Setting and participants
The study will be conducted in six clinical centers in Shanghai, China. The leading center is Shanghai Ren Ji Hospital, which is one of the main teaching hospitals of the Shanghai Jiao Tong University School of medicine. Ren Ji Hospital is also one of the biggest clinical centers of neuroimmunological disorders in China. The five branch centers are: Shanghai Rui Jin Hospital, Shanghai Xin Hua Hospital, Shanghai General Hospital, Shanghai Sixth People’s Hospital and Shanghai Ninth People’s Hospital. In this study protocol, only the patients who fulfilled the diagnostic criteria of NMOSD established by Wingerchuket al. in 2015 [18] will be included.
The study protocol has been approved by the Ethics Committees of Ren Ji Hospital (2016-071K).
The clinical study will be conducted in accordance with all national regulations. A signed written informed consent form will be obtained from all the participants before enrollment. This clinical trial complies with the principles of the World Medical Association Declaration of Helsinki—Ethical Principles for Medical Research Involving Human Subjects, with all its amendments, as well as with the principles of Good Clinical Practice.
Study design
This is a prospective study for an open-labeled, prospective, multicenter, randomized, placebo-controlled clinical trial. The estimated study time period is from Jan 2017 to Dec 2022. NMOSD patients in the remission phase will be randomized into two groups: the treatment group (hUC-MSC infusion plus regular treatment) and the controlled group (stem cell base solution infusion plus regular treatment). All the patients will be treated and followed up for two years.
The whole clinical trial includes three consecutive stages.
The first stage of the trial, which will last 24 months, is an open-labeled study and will be carried out in the leading center only. The aim of the first stage is to evaluate the safety of the infusion of hUC-MSC in NMOSD patients. Patients (n = 30) will be treated with three different doses of hUC-MSC. According to the process of enrollment, the first to the tenth participant are in the low dose group (n = 10, 1 × 106 MSC / kg·weight); the eleventh to the twentieth participant are in the medium dose group (n = 10, 2 × 106 MSC / kg·weight); the twenty-first to thirtieth participant are in the high dose group (n = 10, 5 × 106 MSC / kg·weight). The study starts from the low dose. Only when the lower dose group ends the observation period of 3 months, the study will proceed to a higher dose group. In case of unacceptable toxic reaction occurs in the dose escalation, the study will be ended immediately. In relation to safety consideration, before enrolling the patients in the low dose group, we will give a single medium dose of hUC-MSC to one patient and follow up the therapy for one year. The outcome of this dosage will be used only for safety evaluation, not to evaluate the therapy effectiveness.
The second and third stages of the study protocol are part of the multi-center double blinded randomized clinical trial. The second stage, which aims to screen out the optimal dosage, will last 24 months and will be carried out in six centers (one leading center and five branch centers). Patients (n = 160) will be randomized into four groups: the low, medium, high dose groups and the controlled group (n = 40 respectively). The aim of the second stage is to screen out the optimal dosage of hUC-MSC for NMOSD patients. The third stage, which aims to evaluate the effectiveness, will lasts for 24 months and will be developed in six centers. Patients (n = 320) will be randomized into two groups: the optimal dose group (n = 160) and the controlled group (n = 160). The aim of the third stage is to evaluate the effectiveness and safety of hUC-MSC for NMOSD patients. The study flow chart is shown in Fig. 1.
