BMJ Open BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from
SCISSOR – Spinal Cord Injury Study on Small molecule derived Rho-inhibition: A Clinical Study Protocol
ForJournal: peerBMJ Open review only Manuscript ID bmjopen-2015-010651
Article Type: Protocol
Date Submitted by the Author: 23-Nov-2015
Complete List of Authors: Kopp, Marcel; Charité Universitätsmedizin Berlin, Department of Neurology & Experimental Neurology, Spinal Cord Injury Research Liebscher, Thomas; Trauma Hospital Berlin, Treatment Centre for Spinal Cord Injury Watzlawick, Ralf; Charité Universitätsmedizin Berlin, Department of Neurology & Experimental Neurology, Spinal Cord Injury Research Martus, Peter; Department of Clinical Epidemiology and Applied Biostatistics, Eberhard Karls Universität Tübingen Laufer, Stefan; Eberhard-Karls-University Tübingen, Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy Blex, Christian; Charité Universitätsmedizin Berlin, Department of Neurology & Experimental Neurology, Spinal Cord Injury Research Schindler, Ralf; Charité-University Medicine Berlin, Division of Nephrology and Intensive Care, Department of Internal Medicine, Campus Virchow-
Klinikum http://bmjopen.bmj.com/ Jungehulsing, Gerhard; Jüdisches Krankenhaus Berlin, Department of Neurology Knüppel, Sven; German Institute of Human Nutrition Potsdam-Rehbrücke, Department of Epidemiology Kreutzträger, Martin; Trauma Hospital Berlin, Treatment Centre for Spinal Cord Injury Ekkernkamp, Axel ; Trauma Hospital Berlin, Trauma Surgery and Orthopedics Clinic
Dirnagl, Ulrich; Charité Universitätsmedizin Berlin, Department of on October 2, 2021 by guest. Protected copyright. Neurology & Experimental Neurology Strittmatter, Stephen; Yale University School of Medicine, Program in Cellular Neuroscience, Neurodegeneration and Repair, and Department of Neurology Niedeggen, Andreas; Trauma Hospital Berlin, Treatment Centre for Spinal Cord Injury Schwab, Jan; Charité Berlin, Department of Neurology & Experimental Neurology, Spinal Cord Injury Research; Department of Neurology, Spinal Cord Injury Division, The Ohio State University, Wexner Medical Center
Primary Subject Neurology Heading:
Secondary Subject Heading: Pharmacology and therapeutics, Research methods
ibuprofen, Neuroprotection, Plasticity, Neuropathic pain, Heterotopic Keywords: ossifications
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1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Study Protocol 4 5 6 7 SCISSOR – Spinal Cord Injury Study on Small molecule derived Rho-inhibition: A 8 9 Clinical Study Protocol 10
11 12 13 1 2 1 3 4 14 Marcel A. Kopp *, Thomas Liebscher * Ralf Watzlawick *, Peter Martus , Stefan Laufer , 15 ChristianFor Blex1, Ralf peer Schindler5, Gerhard review J. Jungehulsing only6,7, Sven Knüppel7, Axel 16 8 9 10 17 Ekkernkamp , Ulrich Dirnagl , Stephen M. Strittmatter , 18 Andreas Niedeggen2, Jan M. Schwab1,2,11,12 19 20 21 1Department of Neurology and Experimental Neurology, Spinal Cord Injury Research, Charité 22 Universitätsmedizin Berlin, Germany. 2Treatment Centre for Spinal Cord Injury, Trauma Hospital Berlin, Germany. 23 3 Department of Clinical Epidemiology and Applied Biostatistics, Eberhard Karls Universität Tübingen, Germany 24 4 25 Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Eberhard Karls University 26 Tübingen, Germany. 5 Division of Nephrology and Intensive Care, Department of Internal Medicine, Campus 27 Virchow Klinikum, Charité University Medicine Berlin, Berlin, Germany. 6 Jüdisches Krankenhaus Berlin, 28 Department of Neurology, Germany. 7 Department of Epidemiology German Institute of Human Nutrition 29 8 30 Potsdam Rehbrücke, Germany. Trauma Surgery and Orthopedics Clinic, Trauma Hospital Berlin, Germany. 31 9 Department of Neurology and Experimental Neurology, Center for Stroke Research Berlin, Charité 32 Universitätsmedizin Berlin, Germany. 10Program in Cellular Neuroscience, Neurodegeneration and Repair, and 33 11 Department of Neurology, Yale University School of Medicine, New Haven, USA. Department of Neurology, 34 http://bmjopen.bmj.com/ 12 35 Spinal Cord Injury Division, The Ohio State University, Wexner Medical Center, Columbus, USA Department of 36 Neuroscience and Center for Brain and Spinal Cord Repair, Department of Physical Medicine and Rehabilitation, 37 The Neuroscience Research Institute, The Ohio State University, Wexner Medical Center, Columbus, USA. 38
39 40 * contributed equally to this work 41
42 on October 2, 2021 by guest. Protected copyright. 43
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49 50 Correspondence 51 Prof. Jan Schwab, MD, PhD 52 Department of Neurology and Experimental Neurology, Spinal Cord Injury Research (Clinical and experimental 53 Neuroparaplegiology) , Charité Universitätsmedizin Berlin Charitéplatz 1, D 10117 Berlin 54 55 Phone: 0049 30 450 660293 56 Fax: 0049 30 450 560942 57 E mail: [email protected] 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 1 Page 3 of 33 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 ABSTRACT 4 5 6 Introduction The approved analgesic and anti inflammatory drugs ibuprofen and 7 8 indometacin target the COX pathway unselectively. Moreover, they block the small GTPase 9 RhoA, a key enzyme within a downstream cascade of extra neuronal signals that impede 10 11 axonal sprouting after axonal damage. Preclinical studies in spinal cord injury (SCI) imply 12 improved motor recovery after ibuprofen/indometacin mediated Rho inhibition. This has been 13 14 re assessed by a meta analysis of the underlying experimental evidence, which indicates an 15 overall effectFor size of 20.2% peer regarding motorreview outcome achieved only after ibuprofen/indometacin 16 17 treatment compared to vehicle controls. Additionally, anti inflammatory actions of 18 19 ibuprofen/indometacin probably limit the development of sickness behavior, non neurogenic 20 systemic inflammatory response syndrome (SIRS), neuropathic pain, and heterotopic 21 22 ossifications after SCI. Consequently, ‘small molecule’ mediated Rho inhibition after acute 23 SCI warrants clinical investigation. 24 25 Methods and analysis We report the protocol of an investigator initiated clinical pilot trial on 26 high dose ibuprofen treatment after acute traumatic, motor complete SCI. The open label 27 28 study has the primary safety endpoint: occurrence of serious adverse events, primarily 29 gastroduodenal bleedings. Secondary endpoints are pharmacokinetics, feasibility and 30 31 preliminary effects on neurological recovery, neuropathic pain and heterotopic ossifications. 32 Underlying experimental evidence was challenged by systematic review of preclinical studies 33 34 on ibuprofen/indometacin effects on motor recovery after SCI. The primary safety analysis is http://bmjopen.bmj.com/ 35 based on the incidence of severe gastrointestinal bleedings. Additional analyses will be 36 37 mainly descriptive and casuistic. 38 Ethics and dissemination The clinical trial protocol was approved by the responsible 39 40 German state Ethics Board, and the Federal Institute for Drugs and Medical Devices. The 41 study complies with the Helsinki Declaration, the principles of Good Clinical Practice and all 42 on October 2, 2021 by guest. Protected copyright. 43 further applicable regulations. The current safety and pharmacokinetics trial informs the 44 45 planning of a subsequent randomized controlled trial. Regardless to the result of the primary 46 and secondary outcome assessments the clinical trial will be reported as publication in a 47 48 peer reviewed journal. 49 Registration ClinicalTrials.gov identifier: NCT02096913. 50 51 52 53 54 (300 words) 55 56 57 Keywords: ibuprofen, neuroprotection, plasticity, neuropathic pain, heterotopic ossifications 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 2 BMJ Open Page 4 of 33
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 INTRODUCTION 4 5 At present, the effective pharmacological treatment of acute traumatic spinal cord injury 6 (SCI) is an unmet medical need.[1] The current opportunities for restitution of neurological 7 8 function after SCI are limited to early surgical decompression, stabilization, and 9 physiotherapy. Neuroprotective or plasticity enhancing therapies are under investigation in 10 11 preclinical studies and early phase clinical trials. As yet, however, none of these approaches 12 could be translated into clinical routine.[2 4] 13 14 A major reason for the poor prognosis of central nervous system (CNS) injury is the 15 incapacity ofFor axons to peer re grow within review the CNS. Molecular only barriers preventing axonal 16 17 regeneration after SCI are situated in the environment of the injured axon i.e. in the scar 18 19 tissue and myelin.[5 6] Those molecules, such as chondroitin sulfate proteoglycans 20 (CSPGs), Nogo A, myelin associated glycoprotein (MAG), oligodendrocyte myelin 21 22 glycoprotein (OMgp), Ephrins, and RGMa are up regulated after CNS injury and interfere 23 with a repertoire of cognate receptors on the axon membrane as reviewed elsewhere.[6 7] 24 25 Signals from those receptors are mediated downstream to RhoA. The small GTPase RhoA is 26 a key molecule in a pathway which, once activated, leads to the collapse of axonal growth 27 28 cones and consequently to failure of axonal plasticity or regeneration.[8] 29 Therefore, the Rho pathway constitutes a target for treatments aiming to overcome 30 31 molecular obstacles to a restoration of neuronal connectivity and subsequent functional 32 recovery. The inhibition of Rho or the downstream located Rho associated coiled kinase 33 34 (ROCK) has been demonstrated to foster axonal sprouting or plasticity,[9 22] to have http://bmjopen.bmj.com/ 35 neuroprotective effects,[9 10 12 13 19 23 24], and to enhance neurological recovery[9 10 12 36 37 15 17 19 21 23 25] after acute SCI (Figure 1). These findings are backed up with evidence 38 from other experimental CNS injury conditions as reviewed elsewhere.[7 26] The reported 39 40 effects of various Rho/ROCK blocking approaches on open field motor recovery after 41 experimental SCI have been re assessed by a systematic review and meta analysis 42 on October 2, 2021 by guest. Protected copyright. 43 including correction for publication bias.[27] Specific Rho inhibition mediated by the 44 45 clostridium botulinum derived enzyme C3 transferase, also referred to as BA 210 or 46 Cethrin™,[28] has been studied in a recently completed Phase I/IIa clinical trial. The 47 48 investigators concluded that topically applied BA 210 is safe and is associated with favorable 49 neurological outcome.[29] However, a confirmatory Phase III trial has not yet been 50 51 conducted. 52 Over the last decade, upcoming evidence has assigned a subset of non steroid anti 53 54 inflammatory drugs (NSAIDs) to the group of unspecific Rho inhibitors. The FDA approved 55 NSAIDs ibuprofen,[10 16 18 20 30 31], indometacin,[18 30] and suldinac sulfide,[30] were 56 57 shown to inhibit Rho activation independently of their ‘classical’ mode of action as inhibitors 58 of Cyclooxygenases (COX). It was subsequently demonstrated that ibuprofen treatment 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 3 Page 5 of 33 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 enhances axonal sprouting,[10 16 18], including that of human model neurons,[20] and 4 improves neurological recovery.[10 18] It is noteworthy that ibuprofen mediated Rho 5 6 inhibition involves Peroxisome Proliferator Activated Receptor γ (PPARγ) activation.[16] It 7 remains unclear, however, what the exact mechanism for this activation is and whether co 8 9 factors are required for PPARγ associated Rho inhibition, because other PPARγ activators 10 such as rosiglitazone also inhibit Rho,[16 32], while other NSAIDs such as Naproxen activate 11 12 PPARγ strongly[33] but do not cause Rho inhibition.[10 16 30] 13 14 Other pharmacological targets of ibuprofen – namely PPARγ activation,[16 33 34], COX 15 1/2 inhibition,[35]For and NF κb inhibition,[36]peer review promise a concomitant only limitation of secondary 16 17 damage by anti inflammatory actions, but might also modify the effects of Rho blockade 18 (Figure 1). In more detail, PPARγ activation reduces the cellular,[37] and soluble 19 20 inflammatory response,[38] that is suggested to alter tissue pathology after SCI as reviewed 21 by McTigue.[39] In the context of experimental systemic inflammation COX 1, which reveals 22 23 sustained upregulation in the spinal cord after SCI,[40] promotes sickness behaviour.[41] 24 COX related pathways also exert immune modulation in terms of immune depression,[42] 25 26 and impaired host defense.[42 43] Those effects might aggravate the maladaptive immune 27 response after SCI,[44 45] that is associated with increased susceptibility to infections, which 28 29 are a risk factor for poorer neurological outcome after SCI.[46] Furthermore, NF κB, which is 30 activated after SCI,[47] contributes in neurodegenerative disease to microglia induced loss of 31 32 motor neurons.[48] Together, anti phlogistic actions of ibuprofen are likely to reduce 33
neurodegeneration driven by CNS inflammation,[48 49], which is triggered through the COX http://bmjopen.bmj.com/ 34 35 and/or NF κB related systemic inflammatory response syndrome,[42 50], or infections.[42 43] 36 Besides, NF κB,[51] and COX metabolites such as prostaglandin E [52] are linked to the 37 2, 38 induction of neuropathic pain. Thus, NSAIDs might be effective in preventing SCI specific 39 40 sequelae such as neuropathic pain,[51 53 56], as well as inflammation related neurogenic 41 heterotopic ossifications (Figure 1).[57 59]
42 on October 2, 2021 by guest. Protected copyright. 43 Ibuprofen is recommended primarily to improve neurological function through the 44 enhanced plasticity conferred by its Rho inhibiting properties. The combination of Rho 45 46 inhibition with anti inflammatory actions of ibuprofen might, however, dissolve conflicting 47 aspects of anti inflammatory therapies after axonal injury. It has been demonstrated that 48 49 secondary axonal damage is reduced when inflammation has been limited, but this occurs at 50 the expense of the regenerative capacity of the spared axons.[60] In this context, an 51 52 increased blockade of axonal re growth capacity as a side effect of anti inflammatory 53 neuroprotective therapy could be prevented by concurrent Rho inhibition (Figure 1). 54 55 In vitro sprouting responses under ibuprofen,[10 16 18 20], or indometacin,[18] treatment 56 in the presence of inhibitory matrix components such as myelin or CSPGs are well 57 58 reproducible. However, in vivo evidence provided by some groups for promoting effects of 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 4 BMJ Open Page 6 of 33
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 ‘small molecule’ Rho inhibitors on axonal sprouting,[10 18], or on neurological recovery,[10 4 18 61 62], have not or have only partially been confirmed by others.[31 63] Reasons for the 5 6 variability in the results could be multiple. One possible reason would be differences in the 7 experimental design, such as in timing of the experiments, the animal model applied, the 8 9 route of drug delivery, and assessment tools. On the other hand the variability could be a 10 product of chance due to small sample sizes, which is a general problem in preclinical 11 12 studies.[64] One approach to address the variability of preclinical studies is to subject them 13 14 to meta analysis.[64] 15 This workFor includes apeer systematic review review and meta analysis only of experiments reporting the 16 17 effect of Rho inhibiting NSAIDs on neurobehavioral recovery after SCI. The published 18 preclinical evidence and its positive predictive value, as representing the justification of the 19 20 current clinical investigation, were challenged by the meta analysis. The study protocol of the 21 first clinical trial on high dose ibuprofen as a Rho inhibitor after acute SCI is addressing 22 23 safety, feasibility and pharmacokinetics. Additionally, the study explores preliminary efficacy 24 including aspects of repurposing ibuprofen,[65] as a compound with multiple pharmacological 25 26 targets for the treatment of SCI. 27 28 29 30 31 32 33 34 http://bmjopen.bmj.com/ 35 36 37 38 39 40 41
42 on October 2, 2021 by guest. Protected copyright. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 5 Page 7 of 33 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 METHODS AND ANALYSIS 4 5 Ibuprofen is a drug that has been FDA approved and is available worldwide for decades. 6 However, in the context of traumatic injuries its use is generally restricted to short term low 7 8 dose administration as an analgesic. The mid term high dose application of ibuprofen, as a 9 10 Rho inhibiting and anti inflammatory treatment after SCI, is not an approved indication and 11 information on its tolerability is not available for the population of acute SCI patients. This is 12 13 relevant because critically injured SCI patients require treatment in an intensive care unit, 14 which is a risk factor for gastric ulcers,[66] and particularly SCI patients with cervical and high 15 For peer review only 16 thoracic levels might be at risk for damage to the gastric mucosa due to a disturbance of 17 autonomous innervation.[67] Furthermore, pharmacological data on CNS permeability are 18 19 available for non trauma patients but little is known about pharmacokinetics of orally 20 administered ibuprofen after SCI. Therefore, the SCISSOR study primarily addresses safety, 21 22 feasibility and pharmacokinetics under the clinical condition of acute traumatic SCI. 23 Secondary objectives are neurological recovery and SCI specific complications. 24 25 26 Assessment of underlying evidence 27 28 In order to re asses the preclinical evidence regarding Rho inhibiting NSAIDs and to 29 justify the risks and efforts of the clinical trial, a systematic review was performed. Six 30 31 publications,[10 18 31 61 63], containing 11 single experiments with a total of n=255 animals 32 (Table 1) were included for meta analysis after stepwise study selection (Figure 2). 33 34 http://bmjopen.bmj.com/ 35 36 Table 1: Preclinical study characteristics. 37 ID Publication Drug Species n Dose Motor Injury Follow Type of Applica 38 (Duration) score level -up Injury tion 39 1 Redondo Ibu Rats 16 60 mg/kg/d (42d) BBB T8 42d Contusion s.c. Castro, E 40 2 Sharp, K Ibu Rats 73 60 mg/kg/d (28d) BBB T6/7 42d Hemisection s.c. 41 3 Wang, X Ibu Mice 46 35 70 mg/kg/d BMS T8 35d Transection s.c.
