.

Department of Rheumatology and Immunology, Peking University People's Hospital, Beijing, China.

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Hunan Zheng Qing Pharmaceutical Group Company Limited, Huaihua, China.

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*Correspondence to:

Prof. Liang Liu, State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied

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Research in Medicine and Health, Macau University of Science and Technology, Macau (SAR), China; Email address: [email protected]; Tele: 853-88972238; Fax: 853-2882 2799

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Prof. Qing-chun Huang, Guangdong Provincial Academy of Chinese Medical Sciences, Guangzhou, China; Email address:[email protected]; Tele: +86-20-39318981

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Abstract

Background: A combination of conventional disease-modifying anti-rheumatic drugs improves the

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treatment of rheumatoid arthritis but with high side-effects. Methotrexate (MTX) combination therapy that with high therapeutic efficacy and low toxicity is in demand in many countries to replace the use of

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expensive biological agents. Study Design: This study was an open-label, 24-week, parallel randomized controlled trial conducted between November 2015 and December 2017. Methods: Patients were randomly assigned at a 3:2 ratio to receive MTX combined with sinomenine (SIN) at a dose of 120 mg twice daily, or leflunomide (LEF) at a dose of 20 mg once daily. Efficacy and safety were assessed at weeks 4, 12 and 24. The primary efficacy endpoint was the proportion of patients 1

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achieving an American College of Rheumatology (ACR)50 response and a European League Against Rheumatism (EULAR) good response at week 24. Results: A total of 101/120 (84.2%) patients completed 24 weeks of observation. In the intention-to-treat (ITT) analysis, 65.3% of patients treated with MTX+SIN showed improved disease activity as determined by the ACR50 response at week 24 compared to 69.3% of patients treated with MTX+LEF. A similar

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insignificant pattern was found for the ACR20 and ACR70 responses, as well as the clinical disease activity index, EULAR response, and remission and low disease activity rates between these two treatment groups. The per-protocol analysis showed results consistent with those of the ITT analysis. Notably, significant reductions in gastrointestinal adverse reactions and liver toxicity were found in patients treated with MTX+SIN compared to patients treated with MTX+LEF (P<0.05).

Conclusions: Considering the balance of efficacy and toxicity, the current study provides evidence that

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MTX+SIN combination therapy is probably one of the choices for treating patients with active rheumatoid arthritis in addition to MTX+LEF combination therapy.

Keywords

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Combination therapy; Sinomenine; Rheumatoid arthritis; Efficacy and safety; Randomized controlled

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clinical trial

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Abbreviations

ACR, American College of Rheumatology; anti-CCP, anti-cyclic citrullinated peptide antibody; AE, adverse event; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CRP, C-reactive protein;

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cDAI, clinical disease activity index; DAS28, 28-joint disease activity score; DMARD, disease-modifying anti-rheumatic drug; ESR, erythrocyte sedimentation rate; EULAR, European League Against

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Rheumatism; FAS, full analysis set; HAQ, Health Assessment Questionnaire; ITT, intention-to-treat; LEF, leflunomide; LDA, low disease activity; MTX, methotrexate; NSAIDs, nonsteroidal anti-inflammatory drugs; PaGADA, patient’s global assessment of disease activity; PGE2, prostaglandin E2; PhGADA, physician’s global assessment of disease activity; PP: per-protocol; RA, rheumatoid arthritis; RF, rheumatoid factor; RCTs, randomized controlled trials; SJC, swollen joint count; SIN, sinomenine; TJC, tender joint count; WHO, World Health Organization; ZQFTN, Zheng Qing Feng Tong Ning.

