Efficacy and Safety of Mizoribine in comparison with Cyclophosphamide for Treatment of Refractory Nephrotic Syndrome: Protocol for a Multi-center, Controlled, Open-label, Randomized Controlled Trial

Background: Refractory nephrotic syndrome (RNS) is associated with an increased risk of end-stage renal disease (ESRD), and treatments typically consist of corticosteroids and immunosuppressants. Mizoribine (MZR) is a novel agent used in long-term immunosuppressive therapy for different diseases, but data on its long-term use in patients with RNS are unavailable. Objective: We propose to test the efficacy and safety of MZR relative to cyclophosphamide (CYC) in Chinese adult patients with RNS. Methods: This study had a screening phase (1 week) and a treatment phase (52 weeks). On day-1, 238 patients with RNS were enrolled and randomized into a MZR


Table of Contents
Efficacy and Safety of Mizoribine in comparison with Cyclophosphamide for Treatment of Refractory Nephrotic Syndrome: Protocol for a Multicenter, Controlled, Open-label, Randomized Controlled Trial Preprint Settings 1) Would you like to publish your submitted manuscript as preprint? Please make my preprint PDF available to anyone at any time (recommended). Please make my preprint PDF available only to logged-in users; I understand that my title and abstract will remain visible to all users. Only make the preprint title and abstract visible. No, I do not wish to publish my submitted manuscript as a preprint. 2) If accepted for publication in a JMIR journal, would you like the PDF to be visible to the public?
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Original Manuscript
Abstract Background: Nephrotic syndrome that is resistant to steroidal therapy is termed refractory nephrotic syndrome (RNS), a condition that is associated with an increased risk of end-stage renal disease (ESRD). Immunosuppressants are utilized to treat RNS, however prolonged may lead to significant adverse effects. Mizoribine (MZR) is a novel agent used in long-term immunosuppressive therapy that has few adverse effects, but data on its long-term use in patients with RNS are unavailable.

Introduction
Patients with nephrotic syndrome (NS) have a classic triad of symptoms: heavy proteinuria, edema, and hypoalbuminemia [1]. Additional complications in these patients, such as venous thromboembolism, infection, and less commonly acute renal failure, can lead to catastrophic outcomes [2] Patients with NS who are resistant to or dependent on corticosteroids for more than 4 to 6 months are considered to have refractory nephrotic syndrome (RNS), also referred to as steroidresistant nephrotic syndrome (SRNS), although RNS is a complex condition and there have been differences in how this term is used [3,4]. Almost all patients with NS who have proteinuria eventually progress to ESRD [3,5]. Because prednisone therapy often fails to provide complete remission of proteinuria, immunosuppressants may also be used to prevent or treat RNS [6].
Although several immunosuppressants (cyclophosphamide [CYC], azathioprine, cyclosporine, and rituximab) can effectively treat proteinuria in patients with RNS [7,8], prolonged use of these drugs can lead to severe adverse effects (SAEs), such as sexual dysfunction, tumorigenesis, and renal dysfunction, and patients may also experience recurrence after discontinuation [9]. A novel immunosuppressant therapy that causes fewer side effects and that is suitable for long-term therapy is therefore needed for treatment of RNS.
Initial studies reported it had weak activity against Candida albicans, and subsequent studies confirmed it suppressed lymphocyte proliferation and humoral and cellular immunity [10][11][12]. Other studies had examined its immunosuppressive effects and its use with other immunosuppressants for preventing rejection after kidney transplantation. Japan has approved MZB for treatment of lupus nephritis, rheumatoid arthritis, and primary NS. Previous studies showed that MZR led to fewer SAEs, such as myelosuppression and hepatotoxicity, than other immunosuppressants [10,12].
Several clinical trials confirmed the clinical efficacy of MZR when it was used as a long-term therapy for treatment of certain autoimmune diseases, such as lupus nephritis, rheumatoid arthritis, and primary NS [10,12]. Koshikawa et al. also reported that MZR was superior to corticosteroids in reducing proteinuria in patients with RNS, and that the adverse effects were similar in a MZR group and a control group [10]. However, due to its relatively low efficacy, there is only limited data regarding the effect of MZR in patients with RNS, especially in adults with complex etiologies.
We performed a randomized, multi-center, and controlled study to evaluate the effectiveness and safety of MZR compared to CYC for treatment of adults with RNS.

