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STUDY PROTOCOL
Asia Pac J Clin Trials Nerv Syst Dis 2017,  2:129

Safety and efficacy of allogeneic umbilical cord blood therapy combined with erythropoietin in children with cerebral palsy: study protocol for a double-blind, randomized, placebo-controlled trial


Department of Rehabilitation Medicine, CHA Bundang Medical Center, CHA University, Republic of Korea

Date of Web Publication8-Nov-2017

Correspondence Address:
MinYoung Kim
Department of Rehabilitation Medicine, CHA Bundang Medical Center, CHA University
Republic of Korea
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Source of Support: The study was supported by Korean Ministry of Health and Welfare (HI 13C1204)., Conflict of Interest: None


DOI: 10.4103/2542-3932.217491

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  Abstract 

Background and objectives: Cerebral palsy typically results from brain injury during prenatal, perinatal, or infant period. Established medical management involves rehabilitation therapy with comprehensive multidisciplinary approach to minimize various complications and to improve brain plasticity. Recently, direct brain tissue modifying therapy using stem cell therapy has been investigated as a new therapeutic modality. In this study, the combination therapy of allogeneic umbilical cord blood and/or erythropoietin is investigated to find out the efficacy and safety in children with cerebral palsy over empirical rehabilitation therapy.
Design: A randomized, double blind, controlled trial.
Methods: Patients with cerebral palsy who visited Rehabilitation medicine center in CHA Bundang Medical Center, Republic of Korea from October 2013 to October 2015 are included in this trial. A sample size of 120 patients is needed. Patients will be randomly assigned to three groups of cord blood treatment or a control group. Cord blood treatment group will receive either cord blood and erythropoietin, or cord blood and erythropoietin placebo, or placebo cord blood and erythropoietin in addition to conservative rehabilitation therapy.
Outcome measures: The primary outcome of the study is the efficacy of combination therapy of allogeneic cord blood stem cell and erythropoietin and either monotherapy in terms of the change from baseline motor function assessments including Gross Motor Function Measure, Gross Motor Performance Measure, motor and mental subscales of Korean version Bayley Scales of Infant Development, 2nd edition. The secondary outcomes of the study include other functional assessment tools, imaging studies (fractional anisotropy of diffusion tensor imaging and positron emission tomography), electroencephalography, and evoked potentials.
Discussion: The results of this trial will provide optimal evidence of cord blood transplantation with or without erythropoietin as a therapeutic modality in children with cerebral palsy.
Ethics and dissemination: The study will be reviewed by IRB. The results of the trial will be published in a peer-reviewed journal and will be disseminated via various forms of media.
Trial registration: This trial had been registered in the ClinicalTrials.gov (identifier: NCT0199114) on 17 November 2013.

Keywords: umbilical cord blood; transplantation; erythropoietin; combination therapy; cerebral palsy


How to cite this article:
Cho KH, Min K, Lee SH, Kim M. Safety and efficacy of allogeneic umbilical cord blood therapy combined with erythropoietin in children with cerebral palsy: study protocol for a double-blind, randomized, placebo-controlled trial. Asia Pac J Clin Trials Nerv Syst Dis 2017;2:129-39

How to cite this URL:
Cho KH, Min K, Lee SH, Kim M. Safety and efficacy of allogeneic umbilical cord blood therapy combined with erythropoietin in children with cerebral palsy: study protocol for a double-blind, randomized, placebo-controlled trial. Asia Pac J Clin Trials Nerv Syst Dis [serial online] 2017 [cited 2018 Aug 21];2:129-39. Available from: http://www.actnjournal.com/text.asp?2017/2/4/129/217491


  Introduction Top


Background

Cerebral palsy is a non-progressive disorder that occurs in the developing brain characterized by abnormal movement and posture, eventually leading to impairments of normal daily activity including gait disturbance. Children with cerebral palsy are often accompanied by mental retardation, dysphasia, gastroesophageal reflux disease, neurogenic bladder, visuo-perceptual impairment, strabismus, convulsion, and, in severe cases, significant relevant disorders including dyspnea (Bax et al., 2005), which may increase the risk of morbidity at a young age depending of the severity of the complication and they and their care-givers would have difficulties in maintaining the life. In addition, the incidence of cerebral palsy rises with the increased survival of high-risk newborns resulting from medical improvements (Vohr et al., 2005). Therapeutic modalities, however, is still limited to medical care or treatment for seizures. Despite consistent rehabilitation therapy, recovery of functions from brain lesion is very slow and some impairment becomes consolidated. The causes of cerebral palsy are various, but the typical pathology is hypoxic-ischemic encephalopathy resulting from low birth weight and premature delivery. Although clinical results have been improved by early detection and initiation of treatment in high-risk infants, incidence of disability has not been reduced.