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Background

Rheumatoid arthritis (RA) is a common autoimmune disease associated with progressive disability,

systemic complications, early death, and socioeconomic costs (Aletaha and Smolen, 2018; Smolen et al.,

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2016). Methotrexate (MTX) is the most effective disease-modifying anti-rheumatic drug (DMARD) for treating RA (Smolen et al., 2017). However, MTX alone sometimes fails or only produces a partial

response (Chatzidionysiou et al., 2017; Ortendahl et al., 2002). When RA patients treated with MTX show an inadequate response or a poor prognosis, the addition of conventional synthetic DMARDs is recommended by both the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) (Smolen et al., 2017). The combination of leflunomide (LEF) and MTX

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(MTX+LEF) is one of the most widely used MTX combination therapies, and while it shows improved efficacy, there is also an increased risk of gastrointestinal side effects and liver toxicity (Lee et al., 2009; Smolen et al., 1999; Wang et al., 2018). The determination of an MTX combination therapy with high

biological agents.

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therapeutic efficacy and low toxicity is in high demand in many countries to avoid the use of expensive

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Sinomenine (SIN) is an active ingredient extracted from Caulis Sinomenii (Huang et al., 2017). It has been demonstrated both in vivo and in vitro studies with significant anti-inflammatory and immunosuppressive

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activities (Wang and Li, 2011; Yi et al., 2015; Zhou et al., 2008) through the inhibition of T and B lymphocyte activation, proliferation and function, which indicates its potential role for the treatment of RA (Tong et al., 2016; Wang and Li, 2011). Zheng Qing Feng Tong Ning (ZQFTN), a drug marketed in China

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consisting of SIN, is approved by the State Food and Drug Administration and the National Health Insurance Directory of China for treating RA due to its high clinical efficacy and low toxicity in treating RA. Importantly, a meta-analysis of ten randomized controlled trials (RCTs) revealed that combination treatment with MTX and SIN (MTX+SIN) was more effective than MTX alone for treating RA; however, the majority of the included RCTs showed low methodological quality(Chen et al., 2015; Feng Z). The possibility of MTX+SIN being a preferred MTX-DMARD combination with enhanced efficacy and 3

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without an increased risk of side effects prompted us to conduct an investigator-initiated, 24-week RCT to determine the efficacy and safety of MTX+SIN compared with MTX+LEF.

Methods

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The study was an open-label, 24-week RCT that was handled between November 2015 and December 2017. Patients were recruited from the Second Affiliated Hospital of Guangzhou University of Chinese Medicine and written informed consent was achieved from all the patients recruited in this study. The

protocol was approved by the Medical Ethics Committee of the Second Affiliated Hospital of Guangzhou University of Chinese Medicine (No. B2015-133-01) and registered with the WHO clinical trial registry (No. ChiCTR-IPR-15007415) on 19th November 2015.

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Setting and participants

Eligible patients had to meet the following criteria for this trial: (1) aged 18-65 years; (2) diagnosed with RA as determined by the 2010 ACR/EULAR classification criteria (Kay and Upchurch, 2012); (3) had active disease, as defined by a 28-joint disease activity score (DAS28)-C-reactive protein (CRP) score greater than 3.2; (4) discontinued all disallowed concomitant medications for at least one month prior to the

(6) provided written informed consent.

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first dose of study drug; (5) permitted the use of glucocorticoids at a dose of less than 10 mg per day; and

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Patients were excluded if they had a history of severe, progressive or uncontrolled cardiac, hepatic, renal, or mental diseases, other rheumatic autoimmune diseases, any current infection, or any cancer. Patients in

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pregnant or lactating stage were excluded. Patients who experienced an allergic reaction to the medicine were excluded. Additionally, patients who had previously participated in other clinical studies were not

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allowed in this study.