Ethical considerations
This clinical trial was performed in accordance with the principles in the latest versions of the procedures study were also free of charge. The medical care needed for any adverse events was also covered by the sponsor. All data were de-identified to assure patient confidentiality. The protected health information (PHI) of all participants was entered by authorized staff or investigators into the electronic case report form (eCRF) in an electronic database that was managed by Tigermed Data Management. Any access to the system records or change of data in eCRF after the original entry was automatically recorded, and each patient's PHI was secured. All paper documents, such as consent forms, were collected and locked in the investigators' offices.

Key study objectives
1. Primary objective: To compare the therapeutic effect of MZR with CYC (the current standard therapy) for treatment of RNS by analyzing overall and total remission rates in the different groups.
2. Secondary objectives: To evaluate the efficacy and safety profile of MZR for treatment of RNS, including but not limited to clinical remission rate; progression rate; treatment failure rate (not achieving partial remission); 24 h urine protein and serum albumin; serum creatine; and estimated glomerular filtration rate (eGFR) as endpoints.
3. Exploratory objectives: To evaluate the changes of high-sensitivity C reactive protein (hs-CRP) in patients who receive MZR.

Trial Design
The specific aim of this study was to assess the hypothesis that MZR was not inferior to CYC for treatment of RNS. This study consisted of a 1-week screening phase followed by a 52-week treatment phase (Figure 1). Signed informed consent agreements were collected upon patient enrolment. The investigators identified 238 eligible participants during the screening phase (V0) according to the inclusion and exclusion criteria. Baseline information, including medical records, were obtained at this time. All enrolled participants were then randomized into a MZR group or a CYC group (1:1 ratio).
There were 9 visits by each participant during the treatment phase, during which patients were examined, AEs were evaluated, and samples were collected for laboratory tests performed at a central laboratory (Figure 2). Each visit was within 7 days of the scheduled date. All participants from both groups received tapering oral corticosteroid therapy during the entire treatment phase ( Figure 2). Patients in both groups received specific doses of the treatment drugs in addition to corticosteroids. AEs and symptoms were closely monitored and documented. Participants were able to withdraw voluntarily and investigators were required to remove participants when there was any concern regarding safety, according to the withdrawal protocol (see below).

Inclusion criteria
Patients were eligible for inclusion if they met all the following criteria: 6. Capacity and willingness to sign the informed consent document.

Exclusion criteria
Patients were excluded if they had any of the following conditions: 20. Any other disease that may affect the evaluation of medication efficacy and safety.

Premature withdrawal criteria
Any subject who had signed the informed consent form and initiated the study was able to withdraw prior to the last visit for any of the following reasons: 10. Others: difficulty for a subject to continue the study based on judgment of the principal investigator or co-investigators.

Randomization Strategy
Eligible subjects were randomized to a MZR group or a CYC group in a 1:1 ratio using the Interactive Web Response System (IWRS) on day-1. A unique subject number was assigned to each subject. Stratification was according to pathological classification. When a participant who failed the screening was deemed eligible after rescreening by the investigators, he or she was allowed to enter the randomization before the end of the enrollment.

Screening period (V0)
The investigators obtained written informed consent from all subjects prior to any intervention, and all participants were registered in the IWRS system. During the screening period, subjects were screened by the investigators for collection of relevant information. This information included medical history, medications and previous therapies, and AEs (if any). Height, body weight, and vital signs were measured, and a 12-lead resting electrocardiogram (ECG) was performed. Blood was collected for laboratory tests, including a complete blood count (CBC), biochemistry, estimated glomerular filtration rate (eGFR), HBsAg, HCV Ab, HIV, IgG, and hs-CRP. A routine urine test for 24 h protein was performed. Women of childbearing age were tested for pregnancy.

V1 (Randomization visit)
Each subject's laboratory results, physical exam results, past medical history, medication history, and pathology reports were reviewed for evaluation of eligibility according to the inclusion/exclusion criteria before enrollment and randomization. Participants were then registered in the IWRS system. If they qualified and were enrolled, appropriate doses of the study medication were dispensed, and the investigators provided information on how to take the medication and appropriate storage. The investigators also emphasized the importance of compliance, and requested the return of all unused medications.

V2-9
During this phase, all females of childbearing age returned to the study center for pregnancy tests whenever they thought there was a possibility of pregnancy. All participants were logged in to the IWRS system when they visited the study center. Appropriate doses of the study medications were dispensed, and the investigators reinforced the importance of appropriate dosing and storage.
The importance of compliance and the return of unused medications was also addressed, and detailed information regarding AEs were recorded. Pregnancy tests were routinely performed in all females of childbearing age; laboratory tests including CBC, biochemistry, eGFR, routine urine test, pregnancy test for women of childbearing age (V6 and V9), IgG, 24-h urine protein, and hs-CRP were collected.
Body weight and vital signs were measured at each visit, and a second ECG was performed at V9.