Recently, many studies have focused on cell therapy as a treatment approach to brain injury disorders. A study has reported stem cells administered into animal model for ischemic stroke survived as well as brought functional improvements (Chopp and Li, 2002). An animal study has reported efficacy of umbilical cord blood (UCB) therapy for cerebral palsy. In vitro, UCB cells are known to differentiate into nerve cells, astrocytes, and oligodendrocytes (Buzańska et al., 2006), and, depending on culture conditions, secret cytokines including various growth factors (Neuhoff et al., 2007). Actually, non-hematopoietic UCB cells are known to have properties of nerve system, which is evidenced by the finding that they could differentiate into all of three germ layer cells, particularly dopaminergic neuron (Greschat et al., 2008). In vivo, UCB stem cells have therapeutic effects on neurologic injury from cerebral infarction as well as on the recovery of the nerve from traumatic brain injury or spinal cord injury (Lu et al., 2002; Xiao et al., 2005; Shim et al., 2008). When administered surgically into Alzheimer's disease mouse model, UCB-derived human mesenchymal stem cells have been reported to improve cognitive function and pathophysiology (Lee et al., 2012). After the first UCB transplantation case in Fanconi's anemia in 1988, these cells have been used in hematologic, immunologic, oncologic, and inherent metabolic diseases (Sirchia and Rebulla, 1999; Kurtzberg et al., 2005; Martin et al., 2006).



The mechanism of action has been known to be neuroprotection through anti-inflammatory effect and anti-apoptosis, as well as effects on cell signaling pathway (Dasari et al., 2008). Most of all, the ability to regenerate damaged tissues including regeneration of nerve and blood vessels is thought to have some contribution to it (Taguchi et al., 2004; Greschat et al., 2008). In addition, the following hypothesis is proposed for the finding that intravenous administration of bone marrow-derived mesenchymal cells induced improvement of function, despite “ first-pass effect”, i.e., a significant amount of cells administered are segregated into the lung or spleen; cell stimuli on non-neurologic organ systems like the lung and spleen increase the ratio of anti-inflammatory macrophage to pro-inflammatory macrophage via inducing the alteration of microenvironment in the brain and the control of microglia, leading to neuroprotection (Walker et al., 2012).

Autologous UCB therapy has been reported to be safe and feasible in cerebral palsy, and randomized, placebo-controlled, double-blind studies to investigate efficacy and safety of autologous UCB therapy are on-going (NCT01147653 and NCT01072370 in ClinicalTrials.gov).

Co-administration of UCB therapy with hematopoietic growth factor to potentiate its therapeutic effect has been studied in animal and clinical studies, and, until now, erythropoietin (EPO), granulocyte colony-stimulating growth factor (G-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF) have become available (Maurer et al., 2008). Among them, since 2007 and 2008, a number of studies investing effect of EPO on nervous system have been reported (Lapchak et al., 2008; Maiese et al., 2008; van der Kooij et al., 2008). In infant rats with hypoxic brain injury, EPO showed the neurobehavioral improvement through angiogenesis and nerve regeneration (Iwai et al., 2007), and induced decrease of infarction volume, with reduced neurologic impairment (Wang et al., 2007). In particular, recovery of neurologic injury derived from EPO treatment was reported in cerebral palsy animal model (Meier et al., 2006; Liu et al., 2011). Mode of mechanism of EPO is believed that it inhibits neuronal degeneration and activation of pro-inflammatory cells as well as induces angiogenesis. Particularly, prevention of apoptosis which is believed to be one of major causes for brain necrosis during hypoxic-ischemic brain injury due to premature delivery is known to be one of EPO's effects (Goñi-de-Cerio et al., 2007). In addition, EPO is believed to play a role in proliferation and differentiation of injured nerve cells and endogenous or grafted stem cells, which is evidenced by the finding that EPO showed to have effect on cell development and cell protection via activation of STAT 3, STAT 5, and ERK1/2 and regulation of caspase activity in cellular signal transduction and the finding that activations of Wnt pathway, Atk signaling upon which the pathway is dependent, nuclear factor-κB which is expressed during brain injury, and HIF-1 which is related to these effect as well as plays an important role in nerve regeneration of stem cells occur under the presence of EPO (Maiese et al., 2008). Furthermore, it has been demonstrated that EPO can be transported into and even stabilize the blood-brain barrier which is believed to be one of major obstacles to treatment with stem cells (Chi et al., 2008). Especially, EPO has been known to have long-term neuroprotective effect (Gonzalez et al., 2007) and functional recovery through angiogenesis in perinatal hypoxic brain injury (Gonzalez et al., 2007).

