Efficacy and safety of iguratimod plus corticosteroid as bridge therapy in treating mild IgG4‐related diseases: A prospective clinical trial

Abstract Aim The purpose of this study is to evaluate the therapeutic efficacy and safety of iguratimod plus corticosteroid as bridge therapy in the treatment of mild immunoglobulin G4‐related disease (IgG4‐RD). Methods Newly diagnosed IgG4‐RD patients, without internal organ involvement were enrolled. Patients were given one dose of diprospan, intramuscular injection, and iguratimod, 25 mg, twice daily, for 24 weeks and were followed up at 0, 12 and 24 weeks. Follow‐up indexes included IgG4‐RD responder index (IgG4‐RD RI), serology and imaging, plasma cytokines and adverse drug effect. Flow cytometry was performed for T, B cell subsets and plasma was collected for liquid chromatography mass spectrometry (LC‐MS)‐based metabolomic profiling and data processing. Results Thirty patients were enrolled. At week 24, 9 (30.0%) patients achieved complete response, 17 (56.7%) patients with partial response, and 4 (13.3%) patients had no response to treatment. IgG4‐RD RI, serum IgG and IgG4 levels decreased significantly at weeks 12 and 24 after treatment, as well as CD3+ CD8+ T cells, plasmablast/plasma cells and memory B cells. The LC‐MS based plasma metabolomic profiles revealed significant changes between untreated patients and healthy donors, which became much similar to normal states after treatment. Conclusion Iguratimod plus corticosteroid as bridge therapy is effective for the treatment of mild IgG4‐RD, it can improve the clinical symptoms, reduce serum IgG and IgG4 levels, especially plasmablasts/plasma cells and memory B cells. In addition, the metabolite profiling became similar to normal controls after treatment.


| INTRODUC TI ON
Immunoglobulin G4-related disease (IgG4-RD) is a newly defined chronic fibro-inflammatory disorder characterized by elevated serum IgG4 levels, tumefactive lesions with a dense lymphoplasmacytic infiltration rich in IgG4 positive plasma cells and storiform fibrosis of related organs. [1][2][3] Glucocorticoids are the first-line agents for the treatment of IgG4-RD; [4][5][6][7] however, in order to maintain long-term disease stability and avoid disease relapse, usually glucocorticoids maintenance therapy should last for a long period, which may induce various glucocorticoid-associated adverse reactions. For some IgG4-RD patients with mild symptoms, such as swelling of the lacrimal glands, submandibular glands, parotid glands and nasal sinus, without internal organ damage, long-term glucocorticoids therapy for mild symptoms may have a low benefit/risk ratio. Further, a substantial proportion of patients cannot tolerate glucocorticoids.
Iguratimod (T-614) is a new small molecule compound with anti-inflammation and immune regulation functions which have a definite disease-modifying effect in both animal rheumatoid arthritis (RA) models and patients. [8][9][10][11] In vitro studies had demonstrated that iguratimod has an anti-inflammatory role by inhibiting tumor necrosis factor (TNF), interleukin (IL)-1β, IL-6, IL-8, and monocyte chemotactic protein-1 production. 10 Furthermore, iguratimod could reduce the levels of serum IgG, IgM, and IgA in RA patients. 9,10 Iguratimod could also attenuate proteinuria and kidney injury in MRL/lpr lupus mice, and improve serum markers such as anti-double-stranded DNA (anti-dsDNA) and serum C3. 12 In order to investigate an optimum treatment regime for mild IgG4-RD patients, we launched an investigator-initiated study; after treatment with one dose of diprospan, patients were given iguratimod 25 mg, twice a day, orally. The aim of this study was to evaluate the therapeutic efficacy and safety of iguratimod plus corticosteroid as bridge therapy in the treatment of IgG4-RD with mild symptoms. Newly diagnosed IgG4-RD patients, with mild but progressive superficial glands swelling were enrolled in this study, and were treated with 1 dose of diprospan, 1 mL (containing 5 mg betamethasone dipropionate and 2 mg betamethasone sodium phosphate), and concomitantly took iguratimod 25 mg, twice a day, orally. Patients were evaluated at baseline, 12 and 24 weeks of follow-up. Blood and urine routine tests, erythrocyte sedimentation rate (ESR), highsensitivity C-reactive protein (hsCRP), serum immunoglobulin, IgG subclass and IgE were measured; organ involvement was evaluated by ultrasound scanning, computed tomography (CT), or magnetic resonance imaging, or positron emission tomography/CT (PET/CT).

| Study design
The treatment efficacy was evaluated by IgG4-RD responder index (IgG4-RD RI, 2012 version, each affected organ was individually scored) 13 and physician global assessment (PGA, scored as 0-10 cm).
T cell subpopulations, B cell subpopulations plasma cells, as well as serum cytokines, including IL-1β, IL-17A, TNF-α, transforming growth factor (TGF)-β1, interferon (IFN)-γ were measured at baseline and at 3 months by flow cytometry and cytometric beads array (CBA). In addition, patients' serum metabolomics before and 3 months after treatment were analyzed.
Peripheral blood from healthy donors was collected for comparison of cytokines and T cell subpopulations.