Randomization and interventions

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Patients were assigned at a 3:2 ratio to receive either SIN at a dose of 120 mg twice daily or LEF at a dose of 20 mg once daily in random; all of the included patients received MTX, starting from 10 mg once a week and increasing to 15 mg once a week within 4 weeks, with 5 mg of folic acid two or three times a day. SIN were provided by Hunan Zheng Qing Pharmaceutical Group Company Limited (China Food and Drug Administration: Z20010174) calling ZQFTN and the purity of SIN in each pill of ZQFTN was at least 99.7%. The medicines were taken after a meal for 24 weeks. Participants were allowed to take glucocorticoids at 2.5-10 mg/day if they had intolerable pain, and nonsteroidal anti-inflammatory drugs 4

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(NSAIDs), calcitriol/calcium carbonate and antacids were allowed during the study. Patients could discontinue the assigned treatment at any time for lack of effectiveness or adverse effects or by their own choice. Randomization was performed by an independent statistician using SPSS software. Allocation concealment was achieved using a centralized service. The statistician who prepared the list had no further role in the

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study. We did not use stratification or blocking. Blinding was not practical for this investigator-initiated clinical trial, and the treatments were open to both the researchers and participants. Outcomes and measurements

Outcomes were assessed at baseline and after 4, 12 and 24 weeks. The primary outcome measure was the proportion of patients achieving an ACR50 response. The ACR response was defined by the following

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criteria: 1) improvement from baseline in the tender joint count (TJC)28; 2) improvement from baseline in the swollen joint count (SJC)28; and 3) improvement from baseline in at least 3 of the following 5 parameters: patient’s assessment of pain, patient’s global assessment of disease activity (PaGADA), physician’s global assessment of disease activity (PhGADA), patient’s assessment of physical function (HAQDI) and CRP level (Felson and LaValley, 2014).

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Secondary efficacy measures were the ACR20 and ACR70 responses, EULAR good and moderate responses, the clinical disease activity index (cDAI), clinical remission (DAS28<2.6), and low disease activity (LDA) (DAS28<3.2). The EULAR response criteria were assessed based on the individual amount

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of change in the DAS and the achieved DAS (low, moderate, or high). A change of 1.2 in the DAS28 was considered indicative of a significant change (Fransen and van Riel, 2009). A good response in terms of the

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cDAI was defined as achieving ≥50% improvement or a cDAI≤2.8. Improvements in other RA biomarkers, including the rheumatoid factor (RF), anti-cyclic citrullinated peptide antibody (anti-CCP) level, CRP level,

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ESR and HAQDI, were also compared between the groups. The incidence and severity of all adverse events (AEs) were kept a record in a predefined form. Clinical laboratory tests, including routine blood tests and liver function tests, vital signs detection, and physical

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examinations, were conducted at every visit, and electrocardiography and chest X-ray examinations were performed at the screening visit and at week 24. Liver toxicity was defined by the abnormally elevation of aspartate aminotransferase (AST) or alanine aminotransferase (ALT) level. Haematological AEs included anaemia (haemoglobin<110 g/l), leucopoenia (<3.5×10 9/l), and thrombocytopaenia (<100×109/l). Statistical analysis

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A full analysis set (FAS) was used to evaluate the baseline data, and intention-to-treat (ITT) and (per-protocol) PP analyses were performed to assess the efficacy and safety of the two treatments. The ITT analysis included all patients who received treatment for at least 1 month. The data of patients who withdrew from the trial prematurely were considered missing and were calculated using the last observation when performing the ITT analysis. The PP analysis included the participants who finished 24week

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observation. Dichotomous variables were analysed using Pearson’s chi-square test for the primary efficacy endpoints,

including the ACR and EULAR responses. The secondary efficacy endpoints, including the TJC28, SJC28, patient’s assessment of pain, PaGADA, PhGADA, CRP, and ESR, were assessed by one-way repeated

measures ANOVA of the mean values from baseline to weeks 4, 12 and 24 for each group. The differences between the two treatment groups in these secondary efficacy endpoints were analysed by the

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Mann-Whitney U test. All statistical tests were 2-sided. P values less than 0.05 were considered significant. An independent statistician who was blinded to the group allocation conducted the data analysis using SPSS and GraphPad Prism.