In the absence of available autologous UCB, allogeneic UCB can be an alternative treatment option. For two decades, allogeneic UCB has replaced bone marrow for a patient with malignant hematologic disease without a matching donor who needs hematopoietic stem cell transplantation (Stavropoulos-Giokas et al., 2012). It has been known that UCB has superior immune tolerance and its incidence rate of graft-versus-host disease is low (Tse and Laughlin, 2005). As such, UCB has a merit in the field of regenerative medicine (Lee et al., 2010). The effect of allogeneic UCB has also been reported in a double-blind study in children with cerebral palsy, where EPO was co-administered (Min et al., 2013b). This combination therapy has showed efficacy in patient with severe traumatic brain injury (Min et al., 2013a).

Objective

The objective of this trial is to investigate the safety and efficacy of combination therapy of allogeneic UCB and EPO, and either monotherapy for children with cerebral palsy.


  Methods/Design Top


Study Design

A randomized, double-blind, placebo controlled trial.

Study setting

This study is being conducted at the Department of Rehabilitation medicine, CHA Bundang Medical Center, Republic of Korea.

Study participants

Patients with cerebral palsy who visited rehabilitation clinic, in a University hospital from October 2013 to October 2015 and fulfill the following criteria are eligible.

Inclusion criteria

  • Diagnosed with cerebral palsy
  • Age of ≥ 10 months and ≤ 6 years
  • Mismatch in HLA-A, -B, and -DR ≤ 2, and total nucleated cell count ≥ 3 × 107/kg. If the cell count is less than given values, more than 1 unit could be used.
  • Written informed to participation in the study obtained from the subject's representative
  • Willingness and ability to be hospitalized according to the schedule specified in the protocol and continue to participate for 12 months after study entry


Exclusion criteria

  • Current aspiration pneumonia
  • Known genetic disease
  • History of hypersensitivity reaction to any study drugs pertinent to the study
  • History of participation in any other study with stem cell
  • Prior treatment with EPO within 3 months prior to study entry
  • Known coagulopathy with family history of thrombosis or medical history of recurrent thrombosis
  • Patient with severe convulsion disease who has clinical convulsion despite combination therapy with three or more agents
  • Uncontrolled hypertension defined as systolic blood pressure > 115 mmHg and/or diastolic blood pressure > 70 mmHg
  • Hepatic impairment defined as aspartate aminotransferase (AST) > 55 IU/L and/or alanine aminotransferase (ALT) > 45 IU/L
  • Renal impairment defined as creatinine (Cr) ≥ 1.2 mg/dL
  • Absolute neutrophil count ≤ 500/dL
  • Presence of diagnosed or suspected malignant tumor and/or hematologic malignancy
  • Non-compliance with study visits specified in the protocol or unwillingness of care-giver due to lack of understanding of the patient


Withdrawal Criteria

Patients may be withdrawn from the study if the Principal Investigator (PI) and subinvestigator believe that they could not continue the study due to safety reasons such as adverse events (AEs), or deterioration of complications or existing diseases. Data obtained until discontinuation of or withdrawal from the study according to the following criteria may be included in final analysis of variables:

  • Violation of inclusion/exclusion criteria of the study
  • Occurrence of the disease or need for surgery which may influence on efficacy evaluation resulting from the subject's inability to comply with the study procedure
  • Inability of the care-giver to continue the study
  • Noncompliance of care-giver with instruction of the study staff (e.g., on the basic understanding for status of the child patient or how to deal with the child patient besides treatment period)
  • The subject experiences an AE or a serious AE (SAE) hindering the participation in the study
  • Essential examination or procedure cannot be done due to subject's status or any other reasons.
  • Request of the subject or the subject's representative to discontinue the study
  • Follow-up loss
  • Other cases where, at the discretion of the investigator, the subject should be discontinued from the study