42 (28d) on October 2, 2021 by guest. Protected copyright. 43 4 Guth, L Indo Rats 12 0.2 mg/d (21d) Tarlov T8 21d Compression i.p. 44 5 Wang, X Ibu Rats 47 70 mg/kg/d (28d) BBB T7 49d Contusion s.c. 45 46 6 Fu, Q Ibu Rats 19 60 mg/kg/d (28d) BBB T6/7 42d Hemisection s.c. 7 12 60 mg/kg/d (28d) Contusion 47 8 Pantovic, R Indo Rabbits 6 0.1 mg/kg/d (9d) Tarlov L2 9d Contusion i.v. 48 9 6 0.3 mg/kg/d (9d) 49 10 6 1.0 mg/kg/d (9d) 50 11 6 3.0 mg/kg/d (9d) 51 6 vehicle Abbreviations: Ibu=Ibuprofen, Indo= Indometacin, n= number of animals, d= days, s.c.= subcutaneous, i.p.= 52 intraperitoneal, i.v.= intravenous 53 54 55 Preclinical study characteristics were extracted for each publication and functional outcome 56 was measured for each experiment in order to perform the meta analysis. The method and 57 58 statistical approach is described in greater detail elsewhere.[27 68] In brief, we used a 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 6 BMJ Open Page 8 of 33
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 random effects weighted mean difference meta analysis to calculate an overall estimate of 4 effect size between treated and untreated (control) animals based on the final time point of 5 6 the assessment of functional recovery. A random effects meta regression was used to 7 determine how much heterogeneity can be explained by study design characteristics using 8 9 STATA13 with a significance level of p<0.05. We checked for possible publication bias using 10 trim and fill method for funnel plots and Egger regression in STATA13. 11 12 The effect size in the open field motor testing of treatments with ibuprofen or 13 14 indometacin after experimental SCI was 20.2% (95% CI, 10.8 – 29.6) in the overall analysis 15 (Figure 3) andFor varied in thepeer single experiments review from 33.2% ( 79,2only – 12.8) to 44.9% (19,5 – 16 17 70.4). Meta regression analyses to identify of subgroup effects regarding the administered 18 drug, the behavioral assessment tool, the SCI model, the route of drug delivery or the study 19 20 quality revealed no statistically significant proportion of between study heterogeneity for any 21 of the stratifications. Likewise the tests to detect possible publication bias implied no missing 22 23 experiment, although statistical significance should not be expected given the study’s small 24 overall size.[69] 25 26 Nevertheless, the design of the studies on ibuprofen was different from those on 27 indometacin treatment in terms of the neurobehavioral scales, the animal models, and the 28 29 route of drug delivery (Table 1). The ibuprofen treated animals had all been assessed with 30 the recent Basso Beattie Bresnahan (BBB) score,[70] or the Basso Mouse Scale (BMS),[71] 31 32 whereas modifications of the outdated Tarlov score,[72] were applied for indometacin treated 33
animals. Furthermore, the ibuprofen treated groups underwent contusion, transection, and http://bmjopen.bmj.com/ 34 35 hemisection models in contrast to the exclusive use of compression or contusion 36 experiments in indometacin treated groups, which received the drug intravenously or 37 38 intraperitoneally compared to subcutaneous administration in the ibuprofen groups. 39 40 Therefore, differences in effect size between the two investigated compounds require careful 41 interpretation and do not allow conclusions on differences in their potential therapeutic
42 on October 2, 2021 by guest. Protected copyright. 43 efficacy. 44 Among the ‘small molecule’ Rho inhibitors, ibuprofen is the most feasible for clinical 45 46 investigation in the indication of acute traumatic SCI due to its greater quantity and the higher 47 quality of its preclinical data. All studies on ibuprofen revealed Rho inhibition in vivo within 48 49 the spinal cord after systemic drug administration,[10 18 31], and comprise experimental 50 models applicable for translational research,[73] as well as recent behavioral scores.[70 71] 51 52 53 Study design 54 55 The SCISSOR study is designed as a prospective non randomized open label phase I 56 study, as this is a preferred design for tolerability and pharmacokinetic investigations.[74 75] 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 7 Page 9 of 33 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Study participants are enrolled consecutively in two treatment cohorts characterized by the 4 duration of therapy as further detailed below and illustrated in Figure 4. 5 6 7 Setting 8 9 The initiating sponsor and coordinator of the trial is the Department of Experimental 10 Neurology, Clinical and Experimental Spinal Cord Injury Research (Neuroparaplegiology) at 11 12 Charité University Hospital, Campus Mitte, Berlin, Germany. Data management and statistics 13 14 are performed by the Department of Clinical Epidemiology and Applied Biostatistics at 15 Eberhard Karls UniversityFor peer Tübingen, Germany. review The recruiting only trial center is the Treatment 16 17 Center for Spinal Cord Injuries at the Trauma Hospital Berlin, Germany. The study 18 investigators are physicians trained and experienced in the management and assessment of 19 20 patients with acute and chronic SCI. 21 The reference center for laboratory safety parameters is the Central Laboratory at the 22 23 trial center, Trauma Hospital Berlin, Germany. The central laboratory is regularly certified for 24 clinical diagnostics. The Department of Pharmaceutical and Medicinal Chemistry, Institute of 25 26 Pharmacy, Eberhard Karls University Tübingen, Germany will perform the measurement of 27 ibuprofen concentrations in plasma and CSF using High Performance Liquid 28 29 Chromatography Mass Spectrometry (HPLC MS). The Labor Berlin - Charité Vivantes 30 GmbH, a certified laboratory for clinical and research diagnostics, will run the nephelometric 31 32 protein measurements in serum and CSF for quantification of post SCI Blood Spinal Cord 33
Barrier breakdown. http://bmjopen.bmj.com/ 34 35 36 Intervention 37 38 The study medication is ibuprofen in the galenic preparation of water soluble lysine salt. 39 40 Ibuprofen lysine salt is absorbed faster, leading to earlier peaks of plasma concentrations 41 compared to the free acid.[76] The brand name of the study medication is Dolormin® extra.
42 on October 2, 2021 by guest. Protected copyright. 43 Ibuprofen is applied as tablets administered orally for 4 weeks in cohort I or 12 weeks in 44 cohort II, respectively (Figure 4). The daily dose of 2400mg is administered as three single 45 46 doses of 800 mg. In the case of swallowing disorders, which occur in 16% of tetraplegic 47 acute SCI patients,[77] it is recommended that the tablets be disaggregated in water and the 48 49 medication administered via stomach tube. 50 During the first 4 weeks of treatment, the proton pump inhibitor pantoprazole is used as 51 52 a concomitant medication in a dosage 40mg/d. This reduces the risk of damage to the 53 gastrointestinal mucosa and, after individual risk benefit assessment, can be reduced to 54 55 20mg/d for the following treatment period (applicable to cohort II). 56
57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 8 BMJ Open Page 10 of 33
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Dose estimation 4 Ibuprofen doses of 60 to 70 mg/kg/d have been used in preclinical trials.[10 18 31 63] To 5 6 estimate the pharmacologically active dose (PAD) in humans, we applied a conversion 7 model which is feasible for systemically administered active substances of a small molecular 8 9 size, provided that further pharmacologic properties of the compound have been taken into 10 account.[78] The human equivalent dose (HED), converted from the PAD in rats, is about 11 12 11.3 mg/kg/d (rat PAD of 70 mg/kg/d / 6.2 = HED 11,3 mg/kg/d). The binding capacity for 13 14 ibuprofen in vitro is higher in human albumin than it is in rat albumin. At identical 15 concentrations,For the free bioactivepeer ibuprofen review fraction in human onlyalbumin solution is lower by a 16 17 factor of about 3.[79 80] We therefore multiplied the HED by that factor to achieve an 18 estimate of comparable bioactive concentration of 34 mg/kg. Assuming an average body 19 20 weight of 70 kg, the daily dose of ibuprofen in this trial was therefore set at 2400 mg/d 21 regardless of individual body weight. This is within the FDA approved range of up to 3200 22 23 mg/d for adults. 24 25 26 Outcome measures 27 The primary endpoint of the study is the safety of high dose ibuprofen application after 28 29 SCI as measured by the occurrence of serious adverse events (SAE) related to the study 30 medication. In particular, severe gastroduodenal bleeding attributable to the study medication 31 32 is the primary safety parameter (Table 2). 33
http://bmjopen.bmj.com/ 34 35 Table 2: Clinical trial outcome measures 36 Parameter Assessments/Measures Timing (see Safety 37 also Figure 5) issue 38 Primary Gastroduodenal Serious adverse event (SAE) report Continuous yes 39 Endpoint bleeding observation 40 41 Secondary Adverse events Adverse event monitoring, SAE/SUSAR Continuous yes Endpoints (serious unexpected suspected adverse observation 42 on October 2, 2021 by guest. Protected copyright. 43 reaction) report 44 Spasticity Modified Ashworth Scale, anti spastic Follow up 1 & 2 yes medication 45 Neuropathic Neuropathic Pain Scale, pain medication Baseline, yes 46 pain Follow up 1 & 2 47 Severity of SCI ASIA impairment scale Baseline, no 48 Follow up 1 & 2 49 Motor function Upper and lower extremity motor score Baseline, no 50 Follow up 1 & 2 51 Sensory Pin prick, Light touch Baseline, no 52 function Follow up 1 & 2 53 Lesion height Motor and sensory level, zone of partial Baseline, no 54 preservation, if applicable Follow up 1 & 2 55 Ibuprofen levels Blood & Cerebrospinal fluid (CSF) Pharmacokineti no 56 collection cs 1, 2, & 3* 57 Serum/CSF Blood & CSF collection Pharmacokineti no 58 protein levels cs 1, 2, & 3* 59 Heterotopic Ultrasound of the hip joints, Magnetic Baseline, yes 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 9 Page 11 of 33 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 ossifications resonance imaging, if applicable Follow up 1 & 2
4 Other Laboratory Blood & urine collection Safety 1, 2, 3 & yes 5 Endpoints abnormalities 4* 6 Cardiac Electrocardiography Baseline, yes 7 arrhythmia Follow up 1 & 2 8 Deep vein Ultrasound of pelvic & lower extremity Baseline, yes 9 thrombosis veins Follow up 1 & 2 10 Circulatory Blood pressure & heart rate Baseline, Safety yes 11 disturbance 1, 2 12 Clinical Epigastric pain / Pain projected to the Baseline, Safety yes 13 observation shoulder tip 1, 2, & 3* 14 Feasibility of Screening protocol Screening no 15 Forrecruitment peer review only 16 Differences between the cohorts are based on the course of an extended intervention. In cohort II 17 additional pharmacokinetic and safety assessments are scheduled (indicated by asterisks). 18 19 Secondary endpoints are all further adverse events (AE) including SAE and suspected 20 21 unexpected serious adverse reactions (SUSAR). Clinical, laboratory and technical safety 22 examinations facilitate the detection of AEs that can be expected and as well as the 23 24 assessment of their causality (Table 2). In addition, the sensitive measurement of 25 neuropathic pain and spasticity is also relevant for safety reasons, since the course of those 26 27 very frequent SCI specific sequelae might be altered by plasticity enhancing therapies. The 28 Neuropathic Pain Scale,[81 82], and the Modified Ashworth Scale,[83] are therefore applied 29 30 for assessment of pain and muscle tone, respectively. The pharmacological laboratory 31 endpoints are ibuprofen levels in plasma and CSF as measured at the time of expected peak 32 33 levels.[84] The neurological examination is performed according to the International 34 http://bmjopen.bmj.com/ Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) definitions of 35 36 2011.[85] The ISNCSCI comprises the ASIA impairment scale (AIS) as a measure for 37 completeness and severity of SCI, the ASIA motor scores for upper and lower extremity 38 39 motor function, the ASIA sensory scores for residual pin prick and light touch sensation, the 40 41 motor and sensory neurological level, as well as the zone of partial preservation, if
42 applicable. Optional elements such as non key muscles for determination of the AIS are not on October 2, 2021 by guest. Protected copyright. 43 44 applied in this study.[85] Neurogenic heterotopic ossifications constitute a further clinical 45 endpoint. These will be identified with an ultrasound screening of the hip joints,[86] followed 46 47 by MRI if heterotopic ossifications are suspected (Table 2). 48 49 50 Enrolment 51 In the study center we expect to be screening about 40 60 SCI admitted patients per 52 53 year, about 6 8 of whom are expected to meet eligibility criteria. The investigators will 54 evaluate patient eligibility as soon as possible after admission to the trial center. The 55 56 investigators will conduct an interview with each patient to verify the inclusion and exclusion 57 criteria as related to individual medical history as well as to inform the patient about the trial 58 59 and its potential risks and benefits. Prior to inclusion, written informed consent will be 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 10 BMJ Open Page 12 of 33
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 obtained from the patient, and a written announcement of recruitment will be sent out to the 4 sponsor by the investigators. 5 6 7 Eligibility criteria 8 9 The inclusion and exclusion criteria (Table 3) were chosen with regard to scientific, 10 ethical, and practical considerations specific to SCI.[87] Included in the trial up to day 21 11 12 post injury are patients with an acute traumatic motor complete SCI, classified as AIS A or 13 14 AIS B, and with a neurological level of Th4 to C4. Only in this group of patients is a realistic 15 assessment Forof neurogenic peer gastrointestinal review bleedings possible, only because this classification is 16 17 most likely to be associated with an autonomic complete lesion,[88] that in the acute stage 18 can cause damage to the gastroduodenal mucosa.[67] The imbalance between the altered 19 20 sympathetic outflow through the splanchnic nerve and the intact parasympathetic innervation 21 through the vagus nerve,[67] may increase the ‘baseline’ risk posed by the general post 22 23 traumatic and ventilation triggered stress response.[66] 24 In order to limit risk to patients, the exclusion criteria comprise all absolute 25 26 contraindications of the study medication according to the summary of product 27 characteristics. The exclusion criteria also include drug interactions or other conditions 28 29 mandating precaution. To ensure reliable assessment of safety and preliminary efficacy, 30 patients with concomitant injury to the CNS, pre existing neurological diseases, or severe 31 32 psychiatric disorders are excluded from the trial. Other exclusion criteria assure the 33
adherence to legal requirements (Table 3). http://bmjopen.bmj.com/ 34 35 36 Table 3: Clinical trial eligibility criteria 37 38 Inclusion criteria 39 • Acute SCI of the cervical spine due to trauma 40 41 • Time frame of 4 21 days post trauma