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Results Patient characteristics

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A total of 186 patients with RA were screened initially, and 120 eligible patients were finally enrolled in this study. A total of 73 patients were randomly assigned to receive the MTX+SIN combination therapy, and 47 patients received the MTX+LEF combination therapy. As shown in Figure 1, 61 and 40 patients in

(Figure 1).

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the MTX+SIN and MTX+LEF combination groups, respectively, completed the 24-week evaluation period

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The baseline demographic and clinical characteristics of the patients who were enrolled in the study are shown in Table 1. All patients had moderately to highly active disease, as reflected by the TJC28, SJC28,

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patient’s assessment of pain, PaGADA, PhGADA, ESR, DAS-ESR, and cDAI results. There were no significant differences between the two treatment groups in any of the above metrics (P>0.05). We also compared the use of concomitant medications during the intervention between the two treatment groups, and no significant differences were found in the percentages of patients who used NSAIDs, glucocorticoids, folic acid, calcitriol/calcium carbonate or antacids concomitantly. Notably, comparable ratios of patients treated with MTX+SIN and MTX+LEF, i.e., 55.9% and 59%, respectively, used glucocorticoids (Supplementary Table 1). 6

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The ITT analysis showed that 65.3% and 69.3% of patients treated with MTX+ SIN and MTX+LEF, respectively, achieved at least a 50% improvement in disease activity as determined by the ACR50 response at week 24. Over 80% of the patients who completed the trial achieved ACR20, and 43.1% and 47.8% of patients achieved ACR70, indicating significant improvements in both groups (Figure 2). Similarly, a non-inferiority test was conducted to compare the differences in the cDAI good response rate, EULAR response, remission rate and LDA rate between the two treatment groups (MTX+ SIN vs

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MTX+LEF) at week 24; no differences were observed between the two groups (ACR20: 88.9% vs 82.6%; cDAI good response: 83.3% vs 82.6%;EULAR good or moderate response :97.2% vs 91.3%; EULAR good response: 69.4% vs 73.9%; remission rate: 55.6% vs 56.5%; LDA rate: 71.4% vs 71.7%) (Figure 2). Notably, at week 12, a relatively higher remission rate and LDA were shown in MTX+SIN group

compared to the MTX+LEF group, indicating that patients in MTX+ SIN group may have the potential to

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reach remission earlier.

There were no significant differences in the mean change in the DAS28 from baseline to week 24 between the MTX+SIN (-2.18) and MTX+LEF (-2.25) groups. The HAQDI was used to assess the physical function of patients, and no significant differences were found between the two groups (mean improvement: 0.59 and 0.61, respectively; P>0.05). The most commonly used inflammatory markers, ESR and CRP, were also detected with significant reduction after 24 weeks treatments in both of the two groups, though no marked

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differences were shown between groups. Similar insignificant patterns were found for the TJC28, SJC28, patient’s assessment of pain, PaGADA, PhGADA and morning stiffness (P>0.05) (Table 2).

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We also conducted a PP analysis of the data from patients who completed the 24-week evaluation period, and the ACR50 response rate was 77% and 75% in patients treated with MTX+SIN and MTX+LEF, respectively (Figure 3). At week 4, patients in the MTX+LEF group achieved a significantly higher

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EULAR good or moderate response rate than patients in the MTX+SIN group (85% vs 59%, respectively), but similar results were found for other response indicators. The results of the PP analysis were consistent

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with the results of the ITT analysis of the ACR50, EULAR response, ACR20, ACR70, cDAI good response, clinical remission, and LDA rates at week 24 (Figure 3, Supplementary Table 2).