Randomization

The Study Will Enroll Total 120 Subjects Who Will Be Randomly Assigned to Four Treatment Groups, Considering 20% of Dropout Rate. the Target Number for Each Treatment Group Is 30 ([Figure 1]) Based on Central Limit Theorem (Cam, 1986). Active Rehabilitation Should Be Performed With Each Treatment Arm as the Conservative Therapy.
Figure 1: Trial flow chart.
Note: UCB: Umbilical cord blood; EPO: erythropoietin; UCB-P: UCB placebo; EPO-P: EPO placebo; rehab: rehabilitation.


Click here to view


Subjects will be randomized into their treatment arm of A, B, C, or D in a 1:1:1:1 ratio using block randomization code generated with SAS V9.2 software. Randomization will be stratified by 2 stratification factors: age (< 3 years old vs. ≥ 3 years old) and disease severity per GMFCS (mild to moderate [GMFCS grade I to grade III] vs. severe [grade IV to grade V]). Randomization code list is generated and managed by an independent statistical team and allocated randomization code is delivered to the randomization code manager in clinical trials center, CHA Bundang medical center. The manager will identify the code, but have no contact with the study team and be not engaged in study analysis. Each subject will receive treatment with UCB or UCB placebo (UCB-P); EPO or EPO placebo (EPO-P); and cyclosporine or placebo cyclosporine according to their allocated treatment arm. The randomization code manager will not share the randomization code of each subject with the UCB manager and study drug manager; instead, notify the study drug manager of a medication code for the relevant study drug and the UCB manager whether or not to dispense UCB unit. Investigators including the PI and subinvestigators, physicians, therapists who assess and treat subjects, nurses, subjects, care-givers, and study drug manager will be kept blinded.

The PI or the designee will administer the UCB or UCB-P in cell therapy center, CHA Bundang medical center. After thawed and washed, dispensed UCB will be delivered to UCB administrator. UCB-P will be prepared using peripheral blood obtained from the relevant subject on the day of UCB therapy. Subject in all treatment arms will be provided with same treatment and stay in hospital for same duration.

Dose of cyclosporine needs to be adjusted according to blood level. In order that the investigators is kept blinded, for blood level of cyclosporine in treatment arms C and D, the sham result will be referred and provided in a laboratory report with the cooperation of Department of Laboratory Medicine. True value will be known to the investigators when code unblinding occurs after the completion of the study.

Retrieval and disposal of all unused study drugs and any study drugs returned from subjects will be in charge of the manufacturers of each study drug. In case of cyclosporine, unused drug will be delivered to CMG pharmaceuticals, the two detailed bodies of current trial, to be discarded in accordance with SOP on drug disposal of the CMG pharmaceuticals.

Unblinding of treatment codes will not be performed until PI decides to open blinded data review board, prepare statistical analysis plan, and lock the database. After completing data transfer to an independent data management team, the treatment code can be opened. However, treatment code may be unblinded at the discretion of the investigators in emergency conditions such as occurrence of AEs that lead to death or is fatal, occurrence of SAEs or in a case that at the discretion of the investigators the identification of study drug needs to be known to treat significant medical conditions, or in case that the study should be discontinued due to an unexpected condition. In case of unblinding, it should be ensured that the randomization code manager is contacted immediately to identify the treatment arm of subjects; Treatment arms A, B, C, or D. A randomization code manager is responsible for randomization code to be received from independent statistical department and kept appropriately, to notify an UCB preparer and personnel responsible for managing study drugs of randomization number using pre-specified unique number to ensure study treatment to be performed per subject treatment arm and to monitor relevant procedure.

Intervention

Cord blood preparation and administration

Cryopreservation of UCB: Allogeneic UCB cryopreserved in the UCB bank of CHA Bundang Medical Center, which has been authorized from Korean government will be used. After obtaining written informed consent for the donation from the mother, the UCB collected is to be cryopreserved from the ambient condition within 36 hours. White blood cell concentrate generated by Rubinstein method (Rubinstein et al., 1995) where red blood cell and plasma are removed from UCB resulting in WBC layer, to which cryoprotectant dimethylsulfoxide (DMSO) and dextran 40 are added is to be frozen using a controlled-rate freezer and thereafter cryopreserved in a freezer at –198°C. Prior to freezing, small amount of UCB is to be collected to examine total nucleated cell count, cell viability, CD34+ stem cell, and ABO and Rh (D) blood typing. UCB negative for HBsAg, anti-HCV, anti-HIV, VDRL, bacteria culture, and anti-CMV IgM is considered suitable for use and is to be cryopreserved.