Inclusion criteria
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18–75 years of age;
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met the diagnostic criteria of NMOSD (established by the International Panel for NMOSD in 2015) [18];
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in the remission phase, defined as no new or worsening of neurological symptoms and signs in the last 30 days;
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EDSS score ≤ 8;
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taking azathioprine or mycophenolate mofetil as ongoing treatment for NMOSD;
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the results of the hepatic and renal function tests, complete blood count, and urinalysis are unremarkable;
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the coagulation tests are unremarkable;
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be able to end the follow-up period;
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signed the informed consent (by patients or their family);
Exclusion criteria
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Patients with language dysfunction, disturbance of consciousness, or unstable vital signs;
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with thyroid dysfunction or other endocrine dysfunction which cannot be controlled by treatment;
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with severe psychiatric disorders, thus unable to cooperate during the study;
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with severe hepatic or renal disorders ( the results of hepatic function > 2 upper limits; the creatinine clearance rate calculated by Cockcroft-Gault equation< 60 mL/min = or with severe systematic wasting diseases;
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Scoring complete blood count test, white blood cell count test <3.0 × 109/L or >12 × 109/L,or hemoglobin level < 80% lower limits༌or platelet counts <100 × 109/L;
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Scoring coagulation tests, one or multiple indicators (TT、APTT、PT、FIB、TDP、DD) > 2 upper limits;
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Gastrointestinal bleeding or other severe bleeding disorders in the last month;
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Had allergic history to the study-related drugs;
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Had major surgeries in the last week;
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pregnant or breastfeeding;
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With history of hepatitis B, hepatitis C, HIV infection, syphilis, or malignancies;
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With other rheumatic diseases (such as Sjogren’s syndrome, systematic lupus erythematosis), who need to use other immunodepressants other than azathioprine or mycophenolate mofetil;
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with other situations that may affect the patient’s engagement in the study
Withdrawal criteria
Withdrawal criteria are as follows: (1) patient withdraws consent; (2) newly find misdiagnosis at baseline; (3) patients is lost to follow-up; (4) investigator’s decision to withdraw the subject.
For any subject withdrawal, the reasons for withdrawal will be documented.
Intervention
1. The transportation and storage of hUC-MSC
The hUC-MSC will be immersed in liquid nitrogen, carried in a portable liquid nitrogen container and will be transported to the research centers by a cold chain logistic company. There will be a real-time recording of the temperature inside the liquid nitrogen container. The hUC-MSC will be stored in liquid nitrogen until resuscitation and infusion.
2. Resuscitation of hUC-MSC
A thermostat water bath will be preheated to 37 ℃. A metal clip will be precooled in liquid nitrogen and then the freezing bags of MSC will be extracted. The freezing bags will be quickly dropped into the water bath and swayed back and forth gently until totally melting (time duration: 2 minutes).
3. Inspections of hUC-MSC
The subjects can be infused hUC-MSC only after the sample tests meets the following requirements:
(1) Total cell amount 5*107±10% per package; cell viability over 85%;
(2) Bacterial endotoxin test negative; Gram staining negative.
4. Transfusion of hUC-MSC
Subjects in the intervention groups will receive four hUC-MSC infusions every 3 months besides the primary drugs. The dosages are 1, 2 or 5 × 106 MSC per kilogram of body weight. The hUC-MSC will be infused via the subjects’ peripheral veins. The drip rate is 30 drops per minute in the first 15 minutes of infusion and 45 drops per minute in the next 15 minutes. If there are no adverse effects, the drip rate can be gradually increased, but never exceeding 70 drops per minutes. The time of infusion should last no more than one hour. After the infusion of MSC, subjects will be infused 500 ml normal saline.
Subjects in the control group will receive four stem cell base solution infusions (the same packaging bag as hUC-MSC) every 3 months besides the primary drugs.
5. Participant safety protection measures
(1) Subjects will be admitted to the hospital when receiving the infusion;
(2) Intravenous infusion of dexamethasone of 5 mg will be given to subjects 15 minutes before the infusion of hUC-MSC;
(3) Subjects will have electrocardiogram monitoring during the whole process of infusion;
(4) Subjects will be discharged after 2–4 days observation period after the infusion.
Group allocation, randomization and blinding
The first phase of the trial is an open-label, dose-escalation study that will be performed in leading center, the Shanghai Ren Ji Hospital. For stage 2 and 3, permuted blocks randomization (block sizes of four) will be performed centrally using an integrated web response system (IWRS). In the stage 2, there are four arms, which are the low dose group (1 × 106 MSC / kg·weight), the medium dose group (2 × 106 MSC / kg·weight), the high dose group (5 × 106 MSC / kg·weight) and the control group (stem cell base solution). Patients will be assigned randomly in a 1:1:1:1 ratio to hUC-MSC group and control group, respectively. In the stage 3, there are two treatment arms. Patients will be assigned randomly in a 1:1 ratio to hUC-MSC group and control group, respectively.