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Safety and tolerability

There was an obviously increased rate of AEs in the MTX+LEF group compared to the MTX+SIN group. In particular, the rates of gastrointestinal and hepatic side effects of MTX+LEF, such as abdominal discomfort and increased ALT/AST, were significantly higher than those of MTX+SIN (Table 3). In general, most of the AEs in these two treatment groups were mild and could be alleviated quickly. Two (2.74%) patients in the MTX+SIN group suffering from rash and increased ALT/AST and two (4.65%) 7

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patients in the MTX+LEF group suffering from increased ALT/AST and abdominal discomfort discontinued the study because of the AEs. In the MTX+SIN group, rash was the main AE, which could be immediately relieved after treatment with chlorpheniramine, while in the MTX+LEF group, gastrointestinal and hepatic disorders were the main AEs.

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Discussion

Early DMARD treatment for RA has been recommended in the past few years due to the influence of new classification criteria for the diagnosis of RA and evidence that such therapeutic strategies can mitigate the risk of joint damage before the onset of erosion (Ledingham et al., 2017; Smolen et al., 2007). Doctors usually use a combination of two or more drugs to treat RA, especially for patients with severe active

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conditions; thus, the synergistic effect of multiple medications against this disease is recognized (Smolen et al., 2005). However, the greater risk of AEs associated with MTX combination therapies may lead physicians to select a more conservative approach for initial treatment (Katchamart et al., 2009). Biological DMARDs combined with MTX have been shown in many RCTs to be more effective than MTX monotherapy (Daien et al., 2017). However, because of the benefit-to-risk ratio and high cost, this combination therapy is not suitable for many patients, especially those with early RA (Combe et al., 2017).

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Four trials reporting ACR responses showed that MTX in combination with other conventional synthetic DMARDs achieved significantly improved ACR70 response rates compared with MTX monotherapy, but

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with an increased risk of withdrawal due to toxicity (Hetland et al., 2006; Katchamart et al., 2009; Kremer et al., 2002; Marchesoni et al., 2003). Combining LEF with MTX has been shown to improve the therapeutic efficacy but also increase the risk of gastrointestinal AEs and liver toxicity compared to

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treatment with MTX alone (van den Akker et al., 2008). A multicentre, observational, cross-sectional, retrospective safety study including 2975 RA patients reported that 19% of patients in the MTX+LEF

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group suffered liver function abnormalities, which was higher than the 12% of patients suffering similar effects in the MTX monotherapy group (van den Akker et al., 2008). In the current study, the efficacy of MTX+SIN was not inferior to that of MTX+LEF, but much fewer AEs

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occurred in patients treated with MTX+SIN than in patients treated with MTX+LEF, especially regarding gastrointestinal reactions and liver abnormalities (MTX+SIN vs MTX+LEF, 5.48% vs 19.15%, respectively); the toxicity in gastrointestinal reactions and liver abnormalities in the MTX+SIN group is even lower than that previously reported for MTX monotherapy (van den Akker et al., 2008). ZQFTN is an effective Chinese proprietary medicine that was approved by the China Food and Drug Administration for treating RA nearly twenty years ago (Xu et al., 2008), and the drug has been used 8

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widely throughout China. Unlike tripterygium glycosides (Lv et al., 2014), SIN is suitable for the most patients with RA at any stages, and few side effects have been reported to date. The most common side effects for SIN is mild-to-modulate allergy, revealing pruritus, flushing and macula in the skin. According to the reports of the adverse drug reactions of SIN in the Chinese National Knowledge Infrastructure database from 1992 to 2012, the incidence rate of allergy was around 12%(Juan and Yan-hong., 2012). But, the rate for allergic reactions has markedly reduced after optimizing manufacture technology and enhancing

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the purity of SIN by the company.