Selection of UCB: Donated UCB which has been cryopreserved in the UCB bank of study hospital will be used (see Control and Study of Umbilical Cord Blood Act, Article 10). Suitable UCB (i.e., containing total nucleated cell count ≥ 3 × 107/kg with two or less mismatch among HLA-A, -B, and -DR in high resolution typing) will be selected.

Thawing, washing and storage of UCB: Prior to therapy, DMSO is to be thawed in 37°C water bath, from which DMSO used as cryoprotectant is to be washed using diluted solution consisting equal volume of 10% dextran and 5% albumin. The preparation is to be preserved at an ambient condition where more than 95% of mononuclear cells it contains survive for 2 hours (Rubinstein et al., 1995). Frozen UCB will be thawed and washed by the UCB preparer immediately prior to therapy according to randomization code and UCB protocol.

Method for UCB intravenous infusion: (1) Treatment groups A and B: After 4 hours of fasting, subjects will be sedated with chloral hydrate (Pocral®) syrup according to sedation protocol of study hospital. Intravenous infusion will be conducted in stem cell center of study hospital and the therapy will be performed by the PI or a physician delegated from the PI. Oxygen saturation will be monitored during therapy. (2) Treatment groups C and D: After 4 hours of fasting, subjects will be sedated with chloral hydrate (Pocral®) syrup according to sedation protocol of the study hospital. Similarly as treatment groups A and B, subjects will be transferred to stem cell center of study hospital. Thereafter, therapy will be performed by the PI or a physician delegated from the PI. Subjects should stay at the stem cell center for same period as that of treatment groups A and B. Subjects, care-givers, and all study staffs including nurses or therapists who perform evaluation and therapy will be kept unaware of the treatment allocation.

Package and labeling of UCB: Once the PI decide to treat the subject with and order UCB therapy, the preparation of UCB will be packed with the following label ([Figure 2]) attached on it by or under the supervision of the UCB manager in the UCB bank.
Figure 2: Label attached on the preparation of UCB.

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Management and handling of UCB-P

UCB-P will be prepared and managed under the good manufacturing practice (GMP) condition by mixing 1.5–3 mL of subject's peripheral blood with normal saline to be a total volume of 10–15 mL. Subject's peripheral blood to prepare UCB-P should be obtained and sent to a GMP facility prior to first administration of EPO on the day of UCB therapy.

Management and handling of EPO and EPO-P

EPO and EPO-P packed and labeled by the EPO manufacturer with unique treatment number will be supplied to the personnel responsible for managing study drugs.

Personnel responsible for managing study drugs will maintain records for the accountability including supply, dispensing, inventory, and prescription of the study drug and return or disposition of unused study drug to ensure the verification of the accountability and/or storage condition of study drug. These records will include dates, quantities, batch/serial numbers.

The PI or subinvestigators will reconcile the amount of study drugs in accountability records with those in case report forms (CRFs), and, if there is any discrepancy, investigate the cause, document the results, and take appropriate action including correction of the record. The study drug once allocated to a subject cannot be reused to another subject.

The generic name of EPO is erythropoietin alfa manufactured in 10,000 IU/mL using human erythropoietin as active ingredient. EPO will be stored in hermetic container at 2–8°C. The dosage for administration is 500 IU/kg, in total six times.

Normal saline will be used as placebo of EPO provided by erythropoietin alpha manufacturer, LG Lifesciences, Ltd. The package, labeling, and appearance of placebo should be identical with that of active drug. Cyclosporine will be purchased through pharmaceutical department of study institution through the cooperation of research administration support department. Placebo cyclosporine will be provided by the manufacturer, Chongkundang Pharmaceutical Corp., identical in package, labeling and appearance to active drug.


  Outcome Measures Top


Primary outcome measure

The efficacy of combination of allogeneic UCB therapy and EPO and either monotherapy in terms of the change from baseline in gross motor function measure (GMFM) (Russell, 2002), gross motor performance measure (GMPM) (Boyce et al., 1995), raw scores of motor and mental scale of Bayley Scales of Infant Development, 2nd edition (BSID-II) (Bayley, 1993), during rehabilitation therapy for cerebral palsy.