In stage 2 and 3, all participants, investigators and the statistician are blinded to group allocation until the end of the study and data analysis. Only pharmacists who in charge of preparing study drug are not blinded. Double-blinding will be performed via a centralized randomization system.
Sample size estimation
Based on previous epidemiology studies on NMOSD among Asian, the replasing rate for the patients in the first year of the onset event is estimated at 55% [19]. In this study, we expected that the relapsing rate can be reduced to 40% in the MSC group, according to our preliminary data. In the first stage of exploratory study, each dosage cohort will include 10 patients to meet the safety evaluation goal, which is 30 patients in total. In the adaptive design stage 2 and 3, a total sample size of 400 patient would have 80% power to detect 15% relapsing rate reduction at a two-sided 5% significant level, with 20% expected loss to follow-up rate. Therefore, we expected to enroll a total number of 430 subjects in this study.
Discontinuation of Study Intervention
Unexpected discontinuation of study intervention criteria are: (1) deterioration of symptoms leading to MSC infusion intolerance to; (2) occurrence of adverse events or abnormal laboratory test results leading to MSC infusion intolerance; (3) individual wishes of the subjects; (4) any reasonable situations that require the clinical trial to be halted; (5) researchers decisions to halt the clinical trial.
Expulsion criteria are: (1) non-compliance to the study protocol; (2) Other reasons.
Primary and secondary efficacy endpoints
The primary study endpoint is defined as the first time of recurrence. The recurrence should meet all the following four requirements: occurrence of new symptoms or deterioration of original symptoms; symptoms lasting for more than 24 hours; more than 30 days from last recurrence;no other reasons to explain the recurrence.
Secondary endpoints include the times of recurrence, EDSS scores, MRI lesion numbers, OSIS scores, Hauser walking index and SF-36 (quality of life short Form-36) scores.
Exploratory endpoints include: serum lymphocyte subsets, cytokines, complements, serum anti-AQP4 antibody titers etc.
Adverse Events
Adverse events (AEs) refer to any unwanted symptoms or signs in a clinical investigation subject, and do not necessarily have a causal relationship with the applied intervention [20]. All AEs will be coded according to the World Health Organization’s Adverse Reactions Terminology. At the end of the clinical trial, the intensity and relationship of any AEs with the study intervention will be identified.
If any SAE is encountered, even with extremely low incidence, it will be reported to the trial’s the principal investigator, the stem cell research ethics committee, and the state supervision agency within 24 hours of its occurrence.
There are two types of side effects that need intensive monitoring, side effects during infusion and long-term side effects.
Side effects during infusion include allergic reactions (such as fever, tachycardia, dyspnea) and systematic complications (infection, embolism, multiple organ failure);
While monitoring long-term side effects, the following items will be tested every 3 months: tumor markers (AFP, CEA, CA199, CA125, CA153, CA242); complete blood count and urinalysis, liver and kidney function, blood glucose and electrolyte, coagulation tests, tests for infective diseases (HIV, RPR, hepatitis B and C; tested at baseline, 9 months and 2 years of follow-up), urine HCG; electrocardiogram, chest X-ray or CT, and abdominal ultrasound.
Follow-up
Primary, secondary, exploratory endpoint events and side-effects will be evaluated in all participating subjects at baseline and every 3 months. Magnetic resonance scans and optical coherence tomography (OCT) will be evaluated at baseline, year 1 and year 2 of follow-up.
For safety consideration, during stage one of the trial, all subjects will receive a telephone interview within one week after hUC-MSC infusion and then every two weeks until the second infusion. The telephone interviewing will include information on symptoms’ change and side effects.