| Inclusion and exclusion criteria
Inclusion criteria: enrolled mild IgG4-RD patients had to fulfill the fol- plasmablast/plasma cells and memory B cells. The LC-MS based plasma metabolomic profiles revealed significant changes between untreated patients and healthy donors, which became much similar to normal states after treatment. Iguratimod plus corticosteroid as bridge therapy is effective for the treatment of mild IgG4-RD, it can improve the clinical symptoms, reduce serum IgG and IgG4 levels, especially plasmablasts/plasma cells and memory B cells. In addition, the metabolite profiling became similar to normal controls after treatment.

K E Y W O R D S
B lymphocytes, IgG4-RD, iguratimod, treatment (d) pregnant or planning to be pregnant; (e) active infection, including hepatitis B virus, hepatitis C virus, and tuberculosis; (f) leukocytopenia, impairment of liver and kidney functions; and (g) allergy to iguratimod or cannot tolerate iguratimod.
Thirty untreated patients fulfilled the inclusion criteria and without exclusion criteria were enrolled in this clinical trial.

| Primary and secondary outcomes
The primary outcome of this study was to evaluate the response rate at 24 weeks of treatment. Secondary outcome included changes of IgG4-RD RI, PGA, relapse rate, serum IgG, IgG subclass, changes of B cell, T cell subpopulations, cytokines and metabolite profiles. Side effects were recorded as well.

| Efficacy assessment
Treatment response was assessed by evaluating the changes of IgG4-RD RI scores and was divided into three types: complete response (CR), partial response (PR) and no effect (NE, including no improvement or exacerbation). IgG4-RD RI scores <3 and declining ≥2 were recognized as a CR; IgG4-RD RI scores declining ≥2 but remaining ≥3 were recognized as a PR. If a patient's IgG4-RD RI score was 3 points at the beginning, PR was considered as a 1 point decrease after the therapy. Patients with lack of apparent changes in mass sizes and/or clinical manifestations and IgG4-RD RI scores declining <2 were considered to be NE. 4,15 The relapse of IgG4-RD included two types, clinical relapse and serological relapse. 4 Clinical relapse was defined as recurrence or aggravation of clinical symptoms or imaging findings with or without IgG4 concentrations elevation. Serological relapse was defined as clinical symptoms remained stable while serum IgG4 level increased more than 1 score in IgG4-RD RI.

| Safety assessment
Safety was assessed using adverse events reports, physical examinations, and laboratory tests including hematology, liver and kidney function. These safety assessments were undertaken at screening and at all the follow-up periods.  The MS raw data were processed using Progenesis QI software (Waters Corporation) to perform peak detection and using EZinfo

| Metabolomics analysis
Ver. 3.0 software (Waters Corporation) for pattern recognition and principal component analysis. Further information and details are shown in Data S1.

| Statistical analysis
Statistical analyses were performed using SPSS Statistics version 17.0 software (SPSS Inc.) and Prism software version 6.1 (GraphPad Software). Data of normal distribution are reported as mean ± SD. Normal distribution data between two groups were analyzed using independent-samples t tests or paired samples t tests, and one-way analysis of variance was used to compare among groups.
Categorical data were analyzed by Chi-square test. Non-normal distribution data were analyzed by rank sum test. Laboratory and lymphocyte subsets of four patients who withdrew from iguratimod treatment were not included. A 2-tailed P value <0.05 was considered of significance.

| Demographics of IgG4-RD patients
In total, 30 untreated IgG4-RD patients were enrolled in this study. The age was 50.8 ± 10.2 years with disease duration of 31.5 (interquartile range 8-66) months. The male/female ratio was 1:1.
Twenty-five (83.3%) patients finished this study. Four patients withdrew because of no response or exacerbation, one patient with good response stopped iguratimod treatment at 3 months because of economic reasons.
All patients had slow but active disease progression, and needed to be treated; none of them had internal organs involvement.
ESR, hsCRP, serum IgG, IgA, IgM, IgG subclass from IgG1 to IgG4 and total IgE at baseline and after treatment are shown in Table 1.  Figure 1). After 12 weeks treatment, 88.5% of patients' serum IgG4 had a reduction of more than 30%, 42.3% of patients' serum IgG4 had a reduction of more than 50%, and 11.5% of patients' serum IgG4 returned to normal. After treatment for 24 weeks, 69.6% of patients' serum IgG4 had a reduction of more than 30%, 43.5% of patients' serum IgG4 had a reduction of more than 50%, and 13% of patients' serum IgG4 returned to normal. There was no statistical significance of serum IgG and IgG4 between 12 weeks and 24 weeks of treatment.