Secondary outcome measures

  • The change from baseline on gross motor function classification system (GMFCS), functional independence measure for children (WeeFIM), pediatric evaluation of disability inventory (PEDI), quality of upper extremity skills test (QUEST), visual motor integration (VMI), selective control assessment of lower extremity (SCALE), modified Ashworth scale (MAS), modified Tardieu scale (MTS) for hamstring, and manual muscle testing (MMT) during rehabilitation therapy for cerebral palsy
  • The change from baseline in the value of fractional anisotropy (FA) in diffusion tensor imaging (DTI) and the change of glucose metabolism during positron emission tomography (PET)
  • To investigate therapeutic efficacy of study treatments in terms of the change from baseline in electroencephalography (EEG) and evoked potentials (EP)


The above indices are recorded by therapists blinded to the study protocol and group discretion. The trial outcome measurement schedule is shown in [Table 1].
Table 1: Trial outcome measurement schedule

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Safety assessments

During the trial, AEs include 1) AEs which occurred prior to UCB therapy, and 2) AEs which occurred after UCB therapy (treatment emergent AEs; TEAEs), and TEAEs consist of 1) AEs which newly occurred after UCB therapy, and 2) AEs which are exacerbation of severity of underlying sign/symptom after UCB therapy. Safe evaluation in the study will include TEAEs only, all of which will be listed into table. All AE terms will be standardized by system organ classes (SOC), preferred term (PT) using MedDRA® (Medical Dictionary for Regulatory Activities) Version 14.0. When counting the TEAEs, multiple TEAEs which occurred in a subject for a SOC and PT will not be duplicated, and for AEs of which the severity is not identical, their severity will be attributed to more severe one. For TEAEs in a subject with more than a single causality, it will be attributed to the one more related to the study drug(s).

Subjects or legally acceptable representatives will be given a chance to report AE spontaneously by answering an open-ended question: e.g., Do you have had or felt any abnormalities in your health (since last visit)?

The investigator will collect and document any AEs occurring after initiation of study treatments in appropriate CRFs using the following instructions and definitions. It is preferred to describe comprehensive diagnosis or syndrome rather than individual sign/symptom. The investigator will report the AE using standard medical terminology. The data documented in CRFs should be consistent with those in source documents. Date of the AE onset (for clinically significant abnormality in laboratory measures, the day reported as being abnormal) should be stated. For any AEs, their severity must be graded and reported by the following rating scales: Mild: minimal discomfort without any disruption of normal daily activity, which could be readily endured; Moderate: discomfort sufficient to reduce or affect normal daily activity; Severe: incapacitating with inability to work or perform normal daily activity.

Any action taken regarding experimental or control drug will be documented in appropriate CRFs using following criteria: Not applicable: For the AE which occurs during the period when the experimental or control drug dose not used (e.g., pre- or post-treatment period or single-dose study); Interrupted: The experimental or control drug is temporarily interrupted due to the AE based on the subject's will or at the discretion of the investigator that it is the most appropriate considering safety of the subject; Dose not changed: Dose of the experimental or control drug does not changed despite the AE; Discontinued: The experimental or control drug is permanently discontinued due to the AE based on the subject's decision or at the discretion of the investigator that it is the most appropriate considering safety of the subject.

One of following items must be chosen and documented in appropriated CRFs for other actions taken for the AE: None: No measures were taken; Treatment administered: A certain medicinal product (prescription drug or over the counter (OTC) was administered; Hospitalization or prolongation of existing hospitalization: Inpatient hospitalization or prolongation of existing hospitalization was required; Therapeutic or diagnostic procedure: Other therapeutic measures (e.g., icing, electric cushion, orthosis, or plaster cast) or diagnostic procedures (e.g., further laboratory assessments or CXR) were required; or Others. Date when AE is resolved completely should be documented as date of completion. For AEs with different signs/symptoms (syndrome), the duration of the AE is based on sign/symptom for which the duration is the longest.

The course of the AE will be judged using the following criteria: Resolved: There is no AE any more for complete resolution; Resolved with sequelae: The patient has recovered from the AE with observable residual sequelae; Worsened: The AE is present and its severity is worsened. The event should be documented as another AE according to the rule of repetition of AE reporting; Death: The event results in or induce directly the death of the subject; or Unknown.