42 on October 2, 2021 by guest. Protected copyright. • Motor complete injury AIS (ASIA impairment scale) A and B 43 44 • Neurological level of the lesion C4 Th4 45 • No participation in a different clinical trial according to German Medicinal Products Act one 46 month before and during participation in the current trial 47 48 • The patient has been informed and his/her written consent has been obtained 49 • Age: 18 to 65 years 50 51 • For women of reproductive age: Negative pregnancy test and highly effective contraception 52 (defined as Pearl Index < 1) or sexual abstinence during participation in the trial. 53 Exclusion criteria 54 • Multifocal lesions of the spinal cord 55 56 • Accompanying TBI with visible structural lesions including intracranial hemorrhage on 57 diagnostic images 58 • Significant accompanying injury to the peripheral nervous system, particularly plexus lesions 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 11 Page 13 of 33 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 • Acute or chronic systemic diseases accompanied by neurological deficits or that have caused 4 permanent neurological deficits which may overlay or hinder the registration of sensomotor 5 functions (e.g. multiple sclerosis, Guillain Barré syndrome, HIV infection, Lues etc.) 6 • Malignant neoplasms, except if these are in complete remission. 7 8 • Mental diseases or dementia which, in the investigator's opinion, limit the patient's 9 cooperation in respect of the intake of the study medication and/or significantly hinder the registration of follow up parameters 10 11 • Hemophilia 12 • History of myocardial infarction or stroke 13 14 • Current and persistent misuse of illegal drugs or alcohol 15 • HypothermiaFor below peer 35 C° review only 16 17 • Pregnancy and lactation 18 • All further contraindications to the study medication, including other ingredients of the 19 pharmaceutical form according to the Summary of Product Characteristics 20 21 • Known hypersensitivity to the active substance contained in the concomitant medication 22 Pantoprazole or one of the components of the drug. 23 • Intake of ibuprofen or intake of other active substances from the group of NSAIDs; (Non 24 steroid anti inflammatory drugs: e.g., diclofenac, indometacin) or the intake of NSAIDs in 25 maximum recommended daily doses during one week prior to enrolment in the trial 26 • Simultaneous intake of salicylates, particularly acetylsalicylic acid 27 28 • Simultaneous intake of oral anticoagulants, or heparinisation in therapeutic dosage 29 • Simultaneous intake of systemic glucocorticoids 30 31 • Unwilling to consent to storage and transfer of pseudonymized medical data for the purpose 32 of the clinical trial 33 • Admitted to an institution by a court or official order 34 http://bmjopen.bmj.com/ 35 36 Individual timeline 37 38 Patient evaluation and inclusion will be performed within 21 days after SCI. The CRF at 39 baseline comprises the eligibility criteria, the assessment of injury date and time, medical 40 41 history, concomitant injury and surgical interventions. Furthermore, the clinical, laboratory,
42 and technical safety parameters as well as the ISNCSCI are assessed at baseline (Figure 4, on October 2, 2021 by guest. Protected copyright. 43 44 Table 2). 45 Since the intervention in cohort II is of longer duration, more frequent safety and 46 47 pharmacokinetic assessments will be performed in this arm during the intervention and 48 follow up (Figure 4). In addition to the continuous monitoring of AEs, safety data comprise 49 50 laboratory measures and clinical observations will be collected in tightly scheduled safety 51 assessments. Further safety issues such as spasticity and neuropathic pain are part of the 52 53 follow up documentation (Figure 4) that also includes the neurological endpoints and 54 55 possible confounders such as co medications or infections.[46] 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 12 BMJ Open Page 14 of 33
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Overall Duration 4 A recruitment period of 24 months is scheduled. Each patient will be followed up to 24 5 6 weeks post trauma. After completion of recruitment and follow up, a further six month period 7 is planned for clearing the database, the statistical evaluation and preparation of the trial 8 9 report. The trial was activated in June 2013, but not recruiting. After trial registration and 10 completion the recruiting center’s initiation, enrolment started in April 2014. Expected 11 12 enrolment completion date is the second quarter of 2016. Publication of the trial report is 13 14 scheduled for the year 2017. 15 For peer review only 16 17 Sample size estimation 18 The sample size of 12 patients and the analysis strategy are justified by the fact that – 19 20 given that the number of gastrointestinal ulcerations/bleedings after SCI is 3,5% in the first 21 month as reported by Kewalramani 1979[67] – the probability for the occurrence of more 22 23 than one event is 6,1%. Consequently, observation of more than one event provides 24 evidence of safety problems of Ibuprofen in the indication of acute SCI and probably limits its 25 26 use in subsequent phases of the clinical trial. The occurrence of further bleedings in months 27 two and three or during follow up calls for the same consequences. Nevertheless, based on 28 29 the abovementioned frequency of gastrointestinal ulcerations, the probability for the 30 occurrence of an event is low (0.7% probability) in a sample of 12 SCI patients.[67] However, 31 32 the upper bound of the confidence interval for the probability of an event is 38,5%; for zero 33
events it is 26.5%. This mandates implementing additional safety criteria if subsequent study http://bmjopen.bmj.com/ 34 35 phases are considered, and a placebo control should be taken into consideration. In our pilot 36 study a comparison with patients receiving placebo would have a clear lack of statistical 37 38 power, so no placebo group is scheduled. 39 40 41 Data management
42 on October 2, 2021 by guest. Protected copyright. 43 Data are collected on a paper Case Report Form (pCRF). All patient data are managed 44 with a six digit pseudonym. At the study office the trial coordinators check the pCRFs for 45 46 completeness and consistency. Implausible or missing data may be corrected or added after 47 consulting the investigator at the trial site through the sponsor (Queries). The corrected 48 49 documents will be archived together with the completed CRFs. Data are entered twice to 50 allow double check for correctness and are stored electronically in a database. Access to the 51 52 database is restricted, and regular data backups are performed. 53 54 55 Sample handling 56 Peripheral blood and urine samples collected for laboratory safety measures are 57 58 analyzed immediately after sample collection at the central laboratory of the trial center, and 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 13 Page 15 of 33 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 the results are available for the study investigators at once. This facilitates the timely 4 recognition of AEs. 5 6 Blood and CSF samples for pharmacological and protein analyses are collected under 7 sterile conditions. Those samples are labeled with the six digit pseudonym and any personal 8 9 information of the participants is removed. All samples are processed for storage as soon as 10 possible, at the latest within 8 hours of withdrawal by centrifugation at 3000 g for 10 minutes. 11 12 Serum, heparin plasma and CSF supernatants are stored at 80°C, with central temperature 13 14 control up to subsequent batch analysis. 15 For peer review only 16 17 Statistical analysis 18 The analysis will be based on the safety population, as this is a pilot study for safety and 19 20 feasibility designed to enable planning of a subsequent study. The primary analysis is based 21 on the incidence of severe gastrointestinal bleedings. If more than one event is observed in 22 23 the study population (n =12) the principle investigator/sponsor on recommendation of the 24 independent Data Monitoring Safety Board will perform a new risk benefit assessment and 25 26 will decide the interruption or early termination of the trial. Additional safety analyses, mainly 27 descriptive and casuistic, will be performed. The descriptive analysis will be according to the 28 29 scale and distribution of the data, using frequencies and means, medians, quartiles and 30 ranges. Linear regression will be used as appropriate. 31 32 33
Quality Assurance http://bmjopen.bmj.com/ 34 35 Adherence to i) the recruitment rate, ii) the selection criteria iii) the treatment in 36 accordance with the protocol, and iv) the investigation time points is regarded as a quality 37 38 indicator for the course of the trial. An independent monitor is responsible for reviewing study 39 40 progress, verifying adherence to the protocol, compliance to ICH/GCP and national 41 regulations, and furthermore for handling any problems that arise. The monitor will visit the
42 on October 2, 2021 by guest. Protected copyright. 43 clinical study sites on a regular basis, first after start of enrollment, then after completion of 44 recruitment into cohort I, and finally at study completion. 45 46 Key study data will be checked in all patients. This pertains to patients’ demographical 47 data, signed informed consent, adherence to inclusion and exclusion criteria, documentation 48 49 on primary objectives, and adverse events. Source data verification will be performed for 50 approximately 25% of the data. Any unclear and/or incomplete data will elicit increased in 51 52 depth monitoring. 53 54 55 Data Monitoring and Safety Board 56 An independent Data Monitoring and Safety Board (DMSB) addresses patient safety and 57 58 performs risk/benefit assessments to ensure that for the patients there is no unavoidable risk 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 14 BMJ Open Page 16 of 33
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 or harm. According to its operating procedures, the DMSB reviews accumulating data from 4 the trial to fulfill the safety monitoring. Additionally, the DMSB will assess trial progress, study 5 6 integrity, and design aspects. The DMSB provides the sponsor with recommendations 7 regarding study modification, continuation or termination. The DMSB consists of three 8 9 members: a biostatistician, a neurologist, and an internist, all of whom have practical 10 experience in the work of a DMSB. The DMSB will perform an interim review for safety 11 12 reasons when the entire cohort I has completed week 4 follow up and after completion of 13 14 enrollment and, if necessary, upon request of the sponsor and/or principal investigator. 15 For peer review only 16 17 Stopping rules 18 The discontinuation criteria defined for premature drop out of a patient from the trial 19 20 include cases of emergency or circumstances associated with increased risk for the 21 participant, as well as a patient’s individual wish (Table 4). 22 23 24 Table 4: Clinical trial stopping rules 25 26 Premature drop-out of a patient 27 • Gastrointestinal ulceration with or without hemorrhage and/or perforation 28 29 • A drop in hemoglobin levels below 5 mmol/l consistent after receiving more than 8 red blood 30 cell concentrates 31 • Acute renal failure, defined as an increase in creatinine levels by more than 50% of the 32 baseline value and/or oliguria (urine volume <500 ml/d) persisting for several days after 33 exclusion of extra renal causes 34 http://bmjopen.bmj.com/ • Any hypersensitivity reaction that the investigator attributes to the trial medication 35 36 • Neurological progression of SCI with ascending paralysis with a loss of more than 2 motor 37 levels 38 • Cerebrovascular hemorrhage 39 40 • Myocardial infarction or stroke 41 • Any new injury to the spine affecting the spinal cord
42 on October 2, 2021 by guest. Protected copyright. 43 • The additional intake of more than 1200 mg/d ibuprofen for more than 1 week or the intake of 44 maximal daily doses of other NSAIDs (Non steroid anti inflammatory drugs) for more than 2 45 weeks during the intervention. 46 • The patient’s personal wish 47 • Any other situation which, according to the investigator, would be such that further 48 participation in the clinical not be in the best interests of the patient 49 50 • The onset of pregnancy 51 • Later occurrence of exclusion criteria. 52 53 54 Decisions on the discontinuation of the entire trial will be taken, if the risk benefit 55 56 assessment demonstrates unjustifiable risks and toxicities or new scientific conclusions 57 during the clinical trial could compromise the safety of the study participants. The decision 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 15 Page 17 of 33 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 making body consists of the sponsor and principal investigator and acts, if appropriate, also 4 upon recommendation of the DMSB. 5 6 7 8 9 10 11 12 13 14 15 For peer review only 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 http://bmjopen.bmj.com/ 35 36 37 38 39 40 41
42 on October 2, 2021 by guest. Protected copyright. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 16 BMJ Open Page 18 of 33
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 ETHICS AND DISSEMINATION 4 5 The study protocol was approved by the German state Ethics Board located at the 6 Landesamt für Gesundheit und Soziales (LaGeSo), Berlin, Germany (13/0127 EK13) and the 7 8 Federal Institute for Drugs and Medical Devices (BfArM). Participants will be informed about 9 the trial and its anticipated risks and benefits, orally and in written form, using patient 10 11 information sheets. Patients’ written informed consent will be obtained prior to inclusion. This 12 study complies with the Helsinki Declaration, the principles of Good Clinical Practice (GCP), 13 14 the German Medicinal Products Act (AMG), and the Personal Data Protection Act. The study 15 with the full Forofficial title “Thepeer Rho Inhibitor review Ibuprofen for the Treatmentonly of Acute Spinal Cord 16 17 Injury: Investigation of Safety, Feasibility and Pharmacokinetics” has been registered in the 18 19 ClinicalTrials.gov database (NCT02096913). 20 21 22 Risk benefit assessment 23 In a large number of patients, traumatic SCI signifies a severe lifelong physical disability. 24 25 A standard treatment to promote neuronal plasticity after SCI is not yet available. Based on 26 preclinical investigations in established animal models, a better recovery of neurological 27 28 function in cases of acute SCI is anticipated from making use of ‘small molecule’ Rho 29 inhibition. The systematic review of preclinical data revealed 11 eligible studies on effects of 30 31 Rho inhibiting NSAIDs with motor function as behavioral endpoint. These studies were 32 conducted in six laboratories and used four different SCI models in three rodent species. The 33 34 meta analysis demonstrated an overall effect size of 20.2%. This is backed up by pervious http://bmjopen.bmj.com/ 35 analyses including studies on specific Rho/ROCK inhibitors that have demonstrated overall 36 37 effect sizes of 21% or 15% after correction for publication bias, respectively.[27] Ibuprofen is 38 an established, globally approved drug available for clinical investigation of its ability to 39 40 improve neurological function by Rho inhibition. Furthermore, preventive treatments for 41 inflammation triggered SCI specific complications in terms of neuropathic pain[51 56] and 42 on October 2, 2021 by guest. Protected copyright. 43 neurogenic heterotopic ossifications after SCI,[57 59], are not well established. Favorable 44 45 effects on these threatening sequelae can be anticipated from ibuprofen treatment by the 46 reduction of COX and NFκB mediated inflammation in the CNS and the peripheral soft 47 48 tissue. 49 The appraised benefits of the intervention have to be weighed against its potential risks, 50 51 some of which may be serious. Gastrointestinal ulcers accompanied by hemorrhage or by 52 perforation are the most prominent side effect of NSAIDs. According to FDA estimates from 53 54 1987, gastrointestinal hemorrhage due to peptic ulcers or perforation occurred in 1 2% of 55 patients under sustained three month intake of NSAIDs.[89] The factors that increase the risk 56 57 of gastrointestinal hemorrhage are: advanced age, high daily doses, a medical history of 58 ulcers, simultaneous intake of systemic corticosteroids and the intake of anticoagulants.[90] 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 17 Page 19 of 33 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Within the group of NSAIDs, ibuprofen has a comparatively low gastrointestinal toxicity.[90] A 4 recent Cochrane database review summarized results from recent clinical trials on long term 5 6 high dose ibuprofen administered to reduce respiratory complications in cystic fibrosis. The 7 studies showed a positive overall benefit risk profile.[91] However, a clinical data base 8 9 analysis comparing 1365 ibuprofen treated patients with 8960 controls demonstrated a low 10 overall risk but a higher annual incidence of gastrointestinal bleeding in the ibuprofen group 11 12 of 0.37% vs. 0.14%.[92] In the acute phase, acute injury to the cervical and upper thoracic 13 14 segments of the spinal cord is probably an additional risk factor for gastroduodenal 15 ulceration,[67]For which is whypeer the gastrointestinal review safety of ibuprofen only treatment in the context of 16 17 SCI is the primary endpoint of this trial. 18 Under normal conditions acute renal failure due to NSAIDs is a rare but serious adverse 19 20 reaction. The risk for acute renal failure increases in critically ill patients with a volume 21 deficiency, myocardial insufficiency, or pre existing renal insufficiency; the same holds true 22 23 for simultaneous administration of other nephrotoxic substances such as aminoglycosides, 24 angiotensin converting enzyme inhibitors, and Angiotensin II receptor antagonists.[93] Acute 25 26 renal failure caused by NSAIDs such as ibuprofen, a substance with a short half life and 27 rapid achievement of effective levels, commonly manifests within a few days. After early 28 29 diagnosis and discontinuation of the treatment, renal function usually returns to normal within 30 one week. Only if renal failure is not diagnosed in time may the condition progress rapidly to 31 32 dependence on dialysis. Compared to other NSAIDs, an intermediate level of nephrotoxicity 33