An analysis of 11 RCTs including 956 RA patients demonstrated that the co-administration of SIN and

MTX improved the efficacy of MTX monotherapy and was superior in terms of mitigating adverse drug reactions (Chen et al., 2015). Importantly, SIN not only inhibits the activation of immune-related cells

(Feng et al., 2006) and the secretion of inflammatory mediators and pro-inflammatory cytokines (Feng et

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al., 2017; Huang et al., 2017; Yi et al., 2015; Zhou et al., 2008) but also selectively inhibits synthesis of the membrane-bound prostaglandin E synthase 1 (mPGES-1), an enzyme involved in the last step of prostaglandin E2 (PGE2) synthesis; these effects prevent possible side effects, such as those of cytochrome c oxidase subunit II (COX2) inhibitors, on the cardiovascular system (Zhou et al., 2017). The results of such mechanistic studies suggest that ZQFTN might have less risk of inducing cardiovascular AEs than some COX2 inhibitors in treating rheumatoid arthritis. Additionally, the safety evaluation in this study

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demonstrates a markedly lower frequency of total and severe AEs in the MTX+SIN group than in the MTX+LEF group, especially regarding gastrointestinal intolerance and liver toxicity. These results may be

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related to the antioxidant and anti-inflammatory effects of SIN on such organs like colon, partly via activating the nuclear factor-erythroid 2-related factor 2 (Nrf2)/ NADP(H) quinone oxidoreductase 1 (NQO-1) pathway(Zhou et al., 2018). Thus, MTX+SIN combination therapy has a significant advantage

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over MTX+LEF combination therapy in terms of the benefit/risk ratio, especially for RA patients in the early stage or with gastrointestinal and liver disorders.

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The current study has some limitations. This study was an investigator-initiated open-label clinical trial with limited patient. For a completely objective assessment, a double-blind RCT with multi-centre, large sample would be necessary in the future. Furthermore, as this was a 24-week clinical trial, the radiological

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progression of the affected joints in X ray was not evaluated. Finally, our study did not compare results of the MTX monotherapy approach with the MTX combination therapy, which would be conducted in the ongoing studies. In summary, our findings provide clinical findings of MTX+SIN combination therapy on RA. This approach has therapeutic efficacy comparable to that of MTX+LEF combination therapy, but with much fewer gastrointestinal and hepatic AEs. Considering both the efficacy and toxicity of these drugs in terms 9

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of the benefit/risk ratio, MTX+SIN combination therapy might be one of the choices for clinician to treat active RA patients in addition to MTX + LEF.

Funding: This work was supported by the Key Program in Emerging Industry of Hunan Department of

(102/2016/A3).

Conflict of interest

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The authors have declared that there is no conflict of interest.

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Science & Technology (2014GK1058) and the Macau Science and Technology Development Fund

Acknowledgements: We thank Professor Wei Liu and Professor Yongfei Fang for their suggestions on this

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study.

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sodiuminduced colitis via the Nrf2/NQO1 signaling pathway. Molecular medicine reports 18, 3691-3698.

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Figure 1 Study flow and progress.

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Figure 2 Primary and secondary efficacy measures over time in the ITT analysis. The combination

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treatments were compared using the chi-square test.

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Figure 3 Primary and secondary efficacy measures over time in the PP analysis. The combination treatments were compared using the chi-square test.

Table 1 Clinical and demographic data at baseline in FAS.

MTX+SIN(n=73）

Age(SD), years

48.97(10.79)

Female, n (%)

55 (75%) 32.99 (44.21)

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Disease duration (SD), months TJC (SD), n SJC (SD), n Patient's assessment of pain (SD), mm

48.53(12.10) 40 (85%)

40.56(54.52)

8.77(5.25)

9.96(6.13)

2.92 (2.94)

3.53(3.21)

65.82(16.65)

67.13(18.44)

60.75(16.72)

60.85(18.19) 65.43(18.76)

0.94(0.84)

1.41(1.68)

0.82(0.47)

0.80(0.53)

CRP (SD), mg/L

16.37(17.13)

24.94(35.43)

ESR

49.60(26.93)

57.24(29.10)

RF# (SD), U/mL

164.93(223.20)

181.02(179.30)

Anti-CCP#,positive rate

47/52(90.38)

30/34(88.23)