The investigator will evaluate the relationship of the AE to study drugs and describe his/her opinion on it according to the following criteria. Definitely related: Exposure to the test drug is evident with temporal sequence of the AE onset relative to the administration of the test drug. The AE is more likely explained by the test drug than by another cause. Dechallenge is positive. Rechallenge (if feasible) is positive. The AE shows consistent pattern with previous knowledge of the test drug or test drug class; Probably related: Evident exposure to the test drug. The temporal sequence of the AE onset relative to administration of the test drug is reasonable. The AE is more likely to be explained by the test drug than by another cause. Dechallenge is positive; Possibly related: There is evidence of exposure to the test drug. A temporal relationship between administration and occurrence of the event is suspected. The AE could have been due to another equally likely cause. Dechallenge is positive; Probably not related: There is evidence of exposure to the test drug. There is another more likely cause of the AE. Dechallenge (if performed) is negative or ambiguous. Rechallenge (if performed) is negative or ambiguous; Definitely not related: The subject/patient did not receive the test drug. Or temporal sequence of the AE onset relative to administration of the test drug is not reasonable. Or there is another obvious cause of the AE; Unknown: Information is conflicting or not sufficient to determine and could not be supplemented or verified.

Management of AEs during and after the trial will be performed as follows. Any action taken regarding experimental or control drug will be documented in appropriate CRFs using following criteria. Not applicable: for the AE which occurs during the period when the experimental or control drug dose was not used (e.g., pre-/post-treatment period or single-dose study); Interrupted: the experimental or control drug is temporarily interrupted due to the AE based on the subject's will or at the discretion of the investigator that it is the most appropriate considering safety of the subject; Dose not changed: dose of the experimental or control drug does not changed despite the AE; Discontinued: the experimental or control drug is permanently discontinued due to the AE based on the subject's decision or at the discretion of the investigator that it is most appropriate considering the safety of the subject.

Data collection, management, and statistical analysis

The medical records acquired from the trial will be collected by staff members who will ensure that the records are high-quality and accurate. The number of AE cases and their proportions will be presented in SOC and PT with 95% confidence interval for the subject with at least one AE. Frequency and percentage of GMFCS at months 1, 3, 6, and 12 will be compared to those of baseline by each treatment group. Inter- and intra-treatment group comparisons will be based on chi-square test or Fisher's exact-test, and McNemar test, respectively. Descriptive statistics (mean, standard deviation, median, minimum and maximum) for the changes from baseline in WeeFIM, PEDI, QUEST, VMI, SCALE, MAS, MTS, and MMT at months 1, 3, 6, and 12 will be presented by each treatment group. Comparisons between treatment groups and each time-point will be based on repeated measured analysis of variance (ANOVA). Descriptive statistics (mean, standard deviation, median, minimum and maximum) for raw, scores, and standard scores of the three VMI parameters, i.e., Beery VMI, Visual Perception, and Motor Coordination will be presented by each treatment group. Comparisons of raw scores and standard scores for the three parameters between treatment groups and each time-point will be based on repeated measured ANOVA. Descriptive statistics (mean, standard deviation, median, minimum and maximum) for the change from baseline in FA values of DTI by regions at month 12 will be presented by each treatment group. Comparisons between treatment groups and each time-point will be based on ANOVA or Kruskal-Wallis according to the normality of the data. Descriptive statistics (mean, standard deviation, median, minimum and maximum) for the change from baseline in PET at month 12 will be presented by each treatment group. Comparisons between treatment groups will be based on ANOVA or Kruskal-Wallis according to the normality of the data. Frequency and percentage of EEG and EP at month 12 will be compared to those of baseline by each treatment group. Inter- and intra-treatment group comparisons will be based on chi-square test or Fisher's exact-test, and McNemar test, respectively.

All data is to be recorded on online CRFs provided by Korean National Institute of Health by PI or subinvestigators. Also Korean National Institute of Health will conduct data management to analyze the results. Therefore, data will be kept independent from the sponsor.

Ethical Requirements and Dissemination

The current study adheres to Good Clinical Practice (GCP) and to the ethical principles that have their origin in the Declaration of Helsinki (recommendation for medical research involving human subjects).