The study schedule is shown in Table 1.
Table 1
Schedule of Activities of Human Umbilical Cord Mesenchymal Stem Cells to Treat Neuromyelitis Optica Spectrum Disorder (hUC-MSC-NMOSD) Study
Follow-up Period
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V0
(Screening )
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V1(Baseline)
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V2
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V3
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V4
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V5
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V6
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V7
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V8
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V9
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Time
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-30ཞ-1d
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0
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3M ± 7d
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6M ± 7d
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9M ± 7d
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12M ± 7d
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15M ± 7d
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18M ± 7d
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21M ± 7d
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24M ± 7d
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General Characteristics
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Inclusion and exclusion criteria
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Informed consent
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Physical Examination
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EDSS score
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OSIS score
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Hauser walking index
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SF-36 score
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Exploratory endpoints associated tests
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Complete blood counts
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Urinalysis
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Coagulation function
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Liver and kidney function
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Serum Glucose
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Serum electrolyte
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Thyroid function
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Urine HCG
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Tests for infective diseases
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Tumor markers
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EKG
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Abdominal ultrasound.
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chest X-ray or CT
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MRI(head)
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MRI(spinal cord)
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MRI(Optical Nerve)
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OCT
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Primary treatment
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hUC-MSC or placebo treatment
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Record of AE
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Record of combined medications
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Data management
All study documents from all the centers will be considered highly confidential and will be stored in locked filing cabinets in a room with restricted access. All data will be translated into electronic format and stored in a database. Access to the database will be strongly restricted. The principal investigator and the biostatistician will be able to log into the data set and get full access to the information only upon permission from the head of this study. Data backups will be performed regularly by trial coordinators. If required, data transfer between centers will be encrypted, and any information capable of identifying individuals will be removed.
Trial quality assurance
This study will be implemented according to high quality standards and will be delivered in accordance with the present trial protocol, which will not be amended unless any SAE occurs during its implementation. All research staff, including investigators, research assistants, and outcome evaluators, will be trained in advance to be able to competently administer all the items as per the protocol. Once the clinical trial begins, an independent trial inspector will visit each study site monthly and will be responsible for reviewing the following: overall research progress and integrity, adherence to the selection criteria for all the included subjects, compliance with the scheduled intervention for each participant, compliance with national regulations, and the handling of practical problems. Moreover, this trial inspector may occasionally provide suggestions to the principal investigator, who will make any final decisions about trial modifications, continuation, or termination.
Statistical analysis
The Kolmogrov-Smirnov Z test will be used for checking data normality. Categorical variables will be summarized as counts (percentage) and continuous variables as the means (standard error, SE) or medians (interquartile ranges, IQR), if not normally distributed. Statistical comparisons between the groups will be performed using Student’s t-test or the Mann–Whitney U test for continuous variables, as well as the χ2 and Fisher’s exact tests for categorical variables, as deemed appropriate. Correlations between continuous variables will be assessed by Pearson’s correlation coefficient or Spearman’s correlation coefficient.
In this study, two analysis sets will be required. For the safety set (SS) analysis, all safety indicators will be obtained from those who have undergone at least one dose of hUC-MSC infusions. For the effectiveness analysis set, intention to treat (ITT) analysis set will be used.
Lan–DeMets alpha-spending function with an O’Brien–Fleming boundary will be used to control the family-wise of type I error. Inverse normal combination test will be used to combined data from stage 2 and 3 in the final analysis. An independent Data and Safety Monitoring Boards (DSMB) will review and evaluate the accumulated study data for participant safety, study conduct and progress at each stage of study. Only members of DSMB could have access to the data and results of analysis. DSMB can make recommendations to terminate the study on the basis of one of the following conditions: a significant difference in terms of effectiveness between groups; a significant risk-benefit ratio in one group; low degree of success in a reasonable period (such as poor compliance, low incidence of endpoint events). The advice of the DSMB meeting will be shared with the steering committee of the trial.