is reported for ibuprofen.[94] Acute SCI is generally not associated with a disturbance of http://bmjopen.bmj.com/ 34 35 renal function. However, due to the traumatic etiology of paraplegia, renal function may be 36 transiently limited in some cases due to a volume deficiency or rhabdomyolysis. In those 37 38 cases renal side effects of ibuprofen might be observed more frequently. 39 40 In order to limit the anticipated risks in the Ibuprofen SCI Safety trial, its exclusion 41 criteria comprise known risk factors such as age > 65 years, relevant co morbidities, history
42 on October 2, 2021 by guest. Protected copyright. 43 of critical events, particularly peptic ulcerations, as well as drug interactions. In addition, the 44 trial will be conducted under in hospital conditions of acute care and rehabilitation. In hospital 45 46 monitoring and carefully scheduled laboratory investigations allow for early awareness of AE 47 and their immediate medical treatment. 48 49 50 Limitations 51 52 Limitations of the clinical trial protocol are its small sample size, the lack of a placebo 53 control group and a relatively wide timeframe for inclusion. This design, chosen with regard 54 55 to the primary safety endpoint and feasibility of the pharmacokinetic issues, restricts efficacy 56 evaluation. The time frame of inclusion extended until day 21 after SCI was incorporated for 57 58 ethical reasons in order to enable the patients giving informed to consent before start of the 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 18 BMJ Open Page 20 of 33
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 intervention. However, a late start of intervention might diminish therapeutic efficacy because 4 recovery promoting effects of Rho inhibiton,[22] as well as anti inflammatory effects of 5 6 ibuprofen,[95] depend on the timing of the intervention, and an early start of treatment seems 7 favorable. 8 9 The meta analysis of published preclinical experiments is limited by the relative low 10 number of studies specific to ibuprofen/Indometacin mediated Rho inhibition, and they thus 11 12 hardly enable meta regression or adjustment for publication bias. Still, our analysis is in line 13 14 with a larger previous meta analysis that also includes studies on specific Rho/ROCK 15 inhibitors thatFor demonstrated peer relevant effect review sizes after correction only for publication bias.[27] A 16 17 limitation of the single in vivo experiments on ibuprofen is that they lack dose response 18 curves, and all research groups have applied the drug in comparable dosages. 19 20 Administration of even higher doses would still be within FDA approved range for application 21 in humans and might have larger effects. Confirmative preclinical analyses should therefore 22 23 also consider dose response curves to show functional recovery. 24 25 26 Possible consequences 27 The explorative safety evaluation, feasibility aspects of recruitment and treatment regime 28 29 in the acute phase after SCI are of interest for the planning of a subsequent randomized 30 controlled trial (RCT) in a larger sample. Of particular relevance in the clinical trial are 31 32 treatment timing and the CNS availability of the systemically delivered compound behind the 33
blood spinal cord barrier after acute SCI. An interim bed to bench side translation based on http://bmjopen.bmj.com/ 34 35 the clinical pharmacological data and preliminary efficacy endpoints could be valuable for 36 adjustment of the treatment schedule before embarking on a RCT. Regardless to the result 37 38 of the primary and secondary outcome assessments the clinical trial will be reported as 39 40 publication in a peer reviewed journal. 41 Improved neurological recovery anticipated after SCI, which is proposed as the main
42 on October 2, 2021 by guest. Protected copyright. 43 objective of a subsequent RCT might lead to an improvement of aspects of daily living, even 44 if the recovery has affected only two segments of the spinal cord. For example, regaining 45 46 more than one neurological motor level can be considered as a notable difference with 47 influence on physical independence,[96] and long term survival.[97] Prevention of SCI 48 49 related complications might contribute additionally to improved quality of life. 50 51 52 Authors’ contributions 53 54 MAK, TL, PM, SMS, JMS designed the trial protocol. TL, RW, PM, SL, CB, RS, GJJ, SK, 55 56 AE, UD, AN reviewed the trial protocol. MAK, RW did the systematic review. RW performed 57 the meta analysis. MAK, RW wrote the manuscript. All authors critically revised the 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 19 Page 21 of 33 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 manuscript for important intellectual content. All authors have read and approved the final 4 version of the manuscript. 5 6 7 8 Funding statement 9 The project has received funding from the Wings for Life Spinal Cord Research 10 11 Foundation (Grant Number WfL DE 006/1) and is funded by the Else Kröner Fresenius 12 Stiftung (Grant Number WfL DE 006/1). JMS is supported by the W.E Hunt and C.M. Miller 13 14 Endowment. 15 For peer review only 16 17 Competing interests 18 19 We declare to have no conflicts of interest. 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 http://bmjopen.bmj.com/ 35 36 37 38 39 40 41
42 on October 2, 2021 by guest. Protected copyright. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 20 BMJ Open Page 22 of 33
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 References 4 5 6 1. Hurlbert RJ, Hadley MN, Walters BC, et al. Pharmacological therapy for acute spinal cord 7 injury. Neurosurgery 2013;72 Suppl 2:93-105 doi: 8 10.1227/NEU.0b013e31827765c6[published Online First: Epub Date]|. 9 2. Siddiqui AM, Khazaei M, Fehlings MG. Translating mechanisms of neuroprotection, 10 regeneration, and repair to treatment of spinal cord injury. Prog Brain Res 11 2015;218:15-54 doi: 10.1016/bs.pbr.2014.12.007[published Online First: Epub Date]|. 12 3. Dietz V, Curt A. Translating preclinical approaches into human application. Handb Clin 13 Neurol 2012;109:399-409 doi: 10.1016/B978-0-444-52137-8.00025-5[published 14 15 OnlineFor First: Epub peer Date]|. review only 16 4. Thuret S, Moon LD, Gage FH. Therapeutic interventions after spinal cord injury. Nat Rev 17 Neurosci 2006;7(8):628-43 doi: 10.1038/nrn1955[published Online First: Epub Date]|. 18 5. Yiu G, He Z. Glial inhibition of CNS axon regeneration. Nat Rev Neurosci 2006;7(8):617- 19 27 doi: 10.1038/nrn1956[published Online First: Epub Date]|. 20 6. Xie F, Zheng B. White matter inhibitors in CNS axon regeneration failure. Exp Neurol 21 2008;209(2):302-12 doi: 10.1016/j.expneurol.2007.07.005[published Online First: 22 Epub Date]|. 23 7. Kopp MA, Liebscher T, Niedeggen A, et al. Small-molecule-induced Rho-inhibition: 24 25 NSAIDs after spinal cord injury. Cell Tissue Res 2012;349(1):119-32 doi: 26 10.1007/s00441-012-1334-7[published Online First: Epub Date]|. 27 8. Schmandke A, Schmandke A, Strittmatter SM. ROCK and Rho: biochemistry and neuronal 28 functions of Rho-associated protein kinases. Neuroscientist 2007;13(5):454-69 doi: 29 10.1177/1073858407303611[published Online First: Epub Date]|. 30 9. Tanaka H, Yamashita T, Yachi K, Fujiwara T, Yoshikawa H, Tohyama M. Cytoplasmic 31 p21(Cip1/WAF1) enhances axonal regeneration and functional recovery after spinal 32 cord injury in rats. Neuroscience 2004;127(1):155-64 doi: 33
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1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Figure Legends 4 5 6 Figure 1: Pharmacological targets of ibuprofen. Intracellular signaling cascades converge 7 8 at the GTPase RhoA, which is activated after SCI by myelin and scar associated proteins (for 9 10 review,[5 7 28]). Downstream to Rho, the activated ROCK inhibits axonal re growth, 11 promotes neurodegeneration, contributes to the development of neuropathic pain and tissue 12 13 loss, and impedes neurorestoration and functional recovery (reviewed by Watzlawick et 14 al.[27]). This pathway can be blocked by the ROCK inhibitors Y 27632 and Fasudil or the 15 For peerCIP1/WAF1 review only 16 specific Rho inhibitors P21 , C3 transferase[27], and by the R( ) and S( ) enantiomers 17 of ibuprofen,[10 16 18 20 30 31], as the most convincing Rho inhibitor among individual 18 19 drugs from the group of NSAIDs. Ibuprofen mediated Rho inhibition depends on the 20 upregulation of PPARγ.[16] Treatment with PPARγ agonists was demonstrated to have anti 21 22 inflammatory effects[37 38] and to protect tissue and thereby motor function in other CNS 23 injury conditions (reviewed by McTigue,[39]). It is not yet clear whether the inhibition of NF 24 25 κB as a further target of R( )/S(+) ibuprofen,[36] is independent of PPARγ. Notably, PPARγ 26 inhibits gene expression by antagonizing the activities of the pro inflammatory transcription 27 28 factors NF κMB.[37] Another pathway, mainly operated by the S enantiomer of ibuprofen, is 29 the inhibition of COX 1/2 and consequently of the prostaglandin E production, which 30 2 31 activates NF κB or counter regulates it at very high concentrations.[52] COX 1/2 or NF κB 32 are associated with inflammation induced neuropathic pain,[51 52], neurodegeneration,[48] 33 34 sickness behavior,[41] and the systemic inflammatory response syndrome.[42 50] Systemic http://bmjopen.bmj.com/ 35 36 inflammation contributes to neurogenic heterotopic ossificastions.[57] 37 Taken together, Rho blocking NSAIDs have the potential to decrease the systemic and 38 39 acute CNS inflammatory response by targeting at least two separate pathways, PPARγ and 40 COX 1/2. The suspected side effect of neuroprotective anti inflammatory therapy, that is, that 41
42 it further limits the regeneration capacity of spared axons,[60] is suggested to be abrogated on October 2, 2021 by guest. Protected copyright. 43 by Rho Inhibition. Abbreviations: CNS = central nervous system; COX = cyclooxygenase; 44 45 NSAIDs = non steroid anti inflammatory drugs; PPARγ = peroxisome proliferator activated 46 receptor γ; NF κB = nuclear factor κB; ROCK = Rho associated coiled kinase; SCI = spinal 47 48 cord injury. 49 50 51 52 Figure 2: Systematic review preclinical study selection chart. To identify animal 53 54 studies reporting the effect of ibuprofen or indometacin treatment for neurobehavioral 55 recovery after SCI the following search term was used for PubMed, EMBASE, and ISI Web 56 57 of science (search conducted May 18, 2015): (Ibuprofen OR Indometacin OR NSAID OR 58 nonsteroidal anti inflammatory drugs) AND (spinal cord injury OR hemisection OR contusion 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 28 BMJ Open Page 30 of 33
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 OR dorsal column injury OR transection OR corticospinal tract injury OR compression OR 4 spinal cord lesion). Search results were limited to animals. Studies were included if they 5 6 reported the effects of ibuprofen or indometacin in animal models after various types of SCI. 7 We included SCI experiments comparing functional motor outcome between a group of 8 9 animals receiving treatment and a control group receiving no treatment (sham group). Non 10 traumatic models of SCI were excluded, as well as studies reporting only combined 11 12 treatments. Studies had to report the number of animals for each group, the mean effect size 13 14 and its variance. Studies were excluded due to inappropriate outcome scales, combination of 15 treatments andFor statistical peer inconsistencies. review only 16 17 18 19 20 Figure 3: Meta analysis of preclinical effects on motor recovery. Improvement in 21 neurobehavioral score ranked by effect size. The overall number of included animals was 22 23 n=255 (median n=12, range: 8 – 73). Black dots represent studies using Ibuprofen; white 24 dots show Indometacin studies. The horizontal bar represents the 95% CI of the effect size 25 26 (ES). Details on the design of the included studies are summarized in Table 1. 27 28 29 30 Figure 4: Longitudinal clinical trial design. Diagram of frequency and scope of trial 31 32 procedures. The evaluation for eligibility should start as early as possible after acute SCI. 33
The baseline will be obtained at the day of the inclusion from day 4 and latest at day 21 post http://bmjopen.bmj.com/ 34 35 trauma, in any case as early as possible. Start of the study medication is directly after the 36 baseline assessment. The duration of the intervention is 4 weeks for cohort I, and 12 weeks 37 38 for cohort II. Frequent safety laboratory measurements are performed. Samples for 39 40 pharmacokinetic measurements are collected two times in cohort I and three times in cohort 41 II. The follow up visits for determination of secondary endpoints are performed at week 4 (± 3
42 on October 2, 2021 by guest. Protected copyright. 43 days) and after the end of intervention at week 24 (± 14 days). Final safety laboratory 44 measurements will be performed 4 weeks after the end of the study medication. 45 46 Abbreviations: SCI = spinal cord injury. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 29 Page 31 of 33 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 4 5 6 7 8 9 10 11 12 13 14 15 For peer review only 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Figure 1: Pharmacological targets of ibuprofen. Intracellular signaling cascades converge at the GTPase 30 RhoA, which is activated after SCI by myelin and scar associated proteins (for review,5 7 29). Downstream 31 to Rho, the activated ROCK inhibits axonal re growth, promotes neurodegeneration, contributes to the 32 development of neuropathic pain and tissue loss, and impedes neurorestoration and functional recovery 33 (reviewed by Watzlawick et al.28). This pathway can be blocked by the ROCK inhibitors Y 27632 and Fasudil 34 or the specific Rho inhibitors P21CIP1/WAF1, C3 transferase28, and by the R( ) and S( ) enantiomers of http://bmjopen.bmj.com/ 35 ibuprofen,11 17 19 21 31 32, as the most convincing Rho inhibitor among individual drugs from the group of NSAIDs. Ibuprofen mediated Rho inhibition depends on the upregulation of PPARγ.17 Treatment with 36 PPARγ agonists was demonstrated to have anti inflammatory effects39 40 and to protect tissue and thereby 37 motor function in other CNS injury conditions (reviewed by McTigue,41). It is not yet clear whether the 38 inhibition of NF κB as a further target of R( )/S(+) ibuprofen,38 is independent of PPARγ. Notably, PPARγ 39 inhibits gene expression by antagonizing the activities of the pro inflammatory transcription factors NF 40 κ�B.39 Another pathway, mainly operated by the S enantiomer of ibuprofen, is the inhibition of COX 1/2 41 and consequently of the prostaglandin E2 production, which activates NF κB or counter regulates it at very high concentrations.54 COX 1/2 or NF κB are associated with inflammation induced neuropathic pain,53 54, 42 on October 2, 2021 by guest. Protected copyright. 43 neurodegeneration,50 sickness behavior,43 and the systemic inflammatory response syndrome.44 52 Systemic inflammation contributes to neurogenic heterotopic ossificastions.59 44 Taken together, Rho blocking NSAIDs have the potential to decrease the systemic and acute CNS 45 inflammatory response by targeting at least two separate pathways, PPARγ and COX 1/2. The suspected 46 side effect of neuroprotective anti inflammatory therapy, that is, that it further limits the regeneration 47 capacity of spared axons,62 is suggested to be abrogated by Rho Inhibition. Abbreviations: CNS = central 48 nervous system; COX = cyclooxygenase; NSAIDs = non steroid anti inflammatory drugs; PPARγ = 49 peroxisome proliferator activated receptor γ; NF κB = nuclear factor κB; ROCK = Rho associated coiled 50 kinase; SCI = spinal cord injury.