DAS28-CRP

4.75(0.88)

5.00(1.05)

cDAI (SD)

24.20(9.25)

26.12(10.30)

Steroid used, n (%)

34 (46.58)

25 (53.19)

Morning stiffness (SD), h

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HAQ, mean ± SD

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PhGADA (SD), mm PaGADA (SD), mm

MTX+LEF（n=47）

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Characteristics

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Data are presented as the mean (SD) or n (%). RF# was measured by immunonephelometry with a cut-off value of 20 U/ml. Anti-CCP# was measured using a commercially available second-generation ELISA kit (Abbott, USA) with a cut-off value of 25

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U/ml.

Table 2 Clinical and laboratory measures of the two groups at each visit in the ITT analysis. MTX+SIN

MTX+LEF

Measure 4w

12 w

24 w

0w

8.78

6.64

4.42

2.89

9.65

(5.28)

(4.54)

(3.95)

2.96

1.61

0.83

(2.94)

(1.71)

(1.52)

66.00

47.99

28.75

(16.66)

(18.57)

60.90

45.49

TJC，n

Morning

4.11

2.43

(4.10)

(2.90)

(1.94)

0.44

3.57

1.98

1.07

0.46

(1.16)

(3.24)

(2.53)

(1.91)

(0.84)

17.32

85.98

44.24

28.15

18.76

(17.66)

(17.11)

(101.55)

(18.77)

(14.54)

(15.27)

26.67

15.57

66.85

41.74

24.46

16.96

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(18.86)

(17.76)

(17.32)

(18.54)

(17.10)

(14.15)

(15.18)

64.31

49.17

28.75

16.97

60.65

45.11

26.20

16.96

(17.95)

(20.50)

(18.70)

(18.28)

(18.34)

(18.21)

(14.46)

(14.93)

0.95 (0.84)

0.44 (0.47)

0.12 (0.23)

0.06 (0.18)

1.43 (1.69)

0.56 (0.69)

0.20 (0.27)

0.04 (0.14)

16.41

11.81

8.43

7.30

25.41

15.00

3.96

11.84

(17.25)

(15.32)

(15.33)

(10.31)

(35.67)

(25.68)

(0.98)

(17.17)

0.82

0.55

0.36

0.23

0.79

0.46

0.31

0.18

(0.47)

(0.37)

(0.31)

(0.30)

(0.53)

(0.38)

(0.30)

(0.18)

49.77

41.35

35.71

31.54

58.29

50.70

43.43

41.04

(27.08)

(27.77)

(25.07)

(21.08)

(28.53)

(29.77)

(26.73)

(28.86)

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stiffness(h)

6.50

(5.82)

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PaGADA (SD), mm

24 w

(16.79)

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PhGADA (SD), mm

12 w

(3.49)

SJC，n Patient's assessment of pain (SD), mm

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CRP, mg/L

HAQ

ESR

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4.76

3.99

3.06

2.58

4.99

3.96

3.25

2.74

(0.88)

(1.01)

(1.11)

(1.14)

(1.06)

(0.98)

(0.91)

(0.97)

DAS28-CRP

Data are presented as the mean (SD).

Adverse events

MTX+SIN

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Table 3 AEs of all participants MTX+LEF

Total events

(n=47)

(n=120)

(n=73)

P value

27 (36.99%)

32 (68.09%)

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ALT/AST

4 (5.48%)

9 (19.15%)

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7 (9.59%)

12 (25.53%)

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Baldness

1 (1.34%)

4 (9.30%)

5

0.149

Rash

8 (10.96%)

1 (2.13%)

9

0.150

Weight loss

0 (0%)

2 (4.26%)

2

0.151

Leukopenia

1 (1.34%)

1 (2.13%)

2

1

3 (6.38%)

9

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elevation Abdominal

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Anemia

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discomfort

6 (8.22%)

0.040

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Data are presented as n (%).

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Graphic abstract

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