The head of study institution is responsible to ensure that an adequate number of qualified staff and adequate facilities required for each phase are available for the foreseen duration of the trial to conduct the trial properly and safely. The study will be reviewed by Institutional Review Board (IRB) of Ethics of CHA Bundang Medical Center on its feasibility in the perspective of the ethical, scientific, and medical aspects.

Prior to a subject's participation in the trial, the subject or the subject's care-giver (or the subject's representative) should be informed of the purpose and procedures of the study, and provided ample time and opportunity to inquire about details of the trial pertinent to the possible risk and benefit of participation in the study.

The written informed consent form which should be signed and personally dated by the subject or by the subject's care-giver (or the subject's legally acceptable representative) should be obtained prior to commencing any study-related procedure.

Subjects will be identified with subject number and their privacy including name and disease will be kept strictly confidential.

The PI or subinvestigator should fully inform the subject's representative and confirm whether he/she fully understand all pertinent aspects of trial, after which obtain the informed consent of him/her to participation in the study based on the free willingness. If the subject's representative is thought to be lack of ability to agree, the patient cannot participate in the study. The subject should receive a copy of the written informed consent form and written information provided to the subjects.

The PI will decide unblinding the treatment code after final results are collected; notify Korean National Institute of Health and individual participants of unblinding. The final results of study will be documented by the PI for publication; reported to Korean National Institute of Health; delivered to each participant in electrical document in addition to an official interview. After publication, data will be open to public fully or partially, depending on medical significance and journals published.

The trial will be reported in line with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) checklist (Additional file 1 [Additional file 1]).

Confidentiality

Identification of all subjects will be kept strictly confidential and they will be examined and recorded using subject number allocated during the study. Subjects will be informed that all data pertinent to the study is stored on the computer and kept strictly confidential. Signed informed consent will be kept by the PI or subinvestigators. By signing the protocol, the investigator attests that all records pertinent to the study must be kept strictly confidential and, if needed, the records must be provided to IRB or regulatory authority after they fully understand the confidentiality of the records.


  Discussion Top


Cerebral palsy typically results from brain injury during prenatal, perinatal, or infant period. Established medical management involves rehabilitation therapy with comprehensive multidisciplinary approach to minimize the possibility of various complication and to improve plasticity of brain (Aisen et al., 2011). There has been no treatment to change brain tissue itself. Upon participation in this study, subjects have a chance to receive cell therapy using UCB as well as conservative rehabilitation therapy. In the above study investigating combination therapy of allogeneic UCB and EPO in 105 patients with cerebral palsy, no SAE probably or definitely related to UCB therapy was reported (Min et al., 2013b). Efficacy of rehabilitation therapy is expected to be enhanced by combination with UCB cell therapy. Given relatively long life expectancy and individual functioning in adulthood, amelioration of overall brain function as well as improvement of motor and mental function through UCB therapy are considered valuable. This is also meaningful when considering mental, physical, and economic stress.

Meanwhile, in condition where allogeneic UCB should be used for any reason including inability to use autologous UCB, immune suppression with immunosuppressant such as cyclosporine is considered to prevent the generation of antibody against infused UCB cells and provide an interval to survive phagocytosis, even though this may increase the infection risk. The use of immunosuppressant is supported further by the previous study conducted in this site where subjects receiving UCB with one mismatch in HLA typing showed favorable efficacy than those with two mismatches (Min et al., 2013b).

The trial results will provide more insight into whether the combination use of UCB and EPO would be more beneficial than either monotherapy in the recovery of children with cerebral palsy.


  Trial Status Top


The last participant finished the last follow-up visit. Investigators are now collecting results to prepare data analysis: study yet in double-blind status.[38]

 
  References Top

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Author contributions
KHC revised the protocol according to version updates. KM drafted the study protocol. SHL documented all formats of CRF. MK designed and finalized the protocol.
Conflicts of interest
All authors declare no conflicts of interest. Principle Investigator is responsible for all process of the trial, and not under discretion of any sponsors.
Research ethics
The study will be reviewed by Institutional Review Board of Ethics of CHA Bundang Medical Center. This trial had been registered in the ClinicalTrials.gov (identifier: NCT0199114) on 17 November 2013.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Data sharing statement
No data is reported in the article.
Plagiarism check
Checked twice by iThenticate.
Peer review
Externally peer reviewed.
Additional file
Additional file 1: SPIRIT Checklist.


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