51 345x227mm (300 x 300 DPI) 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 32 of 33
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 4 5 6 7 8 9 10 11 12 13 14 15 For peer review only 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 http://bmjopen.bmj.com/ 35 36 37 38 39 Figure 2: Systematic review preclinical study selection chart. To identify animal studies reporting the effect of ibuprofen or indometacin treatment for neurobehavioral recovery after SCI the following search term was 40 used for PubMed, EMBASE, and ISI Web of science (search conducted May 18, 2015): (Ibuprofen OR 41 Indometacin OR NSAID OR nonsteroidal anti-inflammatory drugs) AND (spinal cord injury OR hemisection 42 OR contusion OR dorsal column injury OR transection OR corticospinal tract injury OR compression OR spinal on October 2, 2021 by guest. Protected copyright. 43 cord lesion). Search results were limited to animals. Studies were included if they reported the effects of 44 ibuprofen or indometacin in animal models after various types of SCI. We included SCI experiments 45 comparing functional motor outcome between a group of animals receiving treatment and a control group 46 receiving no treatment (sham group). Non-traumatic models of SCI were excluded, as well as studies 47 reporting only combined treatments. Studies had to report the number of animals for each group, the mean effect size and its variance. Studies were excluded due to inappropriate outcome scales, combination of 48 treatments and statistical inconsistencies. 49 102x97mm (300 x 300 DPI) 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 33 of 33 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 4 5 6 7 8 9 10 11 12 13 14 15 For peer review only 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Figure 3: Meta analysis of preclinical effects on motor recovery. Improvement in neurobehavioral score 33 ranked by effect size. The overall number of included animals was n=255 (median n=12, range: 8 – 73). 34 Black dots represent studies using Ibuprofen; white dots show Indometacin studies. The horizontal bar http://bmjopen.bmj.com/ 35 represents the 95% CI of the effect size (ES). Details on the design of the included studies are summarized 36 in Table 1. 420x313mm (72 x 72 DPI) 37 38 39 40 41
42 on October 2, 2021 by guest. Protected copyright. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 34 of 33
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 4 5 6 7 8 9 10 11 12 13 14 15 For peer review only 16 17 18 19 20 21 22 23 Figure 4: Longitudinal clinical trial design. Diagram of frequency and scope of trial procedures. The 24 evaluation for eligibility should start as early as possible after acute SCI. The baseline will be obtained at the 25 day of the inclusion from day 4 and latest at day 21 post-trauma, in any case as early as possible. Start of the study medication is directly after the baseline assessment. The duration of the intervention is 4 weeks 26 for cohort I, and 12 weeks for cohort II. Frequent safety laboratory measurements are performed. Samples 27 for pharmacokinetic measurements are collected two times in cohort I and three times in cohort II. The 28 follow-up visits for determination of secondary endpoints are performed at week 4 (± 3 days) and after the 29 end of intervention at week 24 (± 14 days). Final safety laboratory measurements will be performed 4 30 weeks after the end of the study medication. Abbreviations: SCI = spinal cord injury. 31 135x62mm (300 x 300 DPI) 32 33 34 http://bmjopen.bmj.com/ 35 36 37 38 39 40 41
42 on October 2, 2021 by guest. Protected copyright. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from
SCISSOR – Spinal Cord Injury Study on Small molecule derived Rho-inhibition: A Clinical Study Protocol
ForJournal: peerBMJ Open review only Manuscript ID bmjopen-2015-010651.R1
Article Type: Protocol
Date Submitted by the Author: 25-Feb-2016
Complete List of Authors: Kopp, Marcel; Charité Universitätsmedizin Berlin, Department of Neurology & Experimental Neurology, Spinal Cord Injury Research Liebscher, Thomas; Trauma Hospital Berlin, Treatment Centre for Spinal Cord Injury Watzlawick, Ralf; Charité Universitätsmedizin Berlin, Department of Neurology & Experimental Neurology, Spinal Cord Injury Research Martus, Peter; Department of Clinical Epidemiology and Applied Biostatistics, Eberhard Karls Universität Tübingen Laufer, Stefan; Eberhard-Karls-University Tübingen, Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy Blex, Christian; Charité Universitätsmedizin Berlin, Department of Neurology & Experimental Neurology, Spinal Cord Injury Research Schindler, Ralf; Charité-University Medicine Berlin, Division of Nephrology and Intensive Care, Department of Internal Medicine, Campus Virchow-
Klinikum http://bmjopen.bmj.com/ Jungehulsing, Gerhard; Jüdisches Krankenhaus Berlin, Department of Neurology Knüppel, Sven; German Institute of Human Nutrition Potsdam-Rehbrücke, Department of Epidemiology Kreutzträger, Martin; Trauma Hospital Berlin, Treatment Centre for Spinal Cord Injury Ekkernkamp, Axel ; Trauma Hospital Berlin, Trauma Surgery and Orthopedics Clinic
Dirnagl, Ulrich; Charité Universitätsmedizin Berlin, Department of on October 2, 2021 by guest. Protected copyright. Neurology & Experimental Neurology Strittmatter, Stephen; Yale University School of Medicine, Program in Cellular Neuroscience, Neurodegeneration and Repair, and Department of Neurology Niedeggen, Andreas; Trauma Hospital Berlin, Treatment Centre for Spinal Cord Injury Schwab, Jan; Charité Berlin, Department of Neurology & Experimental Neurology, Spinal Cord Injury Research; Department of Neurology, Spinal Cord Injury Division, The Ohio State University, Wexner Medical Center
Primary Subject Neurology Heading:
Secondary Subject Heading: Pharmacology and therapeutics, Research methods
ibuprofen, Neuroprotection, Plasticity, Neuropathic pain, Heterotopic Keywords: ossifications
For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 1 of 40 BMJ Open
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42 on October 2, 2021 by guest. Protected copyright. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 2 of 40
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Study Protocol 4 5 6 7 SCISSOR – Spinal Cord Injury Study on Small molecule derived Rho-inhibition: A 8 9 Clinical Study Protocol 10
11 12 13 1 2 1 3 4 14 Marcel A. Kopp *, Thomas Liebscher *, Ralf Watzlawick *, Peter Martus , Stefan Laufer , 15 ChristianFor Blex1, Ralf peer Schindler5, Gerhard review J. Jungehulsing 6,7only, Sven Knüppel7, Martin 16 2 8 9 10 17 Kreutzträger , Axel Ekkernkamp , Ulrich Dirnagl , Stephen M. Strittmatter , 18 Andreas Niedeggen2, Jan M. Schwab1,2,11,12 19 20 21 1Department of Neurology and Experimental Neurology, Spinal Cord Injury Research, Charité 22 Universitätsmedizin Berlin, Germany. 2Treatment Centre for Spinal Cord Injury, Trauma Hospital Berlin, Germany. 23 3 Department of Clinical Epidemiology and Applied Biostatistics, Eberhard Karls Universität Tübingen, Germany 24 4 25 Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Eberhard Karls University 26 Tübingen, Germany. 5 Division of Nephrology and Intensive Care, Department of Internal Medicine, Campus 27 Virchow Klinikum, Charité University Medicine Berlin, Berlin, Germany. 6 Jüdisches Krankenhaus Berlin, 28 Department of Neurology, Germany. 7 Department of Epidemiology German Institute of Human Nutrition 29 8 30 Potsdam Rehbrücke, Germany. Trauma Surgery and Orthopedics Clinic, Trauma Hospital Berlin, Germany. 31 9 Department of Neurology and Experimental Neurology, Center for Stroke Research Berlin, Charité 32 Universitätsmedizin Berlin, Germany. 10Program in Cellular Neuroscience, Neurodegeneration and Repair, and 33 11 Department of Neurology, Yale University School of Medicine, New Haven, USA. Department of Neurology, 34 http://bmjopen.bmj.com/ 12 35 Spinal Cord Injury Division, The Ohio State University, Wexner Medical Center, Columbus, USA Department of 36 Neuroscience and Center for Brain and Spinal Cord Repair, Department of Physical Medicine and Rehabilitation, 37 The Neuroscience Research Institute, The Ohio State University, Wexner Medical Center, Columbus, USA. 38
39 40 * contributed equally to this work 41
42 on October 2, 2021 by guest. Protected copyright. 43
44 45 46 47 48
49 50 Correspondence 51 Prof. Jan Schwab, MD, PhD 52 Department of Neurology and Experimental Neurology, Spinal Cord Injury Research (Clinical and experimental 53 Neuroparaplegiology) , Charité Universitätsmedizin Berlin Charitéplatz 1, D 10117 Berlin 54 55 Phone: 0049 30 450 660293 56 Fax: 0049 30 450 560942 57 E mail: [email protected] or [email protected] 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 2 Page 3 of 40 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 ABSTRACT 4 5 6 Introduction The approved analgesic and anti inflammatory drugs ibuprofen and 7 8 indometacin block the small GTPase RhoA, a key enzyme that impedes axonal sprouting 9 after axonal damage. Dosages required to block the Rho Pathway in a central nervous 10 11 system effective manner are higher compared with orthodox cyclooxygenase blocking 12 effects. Preclinical studies in spinal cord injury (SCI) imply improved motor recovery after 13 14 ibuprofen/indometacin mediated Rho inhibition. This has been re assessed by a meta 15 analysis of theFor underlying peer experimental review evidence, which indicates only an overall effect size of 16 17 20.2% regarding motor outcome achieved after ibuprofen/indometacin treatment compared 18 19 to vehicle controls. In addition, ibuprofen/indometacin may also limit sickness behavior, non 20 neurogenic systemic inflammatory response syndrome (SIRS), neuropathic pain, and 21 22 heterotopic ossifications after SCI. Consequently, ‘small molecule’ mediated Rho inhibition 23 after acute SCI warrants clinical investigation. 24 25 Methods and analysis Protocol of an investigator initiated clinical open label pilot trial on 26 high dose ibuprofen treatment after acute traumatic, motor complete SCI. A sample of n=12 27 28 patients will be enrolled in two cohorts treated with 2400mg/d ibuprofen for 4 or 12 weeks, 29 respectively. The primary safety endpoint is: occurrence of serious adverse events, primarily 30 31 gastroduodenal bleedings. Secondary endpoints are pharmacokinetics, feasibility, and 32 preliminary effects on neurological recovery, neuropathic pain and heterotopic ossifications. 33 34 The primary safety analysis is based on the incidence of severe gastrointestinal bleedings. http://bmjopen.bmj.com/ 35 Additional analyses will be mainly descriptive and casuistic. 36 37 Ethics and dissemination The clinical trial protocol was approved by the responsible 38 German state Ethics Board, and the Federal Institute for Drugs and Medical Devices. The 39 40 study complies with the Helsinki Declaration, the principles of Good Clinical Practice and all 41 further applicable regulations. This safety and pharmacokinetics trial informs the planning of 42 on October 2, 2021 by guest. Protected copyright. 43 a subsequent randomized controlled trial. Regardless to the result of the primary and 44 45 secondary outcome assessments the clinical trial will be reported as publication in a peer 46 reviewed journal. 47 48 Registration ClinicalTrials.gov identifier: NCT02096913. 49 50 51 (299 words) 52 53 54 Keywords: ibuprofen, neuroprotection, plasticity, neuropathic pain, heterotopic ossifications 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 3 BMJ Open Page 4 of 40
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 STRENGTHS AND LIMITATIONS 4 5 • The SCISSOR study is the first clinical trial on high dose application of the globally 6 approved NSAID ibuprofen as a “small molecule” Rho Inhibitor after acute traumatic 7 spinal cord injury (SCI) within a concept of drug repurposing. 8 • Preclinical evidence for recovery enhancing effects of ibuprofen mediated Rho 9 inhibition after SCI has been challenged by systematic review and meta analysis. 10 • Limitations of this pilot study inherent to a phase I trial are small sample size, the lack 11 of a placebo control group, and a relatively wide timeframe for inclusion. 12 • The results of the SCISSOR trial might inform an interim bed to bench side 13 translation and subsequent randomized controlled trials. 14 15 For peer review only 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 http://bmjopen.bmj.com/ 35 36 37 38 39 40 41
42 on October 2, 2021 by guest. Protected copyright. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 4 Page 5 of 40 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 INTRODUCTION 4 5 At present, the effective pharmacological treatment of acute traumatic spinal cord injury 6 (SCI) is an unmet medical need.1 The current opportunities for restitution of neurological 7 8 function after SCI are limited to early surgical decompression, stabilization, intensive care, 9 rehabilitation, and the prevention or therapy of SCI specific sequelae.2 Neuroprotective or 10 11 plasticity enhancing therapies are under investigation in preclinical studies and early phase 12 clinical trials. As yet, however, none of these approaches could be translated into clinical 13 14 routine.2 4 15 A majorFor reason for peer the poor prognosis review of central nervous only system (CNS) injury is the 16 17 incapacity of axons to re grow within the CNS. Molecular barriers preventing axonal 18 19 regeneration after SCI are situated in the environment of the injured axon i.e. in the scar 20 tissue and myelin or myelin debris.5 6 Those molecules, such as chondroitin sulfate 21 22 proteoglycans (CSPGs), Nogo A, myelin associated glycoprotein (MAG), oligodendrocyte 23 myelin glycoprotein (OMgp), Ephrins, and RGMa are up regulated after CNS injury and 24 25 interfere with a repertoire of cognate receptors on the axon membrane as reviewed 26 elsewhere.6 7 Signals from those receptors are mediated downstream to RhoA. The small 27 28 GTPase RhoA is a key molecule in a pathway which, once activated, leads to the collapse of 29 axonal growth cones and consequently to failure of axonal plasticity or regeneration.8 30 31 Furthermore, myelin debris inhibits the differentiation of oligodendrocyte precursor cells 32 partially dependent on RhoA associated pathways9 and thus may prevent remyelination of 33 34 spared axons. http://bmjopen.bmj.com/ 35 Therefore, the Rho pathway constitutes a target for treatments aiming to overcome 36 37 molecular obstacles to a restoration of neuronal connectivity and subsequent functional 38 recovery. The inhibition of Rho or the downstream located Rho associated coiled kinase 39 40 (ROCK) has been demonstrated to foster axonal sprouting or plasticity,10 23 to have 41 neuroprotective effects,10 11 13 14 20 24 25, to promote oligodendrocyte precursor cell 42 on October 2, 2021 by guest. Protected copyright. 43 differentiation9 or remyelination,25 and to enhance neurological recovery10 11 13 16 18 20 22 24 26 44 45 after acute SCI (Figure 1). These findings are backed up with evidence from other 46 experimental CNS injury conditions as reviewed elsewhere.7 27 The reported effects of 47 48 various Rho/ROCK blocking approaches on open field motor recovery after experimental SCI 49 have been re assessed by a systematic review and meta analysis including correction for 50 28 51 publication bias. Specific Rho inhibition mediated by the clostridium botulinum derived 52 enzyme C3 transferase, also referred to as BA 210 or Cethrin™,29 has been studied in a 53 54 recently completed Phase I/IIa clinical trial. The investigators concluded that topically applied 55 BA 210 is safe and is associated with favorable neurological outcome.30 However, a 56 57 confirmatory Phase III trial has not yet been conducted. 58 Over the last decade, upcoming evidence has assigned a subset of non steroid anti 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 5 BMJ Open Page 6 of 40
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 inflammatory drugs (NSAIDs) to the group of unspecific Rho inhibitors. The FDA approved 4 NSAIDs ibuprofen,11 17 19 21 31 32, indometacin,19 31 and suldinac sulfide,31 were shown to inhibit 5 6 Rho activation independently of their ‘classical’ mode of action as inhibitors of 7 Cyclooxygenases (COX). It was subsequently demonstrated that ibuprofen treatment 8 9 enhances axonal sprouting,11 17 19, including that of human model neurons,21 and improves 10 neurological recovery.11 19 It is noteworthy that ibuprofen mediated Rho inhibition involves 11 12 Peroxisome Proliferator Activated Receptor γ (PPARγ) activation.17 It remains unclear, 13 14 however, what the exact mechanism for this activation is and whether co factors are required 15 for PPARγ associatedFor Rho inhibition,peer because review other PPARγ activatorsonly such as rosiglitazone 16 17 33 34 17 also inhibit Rho, , while other NSAIDs such as Naproxen activate PPARγ strongly but do 18 not cause Rho inhibition.11 17 31 19 20 Importantly, ibuprofen dose regimes currently applied in the clinical setting are sub 21 therapeutic as being likely unable to block the Rho pathway in the CNS compartment 22 23 sufficiently. Moreover, NSAIDs are usually applied in a later phase after SCI. Thus, 24 retrospective analysis studying the effect of lower dose NSAID as being applied at present 25 26 cannot address the hypothesis sufficiently whether ibuprofen mediated Rho inhibition may 27 elicit improved neurological recovery when being applied in sufficient dosage and appropriate 28 35 29 time frame. 30 Other pharmacological targets of ibuprofen – namely PPARγ activation,17 34 36, COX 1/2 31 32 inhibition,37 and NF κb inhibition,38 promise a concomitant limitation of secondary damage by 33
anti inflammatory actions, but might also modify the effects of Rho blockade (Figure 1). In http://bmjopen.bmj.com/ 34 35 more detail, PPARγ activation reduces the cellular,39 and soluble inflammatory response,40 36 that is suggested to alter tissue pathology after SCI as reviewed by McTigue.41 In the context 37 38 of experimental systemic inflammation COX 1, which reveals sustained upregulation in the 39 42 43 40 spinal cord after SCI, promotes sickness behaviour. COX related pathways also exert 41 immune modulation in terms of immune depression,44 and impaired host defense.44 45 Those
42 46 47 on October 2, 2021 by guest. Protected copyright. 43 effects might aggravate the maladaptive immune response after SCI, that is associated 44 with increased susceptibility to infections, which are a risk factor for poorer neurological 45 48 49 46 outcome after SCI. Furthermore, NF κB, which is activated after SCI, contributes in 47 neurodegenerative disease to microglia induced loss of motor neurons.50 Together, anti 48 49 phlogistic actions of ibuprofen are likely to reduce neurodegeneration driven by CNS 50 inflammation,50 51, which is triggered through the COX and/or NF κB related systemic 51 44 52 44 45 53 52 inflammatory response syndrome, , or infections. Besides, NF κB, and COX 53 54 metabolites such as prostaglandin E2, are linked to the induction of neuropathic pain. Thus, 54 53 55 55 NSAIDs might be effective in preventing SCI specific sequelae such as neuropathic pain, 56 58, as well as inflammation related neurogenic heterotopic ossifications (Figure 1).59 61 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 6 Page 7 of 40 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Ibuprofen is recommended primarily to improve neurological function through the 4 enhanced plasticity conferred by its Rho inhibiting properties. The combination of Rho 5 6 inhibition with anti inflammatory actions of ibuprofen might, however, dissolve conflicting 7 aspects of anti inflammatory therapies after axonal injury. It has been demonstrated that 8 9 secondary axonal damage is reduced when inflammation has been limited, but this occurs at 10 the expense of the regenerative capacity of the spared axons.62 In this context, an increased 11 12 blockade of axonal re growth capacity as a side effect of anti inflammatory neuroprotective 13 14 therapy could be prevented by concurrent Rho inhibition (Figure 1). 15 In vitro sproutingFor responses peer under ibuprofen,review11 17 19 21, or indometacin,only19 treatment in the 16 17 presence of myelin or inhibitory matrix components such as CSPGs are well reproducible. 18 However, in vivo evidence provided by some groups for promoting effects of Rho inhibition 19 11 19 11 19 63 64 20 on axonal sprouting, , or on neurological recovery, , have not or have only partially 21 been confirmed by others.32 65 Reasons for the variability in the results could be multiple. One 22 23 possible reason would be differences in the experimental design, such as in timing of the 24 experiments, the animal model applied, the route of drug delivery, and assessment tools. On 25 26 the other hand the variability could be a product of chance due to small sample sizes, which 27 is a general problem in preclinical studies.66 One approach to address the variability of 28 66 29 preclinical studies is to subject them to meta analysis. 30 This work includes a systematic review and meta analysis of experiments reporting the 31 32 effect of Rho inhibiting NSAIDs on neurobehavioral recovery after SCI. The published 33
preclinical evidence and its positive predictive value, as representing the justification of the http://bmjopen.bmj.com/ 34 35 current clinical investigation, were challenged by the meta analysis. The study protocol of the 36 first clinical trial on high dose ibuprofen as a Rho inhibitor after acute SCI is addressing 37 38 safety, feasibility and pharmacokinetics. Additionally, the study explores preliminary efficacy 39 67 40 including aspects of repurposing ibuprofen, as a compound with multiple pharmacological 41 targets for the treatment of SCI.