Changes of IgG4-RD RI
The disease activity was evaluated by IgG4-RD RI and PGA.  Figure 1).

| Safety assessment
Of all 30 patients enrolled, no severe adverse reactions were found in this study. During follow-up, 6 (20%) patients had mild side effects, including three with oral ulcers, two with stomach discomfort, and one with mild hair loss. No patients showed abnormality in blood routine tests, liver function or renal function. Of patients with stomach discomfort, omeprazole and life-style changes helped alleviate this discomfort (Table S1). Oral ulcers and hair loss were alleviated to some extent by applying vitamin B and reducing the dosage of iguratimod to once a day.
There was no significant changes in serum TGF-β1, IL-1β, IL-17A and TNF-α before and after treatment.

| Changes of T and B cell subsets before and after treatment
T cell and B cell subsets were measured before and 3 months after treatment by flow cytometry (Figures S1 and S2). Of T cell subsets, CD3+ T cells were 56.49% ± 16.28% in lymphocytes, which decreased significantly to 41.88% ± 15.4% after 3 months treatment, P = 0.003 ( Figure 2). CD3 + CD8 + T cells also decreased from 22.61% ± 10.94% to 16.13% ± 8.02%, P = 0.03 ( Figure 2). There was no statistical significance of CD3+ CD4+ T cells, CD4+ CXCR5+ TFH ( Figure 2), or the expression of PD-1 and ICOS on CD4+ CXCR5+ TFH cells before and after treatment. And there was no statistical significance in Th17 cells among healthy controls and IgG4-RD patients before and after treatment.

| Global metabolic profiling
In order to characterize the difference in metabolic profiling between the healthy control group (Group A), IgG4-RD initial group (Group B) and iguratimod treatment IgG4-RD group (Group C), OPLS-DA (orthogonal partial least squares-discriminant analysis) and PLS-DA (partial least squares-discriminant analysis) were performed.
As shown in Figure S3 Figure S4). The results suggested that iguratimod treatment affected systematic metabolic profiles, which restored the metabolic pattern back or near normal levels. To further explore the metabolic pathways involved in IgG4-RD, the identified metabolites were subjected to software IMPaLA (Table   S2). As shown in Figure 4C, there were multiple metabolic pathways (P < 0.05) constructed with the corresponding metabolites, including glycerophospholipid metabolism, sphingolipid metabolism and choline metabolism. We found phosphatidylcholines (PCs), lyso-phospatidylcholines (lyso-PCs), phosphatidylethanolamine, sphingomyelins (SMs) and glycocholic acid were involved in most of these pathways.

| Biomarker identification and metabolic pathway reconstruction
It suggested that these compounds were significantly associated with IgG4-RD and might be potential indicators of IgG4-RD (Table   S3). Thus, heatmap analysis was performed to assess the variation in the three groups ( Figure 4D). Compared with healthy controls, biomarkers were significantly changed in initial IgG4-RD patients. After iguratimod treatment, most of these metabolites were reversed to near normal. The result revealed that iguratimod treatment could ameliorate the abnormal state of metabolism induced by IgG4-RD.

| D ISCUSS I ON
In this study, iguratimod with corticosteroid as bridge treatment for  Iguratimod is a member of the methane sulfonanilide family; although most of the members of this family act as cyclo-oxygenase 2 (COX 2) inhibitors, iguratimod was considered as a novel immunomodulator based on more and more evidence. Further, iguratimod was also recommended as a disease-modifying anti-rheumatic drug by Japan, the Asia-Pacific League of Associations for Rheumatology Congress and the Chinese guidelines on the treatment of RA.
Existing studies revealed that iguratimod reduced the production of cytokines including IFN-1β, IL-6, IL-8 and IL-17A both in vitro and in vivo. 20 Further, iguratimod could reduce immunoglobulin production in both mice and humans with RA. 21 Iguratimod could also attenuate proteinuria and kidney injury in MRL/lpr lupus mice, and improve serum markers such as anti-dsDNA and serum C3. 12   This study had some limitations. First, this was a one-arm study without a control group. As there was no "golden" or "anchor" immunosuppressant drug for treatment of IgG4-RD, we did not set controls for this trial. Second, all patients were given one dose of diprospan; we should take into consideration that diprospan might affect the evaluation of iguratimod. Third, patients and doctors were not blinded which may have contributed to some biases.
In conclusion, iguratimod plus corticosteroid as bridge therapy is effective for the treatment of mild IgG4-RD; it can improve the clinical symptoms of patients, reduce the serum IgG and IgG4 levels, and can also reduce the peripheral CD3+ CD8+ T cells, and especially plasmablasts and memory B cells. Iguratimod is well tolerated in IgG4-RD patients. Clinical trial registration number: NCT03368274.

ACK N OWLED G EM ENTS
None.

CO N FLI C T O F I NTE R E S T
The authors disclose no conflicts.