Unscheduled visit (UNS)
Study subjects who came to the study site for an unscheduled visit were documented as such.
The reason for any unscheduled visit was documented in detail. Laboratory tests were performed if necessary, and recording of vital signs, body weight, ECG etc. were performed as needed; an urgent or emergent situation, such as potential pregnancy or disease progression, was evaluated and addressed by study investigators;

Premature withdrawal from the study (End of Study, EOS)
Any subject who prematurely withdrew from the study after confirmation received an examination within 1 week from the withdrawal date, and this event was documented. The examination consisted of necessary tests and other procedures that were recommended by the investigators and accepted by the participants.

Primary Efficacy Variables
The primary efficacy variable is the total remission rate (TRR, %), and is based from data collected from each group at V9. The TRR will be calculated as the sum of the complete remission rate (CRR, %) and the partial remission rate (PRR, %); complete remission (CR) is defined as 24-h urine protein less than 0.3 g and partial remission (PR) as 24-h urine protein decline of more than 50% from baseline (V0) and less than 3.5 g.

Secondary Efficacy Variables
The secondary efficacy variables are the CRR and PRR of each group at V9; remission rate at V3, V6, V7, V8, and V9; treatment failure, defined as not achieving partial remission; 24-h urine protein, serum albumin, SCr, and eGFR (using the Chronic Kidney Disease Epidemiology Collaboration [CKD-EPI] formula), and blood urea nitrogen (BUN). The endpoint events are progression to ESRD or doubling of the SCr relative to baseline.

Exploratory Variables
The exploratory variables are the hs-CRP levels in each group at the different visits.

Safety Assessment
The safety of participants was actively monitored during the study. All events, including unplanned pregnancy, were documented and properly managed. The reportable events include but were not limited to SAEs (including death), pregnancy, important treatment-related AEs (granulocytopenia, infections, hemorrhagic cystitis, liver function impairment, hyperuricemia, malignancy, amenorrhea, alopecia, nausea, and vomiting), and AEs leading to study discontinuation.
Safety was monitored by asking participants about symptoms, and by assessment of physical examination results and laboratory test results (CBC, blood biochemistry, IgG, hs-CRP, pregnancy test, routine urine test, body weight, vital signs [blood pressure, pulse, and body temperature], chest CT, and 12-lead ECG).

Statistical Plans
The sample size calculation was based on a noninferiority comparison of the MZR and CYC groups regarding TRR after treatment, and was to be calculated using SAS. Studies of refractory NS assumed the TRR in each group was 60% [13][14][15] and the risk ratio (RR) of MZR-to-CYC was 1.0.
Thus, we included 119 participants in each group based on the following assumptions: TRR of 60% for each group, non-inferiority difference ratio of 0.674, power of 80%, one-sided significance level of 2.5%, and a drop-out rate of 20%.
The full analysis set (FAS) will include all subjects who were randomized and treated with at least one dose of a study medication and received at least one post-treatment efficacy assessment.
The per-protocol set (PPS) will include all FAS subjects who had no major protocol violation. The safety set (SS) will include all randomized subjects who received at least one dose of a study medication and had at least one safety assessment.
The primary and secondary efficacy outcomes will be assessed and analyzed. A noninferiority test of TR will be performed in the PPS population by comparing the two groups at V9, and relative RRs and two-sided 95% confidence intervals will be calculated. A secondary analysis of the primary efficacy variable will be performed in the PPS at the end of the study and in the FAS at V9. The number of subjects who achieve TR in the PPS will be calculated in each group at each visit. The secondary efficacy variables will be analyzed using descriptive statistics. The safety assessment will be performed in the SS population. The incidences of AEs in the two groups will be compared.
Subgroup analysis will be performed as necessary, such as exploratory outcome analysis using hs-CRP and other variables.

Results:
The study began in November 2014, and completed in March 2019. A total of 239 participants from 34 hospitals in China were enrolled. Data analysis have been completed. The results are waiting for finalized by Center for Drug Evaluation.