42 on October 2, 2021 by guest. Protected copyright. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 7 BMJ Open Page 8 of 40
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 METHODS AND ANALYSIS 4 5 Ibuprofen is a drug that has been FDA approved and is available worldwide for decades. 6 However, in the context of traumatic injuries its use is generally restricted to short term low 7 8 dose administration as an analgesic. The mid term high dose application of ibuprofen, as a 9 10 Rho inhibiting and anti inflammatory treatment after SCI, is not an approved indication and 11 information on its tolerability is not available for the population of acute SCI patients. This is 12 13 relevant because critically injured SCI patients require treatment in an intensive care unit, 14 which is a risk factor for gastric ulcers,68 and particularly SCI patients with cervical and high 15 For peer review only 16 thoracic levels might be at risk for damage to the gastric mucosa due to a disturbance of 17 autonomous innervation.69 Furthermore, pharmacological data on CNS permeability are 18 19 available for non trauma patients but little is known about pharmacokinetics of orally 20 administered ibuprofen after SCI. Therefore, the SCISSOR study primarily addresses safety, 21 22 feasibility and pharmacokinetics under the clinical condition of acute traumatic SCI. 23 Secondary objectives are neurological recovery and SCI specific complications. 24 25 26 Assessment of underlying evidence 27 28 In order to re asses the preclinical evidence regarding Rho inhibiting NSAIDs and to 29 justify the risks and efforts of the clinical trial, a systematic review was performed. Six 30 31 publications,11 19 32 63 65, containing 11 single experiments with a total of n=255 animals (Table 32 1) were included for meta analysis after stepwise study selection (Figure 2). 33 34 http://bmjopen.bmj.com/ 35 36 Table 1: Preclinical study characteristics. 37 ID Publication Drug Species n Dose (Duration) Motor Injury Follow Type of Appli- 38 score level -up Injury cation 39 1 Redondo Ibu Rats 16 60 mg/kg/d (42d) BBB T8 42d Contusion s.c. Castro, E 40 2 Sharp, K Ibu Rats 73 60 mg/kg/d (28d) BBB T6/7 42d Hemisection s.c. 41 3 Wang, X Ibu Mice 46 35 70 mg/kg/d BMS T8 35d Transection s.c.
42 (28d) on October 2, 2021 by guest. Protected copyright. 43 4 Guth, L Indo Rats 12 0.2 mg/d (21d) Tarlov T8 21d Compression i.p. 44 5 Wang, X Ibu Rats 47 70 mg/kg/d (28d) BBB T7 49d Contusion s.c. 45 46 6 Fu, Q Ibu Rats 19 60 mg/kg/d (28d) BBB T6/7 42d Hemisection s.c. 7 12 60 mg/kg/d (28d) Contusion 47 8 Pantovic, R Indo Rabbits 6 0.1 mg/kg/d (9d) Tarlov L2 9d Contusion i.v. 48 9 6 0.3 mg/kg/d (9d) 49 10 6 1.0 mg/kg/d (9d) 50 11 6 3.0 mg/kg/d (9d) 51 6 vehicle Abbreviations: Ibu=Ibuprofen, Indo= Indometacin, n= number of animals, d= days, s.c.= subcutaneous, i.p.= 52 intraperitoneal, i.v.= intravenous 53 54 55 Preclinical study characteristics were extracted for each publication and functional outcome 56 was measured for each experiment in order to perform the meta analysis. The method and 57 58 statistical approach is described in greater detail elsewhere.28 70 In brief, we used a random 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 8 Page 9 of 40 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 effects weighted mean difference meta analysis to calculate an overall estimate of effect size 4 between treated and untreated (control) animals based on the final time point of the 5 6 assessment of functional recovery. A random effects meta regression was used to determine 7 how much heterogeneity can be explained by study design characteristics using STATA13 8 9 with a significance level of p<0.05. We checked for possible publication bias using trim and 10 fill method for funnel plots and Egger regression in STATA13. 11 12 The effect size in the open field motor testing of treatments with ibuprofen or 13 14 indometacin after experimental SCI was 20.2% (95% CI, 10.8 – 29.6) in the overall analysis 15 (Figure 3) andFor varied in thepeer single experiments review from 33.2% ( 79,2only – 12.8) to 44.9% (19,5 – 16 17 70.4). Meta regression analyses to identify of subgroup effects regarding the administered 18 drug, the behavioral assessment tool, the SCI model, the route of drug delivery or the study 19 20 quality revealed no statistically significant proportion of between study heterogeneity for any 21 of the stratifications. Likewise the tests to detect possible publication bias implied no missing 22 23 experiment, although statistical significance should not be expected given the study’s small 24 overall size.71 25 26 Nevertheless, the design of the studies on ibuprofen was different from those on 27 indometacin treatment in terms of the neurobehavioral scales, the animal models, and the 28 29 route of drug delivery (Table 1). The ibuprofen treated animals had all been assessed with 30 the recent Basso Beattie Bresnahan (BBB) score,72 or the Basso Mouse Scale (BMS),73 31 32 whereas modifications of the outdated Tarlov score,74 were applied for indometacin treated 33
animals. Furthermore, the ibuprofen treated groups underwent contusion, transection, and http://bmjopen.bmj.com/ 34 35 hemisection models in contrast to the exclusive use of compression or contusion 36 experiments in indometacin treated groups, which received the drug intravenously or 37 38 intraperitoneally compared to subcutaneous administration in the ibuprofen groups. 39 40 Therefore, differences in effect size between the two investigated compounds require careful 41 interpretation and do not allow conclusions on differences in their potential therapeutic
42 on October 2, 2021 by guest. Protected copyright. 43 efficacy. 44 Among the ‘small molecule’ Rho inhibitors, ibuprofen is the most feasible for clinical 45 46 investigation in the indication of acute traumatic SCI due to its greater quantity and the higher 47 quality of its preclinical data. All studies on ibuprofen revealed Rho inhibition in vivo within 48 11 19 32 49 the spinal cord after systemic drug administration, , and comprise experimental models 50 applicable for translational research,75 as well as recent behavioral scores.72 73 51 52 53 Study design 54 55 The SCISSOR study is designed as a prospective non randomized open label phase I 56 study, as this is a well established design for tolerability and pharmacokinetic 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 9 BMJ Open Page 10 of 40
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 76 77 3 investigations. Study participants are enrolled consecutively in two treatment cohorts 4 characterized by the duration of therapy as further detailed below and illustrated in Figure 4. 5 6 7 Setting 8 9 The initiating sponsor and coordinator of the trial is the Department of Experimental 10 Neurology, Clinical and Experimental Spinal Cord Injury Research (Neuroparaplegiology) at 11 12 Charité University Hospital, Campus Mitte, Berlin, Germany, represented by Prof. Jan 13 14 Schwab. For contact information see correspondence address. Data management and 15 statistics areFor performed bypeer the Department review of Clinical Epidemiology only and Applied Biostatistics 16 17 at Eberhard Karls University Tübingen, Germany. The recruiting trial center is the Treatment 18 Center for Spinal Cord Injuries at the Trauma Hospital Berlin, Germany. The study 19 20 investigators are physicians trained and experienced in the management and assessment of 21 patients with acute and chronic SCI. 22 23 The reference center for laboratory safety parameters is the Central Laboratory at the 24 trial center, Trauma Hospital Berlin, Germany. The central laboratory is regularly certified for 25 26 clinical diagnostics. The Department of Pharmaceutical and Medicinal Chemistry, Institute of 27 Pharmacy, Eberhard Karls University Tübingen, Germany will perform the measurement of 28 29 ibuprofen concentrations in plasma and CSF using High Performance Liquid 30 Chromatography Mass Spectrometry (HPLC MS). The Labor Berlin - Charité Vivantes 31 32 GmbH, a certified laboratory for clinical and research diagnostics, will run the nephelometric 33
protein measurements in serum and CSF for quantification of post SCI Blood Spinal Cord http://bmjopen.bmj.com/ 34 35 Barrier breakdown. 36
37 38 Intervention 39 40 The study medication is ibuprofen in the galenic preparation of water soluble lysine salt. 41 Ibuprofen lysine salt is absorbed faster, leading to earlier peaks of plasma concentrations
42 78 on October 2, 2021 by guest. Protected copyright. 43 compared to the free acid. The brand name of the study medication is Dolormin® extra. 44 Ibuprofen is applied as tablets administered orally for 4 weeks in cohort I or 12 weeks in 45 46 cohort II, respectively (Figure 4). The daily dose of 2400mg is administered as three single 47 doses of 800 mg. In the case of swallowing disorders, which occur in 16% of tetraplegic 48 79 49 acute SCI patients, it is recommended that the tablets be disaggregated in water and the 50 medication administered via stomach tube. 51 52 The proton pump inhibitor pantoprazole is used as a concomitant medication in a 53 dosage 40mg/d. This reduces the risk of damage to the gastrointestinal mucosa. After 4 54 55 weeks of treatment and individual risk benefit assessment, the dosage of pantoprazole may 56 be reduced to 20mg/d during the following weeks of treatment (applicable to cohort II). 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 10 Page 11 of 40 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Dose estimation 4 Ibuprofen doses of 60 to 70 mg/kg/d have been used in preclinical trials.11 19 32 65 To estimate 5 6 the pharmacologically active dose (PAD) in humans, we applied a conversion model which is 7 feasible for systemically administered active substances of a small molecular size, provided 8 9 that further pharmacologic properties of the compound have been taken into account.80 The 10 human equivalent dose (HED), converted from the PAD in rats, is about 11.3 mg/kg/d (rat 11 12 PAD of 70 mg/kg/d / 6.2 = HED 11,3 mg/kg/d). The binding capacity for ibuprofen in vitro is 13 14 higher in human albumin than it is in rat albumin. At identical concentrations, the free 15 bioactive ibuprofenFor fraction peer in human albumin review solution is lower only by a factor of about 3.81 82 We 16 17 therefore multiplied the HED by that factor to achieve an estimate of comparable bioactive 18 concentrations. Assuming an average body weight of 70 kg, the estimated PAD in humans is 19 20 34 mg/kg regardless of individual body weight. The daily dose of ibuprofen in this trial was 21 therefore set at 2400 mg/d. This is within the FDA approved range of up to 3200 mg/d for 22 23 adults. 24 25 26 Outcome measures 27 The primary endpoint of the study is the safety of high dose ibuprofen application after 28 29 SCI as measured by the occurrence of serious adverse events (SAE) related to the study 30 medication. In particular, severe gastroduodenal bleeding attributable to the study medication 31 32 is the primary safety parameter (Table 2). SAE definitions are in accordance with the ICH 33 83
GCP guidelines . All other adverse events (AE) that do not fulfill these definitions are http://bmjopen.bmj.com/ 34 35 documented on AE documentation sheets and type, severity, relatedness, treatment, and 36 outcome are recorded. 37 38 Secondary endpoints are all further AE including SAE and suspected unexpected 39 40 serious adverse reactions (SUSAR). Clinical, laboratory and technical safety examinations 41 facilitate the detection of AEs that can be expected and as well as the assessment of their
42 on October 2, 2021 by guest. Protected copyright. 43 causality (Table 2). In addition, the sensitive measurement of neuropathic pain and spasticity 44 is also relevant for safety reasons, since the course of those very frequent SCI specific 45 84 46 sequelae might be altered by plasticity enhancing therapies. The Neuropathic Pain Scale, 47 85, and the Modified Ashworth Scale,86 are therefore applied for assessment of pain and 48 49 muscle tone, respectively. The pharmacological laboratory endpoints are ibuprofen levels in 50 plasma and CSF as measured at the time of expected peak levels.87 The neurological 51 52 examination is performed according to the International Standards for Neurological 53 Classification of Spinal Cord Injury (ISNCSCI) definitions of 2011.88 The ISNCSCI comprises 54 55 the ASIA impairment scale (AIS) as a measure for completeness and severity of SCI, the 56 ASIA motor scores for upper and lower extremity motor function, the ASIA sensory scores for 57 58 residual pin prick and light touch sensation, the motor and sensory neurological level, as well 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 11 BMJ Open Page 12 of 40
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 as the zone of partial preservation, if applicable. Optional elements such as non key muscles 4 for determination of the AIS are not applied in this study.88 Neurogenic heterotopic 5 6 ossifications constitute a further clinical endpoint. These will be identified with an ultrasound 7 screening of the hip joints,89 followed by MRI if heterotopic ossifications are suspected (Table 8 9 2). 10
11 12 Table 2: Clinical trial outcome measures 13 Parameter Assessments/Measures Timing (see Safety 14 also Figure 5) issue 15 Primary ForGastroduodenal peer Serious adverse review event (SAE) report only Continuous yes 16 Endpoint bleeding observation 17 18 Secondary Adverse events Adverse event monitoring, SAE/SUSAR Continuous yes 19 Endpoints (serious unexpected suspected adverse observation 20 reaction) report 21 Spasticity Modified Ashworth Scale, anti spastic Follow up 1 & 2 yes 22 medication Neuropathic Neuropathic Pain Scale, pain medication Baseline, yes 23 pain Follow up 1 & 2 24 Severity of SCI ASIA impairment scale Baseline, no 25 Follow up 1 & 2 26 Motor function Upper and lower extremity motor score Baseline, no 27 Follow up 1 & 2 28 Sensory Pin prick, Light touch Baseline, no 29 function Follow up 1 & 2 30 Lesion height Motor and sensory level, zone of partial Baseline, no 31 preservation, if applicable Follow up 1 & 2 32 Ibuprofen levels Blood & Cerebrospinal fluid (CSF) Pharmacokineti no 33 collection cs 1, 2, & 3* 34 Serum/CSF Blood & CSF collection Pharmacokineti no http://bmjopen.bmj.com/ 35 protein levels cs 1, 2, & 3* 36 Heterotopic Ultrasound of the hip joints, Magnetic Baseline, yes 37 ossifications resonance imaging, if applicable Follow up 1 & 2
38 Other Laboratory Blood & urine collection Safety 1, 2, 3 & yes 39 Endpoints abnormalities 4* 40 Cardiac Electrocardiography Baseline, yes 41 arrhythmia Follow up 1 & 2
42 Deep vein Ultrasound of pelvic & lower extremity Baseline, yes on October 2, 2021 by guest. Protected copyright. 43 thrombosis veins Follow up 1 & 2 44 Circulatory Blood pressure & heart rate Baseline, Safety yes 45 disturbance 1, 2 46 Clinical Epigastric pain / Pain projected to the Baseline, Safety yes 47 observation shoulder tip 1, 2, & 3* 48 Feasibility of Screening protocol Screening no 49 recruitment Differences between the cohorts are based on the course of an extended intervention. In cohort II 50 additional pharmacokinetic and safety assessments are scheduled (indicated by asterisks). 51
52 53 Data on adverse effects of perioperative NSAIDs on bone healing in terms of 54 90 55 pseudoarthrosis after spinal fusion have been discussed in the past. These data, however, 56 are based different types of NSAIDs and from retrospective cohort studies, the results of 57 91 92 58 which are sometimes conflicting. In this study all spinal surgeries during the follow up 59 period will be documented. In combination with data from routinely performed spinal imaging 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 12 Page 13 of 40 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 procedures relevant impairment of bone healing can be detected and would be documented 4 as SAE. 5 6 7 Enrolment 8 9 In the study center we expect to be screening about 40 60 SCI admitted patients per 10 year, about 6 8 of whom are expected to meet eligibility criteria. The investigators will 11 12 evaluate patient eligibility as soon as possible after admission to the trial center. The 13 14 investigators will conduct an interview with each patient to verify the inclusion and exclusion 15 criteria as relatedFor to individual peer medical historyreview as well as to inform only the patient about the trial 16 17 and its potential risks and benefits. Prior to inclusion, written informed consent will be 18 obtained from the patient. If the patient is willing to consent but is unable to sign, a witness 19 20 independent from the trial team must confirm the verbal informed consent by providing 21 his/her signature. A written announcement of recruitment will be sent out to the sponsor by 22 23 the investigators. 24 25 26 Eligibility criteria 27 The inclusion and exclusion criteria (Table 3) were chosen with regard to scientific, 28 93 29 ethical, and practical considerations specific to SCI. Inclusion in the trial is possible from 30 day four up to day 21 post injury, but should be performed as soon as possible mainly 31 32 dependent of the patient’s ability to give his/her informed consent. Further inclusion criteria 33
are acute traumatic motor complete SCI, classified as AIS A or AIS B, and a neurological http://bmjopen.bmj.com/ 34 35 level of Th4 to C4. Only in this group of patients is a realistic assessment of neurogenic 36 gastrointestinal bleedings possible, because this classification is most likely to be associated 37 38 with an autonomic complete lesion,94 that in the acute stage can cause damage to the 39 69 40 gastroduodenal mucosa. The imbalance between the altered sympathetic outflow through 41 the splanchnic nerve and the intact parasympathetic innervation through the vagus nerve,69
42 on October 2, 2021 by guest. Protected copyright. 43 may increase the ‘baseline’ risk posed by the general post traumatic and ventilation triggered 44 stress response.68 45 46 In order to limit risk to patients, the exclusion criteria comprise all absolute 47 contraindications of the study medication according to the summary of product 48 49 characteristics. The exclusion criteria also include drug interactions or other conditions 50 mandating precaution. To ensure reliable assessment of safety and preliminary efficacy, 51 52 patients with concomitant injury to the CNS, pre existing neurological diseases, or severe 53 psychiatric disorders are excluded from the trial. Other exclusion criteria assure the 54 55 adherence to legal requirements (Table 3). 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 13 BMJ Open Page 14 of 40
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Table 3: Clinical trial eligibility criteria 4 Inclusion criteria 5 6 • Acute SCI of the cervical spine due to trauma 7 • Time frame of 4-21 days post-trauma 8 • Motor complete injury AIS (ASIA impairment scale) A and B 9 • Neurological level of the lesion C4-Th4 10 • No participation in a different clinical trial according to German Medicinal Products Act one month before and during 11 participation in the current trial 12 • The patient has been informed and his/her written consent has been obtained 13 • Age: 18 to 65 years • For women of reproductive age: Negative pregnancy test and highly effective contraception (defined as Pearl Index < 1) 14 or sexual abstinence during participation in the trial. 15 For peer review only 16 Exclusion criteria 17 • Multifocal lesions of the spinal cord 18 • Penetrating spinal cord injury 19 • Accompanying TBI with visible structural lesions including intracranial hemorrhage on diagnostic images 20 • Significant accompanying injury to the peripheral nervous system, particularly plexus lesions 21 • Acute or chronic systemic diseases accompanied by neurological deficits or that have caused permanent neurological 22 deficits which may overlay or hinder the registration of sensomotor functions (e.g. multiple sclerosis, Guillain-Barré 23 syndrome, HIV infection, Lues etc.) 24 • Malignant neoplasms, except if these are in complete remission. 25 • Mental diseases or dementia which, in the investigator's opinion, limit the patient's cooperation in respect of the intake 26 of the study medication and/or significantly hinder the registration of follow-up parameters 27 • Hemophilia 28 • History of myocardial infarction or stroke 29 • Current and persistent misuse of illegal drugs or alcohol 30 • Hypothermia below 35 C° 31 • Pregnancy and lactation 32 • All further contraindications to the study medication, including other ingredients of the pharmaceutical form according to 33 the Summary of Product Characteristics 34 o known hypersensitivity to the active substance ibuprofen or one of the ingredients of the drug http://bmjopen.bmj.com/ o 35 known reactions by way of bronchospasm, asthma, rhinitis or urticaria after the intake of acetylsalicylic 36 acid or other NSAIDs in the past, o unexplained hematopoietic disorders, 37 o peptic ulcers or hemorrhagia: either at the present time or occurred repetitively in the past (at least 2 38 different episodes of proven ulceration or hemorrhage), 39 o gastrointestinal hemorrhage or perforation in the patient's medical history in connection with previous 40 treatment with NSAIDs, 41 o cerebrovascular or other active hemorrhage,
42 o severe disturbance of liver function (with coagulation disorder due to reduced protein synthesis) on October 2, 2021 by guest. Protected copyright. 43 o severe renal function disorder (defined as chronic renal insufficiency, including post-kidney 44 transplantation or acute renal failure, defined as elevated creatinine values and/or oliguria for several 45 days with a limited GFR) 46 o severe myocardial insufficiency (NYHA grade III-IV) o 47 severe dehydration (caused by vomitus, diarrhea or insufficient volume resuscitation). 48 • Known hypersensitivity to the active substance contained in the concomitant medication Pantoprazole or one of the 49 components of the drug. 50 • Intake of ibuprofen or intake of other active substances from the group of NSAIDs; (Non-steroid anti-inflammatory drugs: e.g., diclofenac, indometacin) or the intake of NSAIDs in maximum recommended daily doses during more than one 51 week prior to enrolment in the trial 52 • Simultaneous intake of salicylates, particularly acetylsalicylic acid 53 • Simultaneous intake of oral anticoagulants, or heparinisation in therapeutic dosage 54 • Simultaneous intake of systemic glucocorticoids 55 • Unwilling to consent to storage and transfer of pseudonymized medical data for the purpose of the clinical trial 56 • Admitted to an institution by a court or official order 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 14 Page 15 of 40 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Individual timeline 4 Patient evaluation and inclusion will be performed within 21 days after SCI. The CRF at 5 6 baseline comprises the eligibility criteria, the assessment of injury date and time, medical 7 history, concomitant injury and surgical interventions. Furthermore, the clinical, laboratory, 8 9 and technical safety parameters as well as the ISNCSCI are assessed at baseline (Figure 4, 10 Table 2). 11 12 Since the intervention in cohort II is of longer duration, more frequent safety and 13 14 pharmacokinetic assessments will be performed in this arm during the intervention and 15 follow up (FigureFor 4). In additionpeer to the continuousreview monitoring onlyof AEs, safety data comprise 16 17 laboratory measures and clinical observations will be collected in tightly scheduled safety 18 assessments up to 24 weeks after inclusion. This timeframe seems reasonable for the 19 20 recognition of the major safety endpoints. Further safety issues such as spasticity and 21 neuropathic pain are part of the follow up documentation (Figure 4) that also includes the 22 48 23 neurological endpoints and possible confounders such as co medications or infections. 24 25 26 Overall Duration 27 A recruitment period of 24 months is scheduled. Each patient will be followed up to 24 28 29 weeks post trauma. After completion of recruitment and follow up, a further six month period 30 is planned for clearing the database, the statistical evaluation and preparation of the trial 31 32 report. The trial was activated in June 2013, but not recruiting. After trial registration and 33
completion the recruiting center’s initiation, enrolment started in April 2014. Expected http://bmjopen.bmj.com/ 34 35 enrolment completion date is the second quarter of 2016. Publication of the trial report is 36 scheduled for the year 2017. 37 38 39 40 Sample size estimation 41 The sample size of 12 patients and the analysis strategy are justified by the fact that –
42 on October 2, 2021 by guest. Protected copyright. 43 given that the number of gastrointestinal ulcerations/bleedings after SCI is 3,5% in the first 44 month as reported by Kewalramani 197969 – the probability for the occurrence of more than 45 46 one event is 6,1%. Consequently, observation of more than one event provides evidence of 47 safety problems of Ibuprofen in the indication of acute SCI and probably limits its use in 48 49 subsequent phases of the clinical trial. The occurrence of further bleedings in months two 50 and three or during follow up calls for the same consequences. Nevertheless, based on the 51 52 abovementioned frequency of gastrointestinal ulcerations, the probability for the occurrence 53 of an event is low (0.7% probability) in a sample of 12 SCI patients.69 However, the upper 54 55 bound of the confidence interval for the probability of an event is 38,5%; for zero events it is 56 26.5%. This mandates implementing additional safety criteria if subsequent study phases are 57 58 considered, and a placebo control should be taken into consideration. In our pilot study a 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 15 BMJ Open Page 16 of 40
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 comparison with patients receiving placebo would have a clear lack of statistical power, so 4 no placebo group is scheduled. 5 6 7 Data management 8 9 All study documents including personal identifiers are stored at the recruiting trial centre 10 in locked file cabinets in a room with restricted access. Data are collected on a paper Case 11 12 Report Form (pCRF) and CRFs and all patient data are managed with a six digit pseudonym. 13 14 At the sponsor’s study office the trial coordinators check the pCRFs for completeness and 15 consistency. For Implausible peer or missing data review may be corrected only or added after consulting the 16 17 investigator at the trial site through the sponsor (Queries). The corrected documents will be 18 archived together with the completed CRFs. Data are entered twice to allow double check for 19 20 correctness and are stored electronically in a database (Oracle). Access to the database is 21 restricted, and regular data backups are performed. The principal investigator / sponsor and 22 23 the trial statistician will have full access to the dataset. 24 25 26 Sample handling 27 Peripheral blood and urine samples collected for laboratory safety measures are 28 29 analyzed immediately after sample collection at the central laboratory of the trial center, and 30 the results are available for the study investigators at once. This facilitates the timely 31 32 recognition of AEs. 33
Blood and CSF samples for pharmacological and protein analyses are collected under http://bmjopen.bmj.com/ 34 35 sterile conditions. Those samples are labeled with the six digit pseudonym and any personal 36 information of the participants is removed. All samples are processed for storage as soon as 37 38 possible, at the latest within 8 hours of withdrawal by centrifugation at 3000 g for 10 minutes. 39 40 Serum, heparin plasma and CSF supernatants are stored at the sponsor’s institution 41 at 80°C, with central temperature control up to subsequent batch analysis.