Discussion
NS is caused by abnormally increased amount of protein leaking from the kidneys into the urine [16]. In adults, proteinuria greater than 3.5 g/1.73 m 2 per day is considered nephrotic. The incidence of NS in the general population is about 1 per 100,000 [17]. The manifestations of NS are more complex in adults than children, because adults experience a wider variety of primary and systemic diseases, making management more difficult.
Diabetes mellitus is the most common cause of secondary NS, and other causes include systemic lupus erythematosus, Hepatitis B or C, amyloidosis, and multiple myeloma [2]. The pathogenesis of secondary NS differs from that of primary NS, in that treatments for primary NS focus on the primary disease. The steroid hormone regimens and immunosuppression strategies also differ for primary and secondary NS. Thus, we excluded patients with secondary NS from this study. A potential limitation of this study is that the pathologic types of RNS may be concentrated in a few common pathologic types in Chinese patients such as MN.
RNS remains poorly understood, but is currently considered immune-mediated because many patients respond to immunosuppressive drugs [22]. The immune mechanisms underlying the pathophysiology of RNS may include systemic T-and/or B-cell dysfunction [23]. In particular, cytokines produced by T cells are thought to act on the slit diaphragms of renal podocytes, and increase protein permeability [24]. Alkylating agents, such as CYC, can inhibit T cells and B cells, have a wide range of applications in RNS, and are often used as positive controls in clinical studies.
However, CYC and other traditional alkylating agents can cause myelosuppression, infertility, bladder cancer, leukemia, skin cancer, and hemorrhagic cystitis [25]. The optimal regimen to be used for immunosuppression in patients with RNS is uncertain, and many clinical trials are evaluating different therapeutic schedules for patients with different pathological types of RNS. Calcineurin inhibitors mainly inhibit T cells and can induce remission, but there is nearly a 50% risk of relapse after cessation of calcineurin inhibitors in patients with IMN [26]. Rituximab is a B-cell-depleting agent, and when used alone it can induce disease remission and causes few SAEs [27].
MZR was originally isolated as an antibiotic agent that had activity against C. albicans, and subsequent studies reported it had strong immunosuppressive activity in various animal experimental models [11]. MZR is an imidazole nucleoside, and its metabolite, MZ-5-P, selectively inhibits inosine monophosphate (IMP) synthetase and guanosine monophosphate (GMP) synthetase, resulting in the complete inhibition of guanine nucleotide synthesis without incorporation into nucleotides. MZR also has selective inhibitory effects on inosine 5-monophosphate dehydrogenase (IMP-DH), an enzyme that functions in de-novo purine nucleotide synthesis. MZR thus suppresses cell-mediated and humoral immune responses by suppressing T-and B-lymphocyte proliferation via the inhibition of GMP synthesis [28,29]. This mechanism is similar to that of CYC, but MZR is associated with fewer AEs. Two previous small head-to-head trials of specific agents compared the relative effectiveness and safety profiles of MZR and CYC in adult patients with MN [30,31]. Both studies showed that MZR reduced albuminuria and had a good safety profile.
Accordingly, the current clinical trial will evaluate the efficacy and safety of MZR in comparison with CYC for treatment of RNS in Chinese adults with glomerular diseases. It is a multicenter, randomized and controlled trial, and we will assess the efficacy of MZR based on changes in total proteinuria, eGFR, and AEs.
There is no universal treatment regimen used for adults with primary NS, although corticosteroids are beneficial in pediatric patients. Adults with NS usually receive conservative management and steroid treatment when conservative therapies fail [17,32]. However, the long-term use of immunosuppressants for treatment of RNS may lead to SAEs with unclear benefits. MZR appears to cause less severe AEs and provides good outcomes when used to treat other immunerelated conditions, such as lupus nephritis, post-kidney-transplant disorders, and primary nephritic syndrome [10,12,33,34]. However, there is currently insufficient evidence regarding its efficacy and safety for treatment of RNS.
The proposed study will examine the safety and efficacy of MZR as a long-term treatment for adults with RNS. It is so far the largest randomized control trial to examine MZR in China. The trial lasted about 1 year, and frequent follow-ups were used to monitor efficacy and AEs. If the results confirm the efficacy and safety of MZR, this could lead to the improved management of adults with RNS and also improve our understanding of the pathology of RNS caused by different renal diseases.

Ethics approval and consent to participate
This study was approved by the Medical Ethics Committee of the Chinese People's Liberation Army General Hospital on 15 September 2014 (C2014-042-01). Written informed consent for publication was obtained from all participants.

Consent for publication
Written informed consent for publication was obtained from all participants.

Availability of data and material
The datasets generated during the current study are not publicly available due to the unfinished clinical analysis, but will be available from the corresponding author on reasonable request.

Figures
Study timeline.