42 on October 2, 2021 by guest. Protected copyright. 43 44 Statistical analysis 45 46 The analysis will be based on the safety population, as this is a pilot study for safety and 47 feasibility designed to enable planning of a subsequent study. The primary analysis is based 48 49 on the incidence of severe gastrointestinal bleedings. If more than one event is observed in 50 the study population (n =12) the principle investigator/sponsor on recommendation of the 51 52 independent Data Monitoring Safety Board will perform a new risk benefit assessment and 53 will decide the interruption or early termination of the trial. Additional safety analyses, mainly 54 55 descriptive and casuistic, will be performed. The descriptive analysis will be according to the 56 scale and distribution of the data, using frequencies and means, medians, quartiles and 57 58 ranges. Linear regression will be used as appropriate. 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 16 Page 17 of 40 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 4 Quality Assurance 5 6 Adherence to i) the recruitment rate, ii) the selection criteria iii) the treatment in 7 accordance with the protocol, and iv) the investigation time points is regarded as a quality 8 9 indicator for the course of the trial. An independent monitor is responsible for reviewing study 10 progress, verifying adherence to the protocol, compliance to ICH/GCP and national 11 12 regulations, and furthermore for handling any problems that arise. The monitor will visit the 13 14 clinical study sites on a regular basis, first after start of enrollment, then after completion of 15 recruitment intoFor cohort I, peerand finally at study review completion. only 16 17 Key study data will be checked in all patients. This pertains to patients’ demographical 18 data, signed informed consent, adherence to inclusion and exclusion criteria, documentation 19 20 on primary objectives, and adverse events. Source data verification will be performed for 21 approximately 25% of the data. Any unclear and/or incomplete data will elicit increased in 22 23 depth monitoring. 24 25 26 Data Monitoring and Safety Board 27 An independent Data Monitoring and Safety Board (DMSB) addresses patient safety and 28 29 performs risk/benefit assessments to ensure that for the patients there is no unavoidable risk 30 or harm. All DMSB members reviewed the trial protocol prior to study activation in order to 31 32 ensure the implementation of safety endpoints and procedures necessary to fulfill the 33
DMSB’s assignment. According with its operating procedures, the DMSB reviews http://bmjopen.bmj.com/ 34 35 accumulating data from the trial to fulfill the safety monitoring. Additionally, the DMSB will 36 assess trial progress, study integrity, and design aspects. The DMSB provides the sponsor 37 38 with recommendations regarding study modification, continuation or termination. The DMSB 39 40 consists of three members: a biostatistician, a neurologist, and an internist, all of whom have 41 practical experience in the work of a DMSB. The DMSB will perform an interim review for
42 on October 2, 2021 by guest. Protected copyright. 43 safety reasons when the entire cohort I has completed week 4 follow up and after completion 44 of enrollment and, if necessary, upon request of the sponsor and/or principal investigator. 45 46 47 Stopping rules 48 49 The discontinuation criteria defined for premature drop out of a patient from the trial 50 include cases of emergency or circumstances associated with increased risk for the 51 52 participant, as well as a patient’s individual wish (Table 4). Patients who have dropped out of 53 the trial prematurely should be examined from the time of discontinuation of treatment 54 55 according to the scheduled program, provided the patient has given his/her consent to such 56 examination. At least the final examination should be performed as far as possible. 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 17 BMJ Open Page 18 of 40
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Decisions on the discontinuation of the entire trial will be taken, if the risk benefit 4 assessment demonstrates unjustifiable risks and toxicities or new scientific conclusions 5 6 during the clinical trial could compromise the safety of the study participants. The decision 7 making body consists of the sponsor and principal investigator and acts, if appropriate, also 8 9 upon recommendation of the DMSB. 10
11 12 Table 4: Clinical trial stopping rules 13 14 Premature drop-out of a patient 15 • GastrointestinalFor ulceration with peer or without hemorrhage review and/or perforation only 16 • A drop in hemoglobin levels below 5 mmol/l consistent after receiving more than 8 red blood cell concentrates 17 • Acute renal failure, defined as an increase in creatinine levels by more than 50% of the baseline value and/or oliguria 18 (urine volume <500 ml/d) persisting for several days after exclusion of extra renal causes 19 • Any hypersensitivity reaction that the investigator attributes to the trial medication 20 • Neurological progression of SCI with ascending paralysis with a loss of more than 2 motor levels 21 • Cerebrovascular hemorrhage 22 • Myocardial infarction or stroke 23 • Any new injury to the spine affecting the spinal cord 24 • The additional intake of more than 1200 mg/d ibuprofen for more than 1 week or the intake of maximal daily doses of 25 other NSAIDs (Non-steroid anti-inflammatory drugs) for more than 2 weeks during the intervention. 26 • The patient’s personal wish 27 • Any other situation which, according to the investigator, would be such that further participation in the clinical not be in the best interests of the patient 28 • The onset of pregnancy 29 • Later occurrence of exclusion criteria. 30 31 32 33 34 http://bmjopen.bmj.com/ 35 36 37 38 39 40 41
42 on October 2, 2021 by guest. Protected copyright. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 18 Page 19 of 40 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 ETHICS AND DISSEMINATION 4 5 The study protocol (protocol version 1.2, date 06.05.2013) was approved by the Ethics 6 Board of the Landesamt für Gesundheit und Soziales (LaGeSo), Berlin, Germany (13/0127 7 8 EK13) and the Federal Institute for Drugs and Medical Devices (BfArM). The protocol 9 amendment (version 2.0, date 12.08.2015) was related to changes of the Summary of 10 11 Product Characteristics of the study medication ibuprofen and on a recent advice of the 12 European Medicines Agency (EMA)95. Two further exclusion criteria i) “severe dehydration” 13 14 and ii) “history of myocardial infarction or stroke” were added to the protocol. The 15 abovementionedFor regulatory peer authorities approvedreview the amendment. only 16 17 Participants will be informed about the trial and its anticipated risks and benefits, orally 18 19 and in written form, using patient information sheets. Patients’ written informed consent will 20 be obtained prior to inclusion. This study complies with the Helsinki Declaration, the 21 22 principles of Good Clinical Practice (GCP), the German Medicinal Products Act (AMG) and 23 the Personal Data Protection Act. The study with the full official title “The Rho Inhibitor 24 25 Ibuprofen for the Treatment of Acute Spinal Cord Injury: Investigation of Safety, Feasibility 26 and Pharmacokinetics” has been registered in the ClinicalTrials.gov database 27 28 (NCT02096913). The registration data is summarized in table 5. 29 30 31 Table 5 32 Data category Information 33
Primary registry and trial ClinicalTrials.gov NCT02096913 http://bmjopen.bmj.com/ 34 identifying number 35 Date of registration in primary 24.03.2014 36 registry 37 Secondary identifying numbers 2011-000584-28 38 Sources of monetary or material Charité Universitätsmedizin Berlin, Else Kröner Fresenius Foundation 39 support 40 Primary sponsor Charitè Universitätsmedizin Berlin, Prof. Jan M. Schwab MD, PhD 41 Contact for public queries Prof. Jan M. Schwab MD, PhD ([email protected]) 42 on October 2, 2021 by guest. Protected copyright. Marcel A. Kopp MD ([email protected]) 43 Contact for scientific queries Prof. Jan M. Schwab MD, PhD ([email protected]) 44 Marcel A. Kopp MD ([email protected]) 45 Public title Safety Study of Ibuprofen to Treat Acute Traumatic Spinal Cord Injury 46 47 Scientific title The Rho-Inhibitor Ibuprofen for the Treatment of Acute Spinal Cord Injury: Investigation of Safety, Feasibility and Pharmacokinetics 48 Countries of recruitment Germany 49 50 Health conditions or problem Spinal Cord Injury 51 studied Interventions Ibuprofen (Dolormin extra), 2400mg/d (400mg 2-2-2) applied orally for 4 weeks (Arm 52 I; n=6) or 12 weeks (Arm II, n=6). 53 54 Key inclusion criteria Acute traumatic SCI; neurologic level C4-T4; AIS A or B; inclusion at day 4 -21 post- 55 injury; no participation in another clinical trial; written consent; age 18-65 years; no 56 pregnancy of female participants during trial conduction 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 19 BMJ Open Page 20 of 40
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Key exclusion criteria Multifocal lesions; penetrating injury; traumatic brain injury (TBI) with visible structural 4 lesions; accompanying injury to the peripheral nervous system (plexus lesions); acute 5 or chronic diseases causing/including neurological deficits; malignant neoplasms; 6 significant mental disease or dementia; hemophilia; history of myocardial infarction / stroke; drug abuse; hypothermia below 35°C; pregnancy/lactation; 7 contraindications/hypersensitivity to study medication; current intake of ibuprofen or 8 other NSAIDs or previous intake of maximum doses one week prior to enrolment; 9 intake of salicylates, systemic glucocorticoids, oral anticoagulants or therapeutic 10 heparinisation; no consent to storage and transfer of trial-based data; admittance to 11 institution by court or official order 12 Study type Interventional; Phase 1; open-label 13 Study activation 20.06.2013 14 First patient in 07.04.2014 15 For peer review only 16 Target sample size 12 17 Recruitment status Recruiting 18 Primary outcomes Severe gastroduodenal bleedings 19 Key secondary outcomes Spasticity; neuropathic pain; ASIA impairment scale; ISNCSCI/ASIA motor and 20 sensory score; documentation of adverse events; plasma and cerebrospinal fluid 21 ibuprofen level; heterotopic ossifications 22 23 24 25 26 Risk benefit assessment 27 In a large number of patients, traumatic SCI signifies a severe lifelong physical disability. 28 29 A standard treatment to promote neuronal plasticity after SCI is not yet available. Based on 30 preclinical investigations in established animal models, a better recovery of neurological 31 32 function in cases of acute SCI is anticipated from making use of ‘small molecule’ Rho 33 inhibition. The systematic review of preclinical data revealed 11 eligible studies on effects of 34 http://bmjopen.bmj.com/ 35 Rho inhibiting NSAIDs with motor function as behavioral endpoint. These studies were 36 conducted in six laboratories and used four different SCI models in three rodent species. The 37 38 meta analysis demonstrated an overall effect size of 20.2%. This is backed up by pervious 39 analyses including studies on specific Rho/ROCK inhibitors that have demonstrated overall 40 41 effect sizes of 21% or 15% after correction for publication bias, respectively.28 Ibuprofen is an
42 on October 2, 2021 by guest. Protected copyright. 43 established, globally approved drug available for clinical investigation of its ability to improve 44 neurological function by Rho inhibition. Furthermore, preventive treatments for inflammation 45 53 58 46 triggered SCI specific complications in terms of neuropathic pain and neurogenic 47 heterotopic ossifications after SCI,59 61, are not well established. Favorable effects on these 48 49 threatening sequelae can be anticipated from ibuprofen treatment by the reduction of COX 50 and NFκB mediated inflammation in the CNS and the peripheral soft tissue. 51 52 The appraised benefits of the intervention have to be weighed against its potential risks, 53 some of which may be serious. Gastrointestinal ulcers accompanied by hemorrhage or by 54 55 perforation are the most prominent side effect of NSAIDs. According to FDA estimates from 56 1987, gastrointestinal hemorrhage due to peptic ulcers or perforation occurred in 1 2% of 57 96 58 patients under sustained three month intake of NSAIDs. The factors that increase the risk 59 of gastrointestinal hemorrhage are: advanced age, high daily doses, a medical history of 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 20 Page 21 of 40 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 97 3 ulcers, simultaneous intake of systemic corticosteroids and the intake of anticoagulants. 4 Within the group of NSAIDs, ibuprofen has a comparatively low gastrointestinal toxicity.97 A 5 6 recent Cochrane database review summarized results from recent clinical trials on long term 7 high dose ibuprofen administered to reduce respiratory complications in cystic fibrosis. The 8 9 studies showed a positive overall benefit risk profile.98 However, a clinical data base analysis 10 comparing 1365 ibuprofen treated patients with 8960 controls demonstrated a low overall risk 11 12 but a higher annual incidence of gastrointestinal bleeding in the ibuprofen group of 0.37% vs. 13 99 14 0.14%. In the acute phase, acute injury to the cervical and upper thoracic segments of the 15 spinal cord isFor probably anpeer additional risk review factor for gastroduodenal only ulceration,69 which is why 16 17 the gastrointestinal safety of ibuprofen treatment in the context of SCI is the primary endpoint 18 of this trial. 19 20 Under normal conditions acute renal failure due to NSAIDs is a rare but serious adverse 21 reaction. The risk for acute renal failure increases in critically ill patients with a volume 22 23 deficiency, myocardial insufficiency, or pre existing renal insufficiency; the same holds true 24 for simultaneous administration of other nephrotoxic substances such as aminoglycosides, 25 100 26 angiotensin converting enzyme inhibitors, and Angiotensin II receptor antagonists. Acute 27 renal failure caused by NSAIDs such as ibuprofen, a substance with a short half life and 28 29 rapid achievement of effective levels, commonly manifests within a few days. After early 30 diagnosis and discontinuation of the treatment, renal function usually returns to normal within 31 32 one week. Only if renal failure is not diagnosed in time may the condition progress rapidly to 33
dependence on dialysis. Compared to other NSAIDs, an intermediate level of nephrotoxicity http://bmjopen.bmj.com/ 34 35 is reported for ibuprofen.101 Acute SCI is generally not associated with a disturbance of renal 36 function. However, due to the traumatic etiology of paraplegia, renal function may be 37 38 transiently limited in some cases due to a volume deficiency or rhabdomyolysis. In those 39 40 cases renal side effects of ibuprofen might be observed more frequently. 41 In order to limit the anticipated risks in the Ibuprofen SCI Safety trial, its exclusion
42 on October 2, 2021 by guest. Protected copyright. 43 criteria comprise known risk factors such as age > 65 years, relevant co morbidities, history 44 of critical events, particularly peptic ulcerations, as well as drug interactions. In addition, the 45 46 trial will be conducted under in hospital conditions of acute care and rehabilitation. In hospital 47 monitoring and carefully scheduled laboratory investigations allow for early awareness of AE 48 49 and their immediate medical treatment. In case that a patient suffers harm from his trial 50 participation, compensation will be covered by a clinical trial specific insurance of the 51 52 sponsor’s institution. After completion of the trial the patients will receive further treatment 53 according to the general principles of long term rehabilitation of SCI and therapy of related 54 55 secondary complications. 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 21 BMJ Open Page 22 of 40
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Limitations 4 Limitations of the clinical trial protocol are its small sample size, the lack of a placebo 5 6 control group and a relatively wide timeframe for inclusion. This design, chosen with regard 7 to the primary safety endpoint and feasibility of the pharmacokinetic issues, restricts efficacy 8 9 evaluation. The time frame of inclusion extended until day 21 after SCI was incorporated for 10 ethical reasons in order to enable the patients giving informed to consent before start of the 11 12 intervention. However, a late start of intervention might diminish therapeutic efficacy because 13 23 14 recovery promoting effects of Rho inhibiton, as well as anti inflammatory effects of 15 ibuprofen,102For depend on thepeer timing of the review intervention, and an onlyearly start of treatment seems 16 17 favorable. 18 The meta analysis of published preclinical experiments is limited by the relative low 19 20 number of studies specific to ibuprofen/Indometacin mediated Rho inhibition, and they thus 21 hardly enable meta regression or adjustment for publication bias. Still, our analysis is in line 22 23 with a larger previous meta analysis that also includes studies on specific Rho/ROCK 24 inhibitors that demonstrated relevant effect sizes after correction for publication bias.28 A 25 26 limitation of the single in vivo experiments on ibuprofen is that they lack dose response 27 curves, and all research groups have applied the drug in comparable dosages. 28 29 Administration of even higher doses would still be within FDA approved range for application 30 in humans and might have larger effects. Confirmative preclinical analyses should therefore 31 32 also consider dose response curves to show functional recovery. 33
http://bmjopen.bmj.com/ 34 35 Possible consequences 36 The explorative safety evaluation, feasibility aspects of recruitment and treatment regime 37 38 in the acute phase after SCI are of interest for the planning of a subsequent randomized 39 40 controlled trial (RCT) in a larger sample. Of particular relevance in the clinical trial are 41 treatment timing and the CNS availability of the systemically delivered compound behind the
42 on October 2, 2021 by guest. Protected copyright. 43 blood spinal cord barrier after acute SCI. An interim bed to bench side translation based on 44 the clinical pharmacological data and preliminary efficacy endpoints could be valuable for 45 46 adjustment of the treatment schedule before embarking on a RCT. 47 Improved neurological recovery anticipated after SCI, which is proposed as the main 48 49 objective of a subsequent RCT might lead to an improvement of aspects of daily living, even 50 if the recovery has affected only two segments of the spinal cord. For example, regaining 51 52 more than one neurological motor level can be considered as a notable difference with 53 influence on physical independence,103 and long term survival.104 Prevention of SCI related 54 55 complications might contribute additionally to improved quality of life. 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 22 Page 23 of 40 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Regardless to the result of the primary and secondary outcome assessments the clinical 4 trial will be reported as publication in a peer reviewed journal compliant with reporting and 5 6 authorship criteria according to the principles of Good Scientific Practice. 7 8 9 10 11 12 13 14 15 For peer review only 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 http://bmjopen.bmj.com/ 35 36 37 38 39 40 41
42 on October 2, 2021 by guest. Protected copyright. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 23 BMJ Open Page 24 of 40
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Authors’ contributions 4 MAK, TL, PM, SMS, JMS designed the trial protocol. TL, RW, PM, SL, CB, RS, GJJ, SK, 5 6 AE, UD, AN reviewed the trial protocol. MAK, RW did the systematic review. RW performed 7 8 the meta analysis. MAK, RW wrote the manuscript. All authors critically revised the 9 manuscript for important intellectual content. All authors have read and approved the final 10 11 version of the manuscript. 12 13 14 Funding statement 15 The projectFor has receivedpeer funding review from the Wings for only Life Spinal Cord Research 16 17 Foundation (Grant Number WfL DE 006/12) and is funded by the Else Kröner Fresenius 18 19 Stiftung (Grant Number EKFS 2012_A32). JMS is supported by the W.E Hunt and C.M. 20 Miller Endowment. The funding agencies had no role in the design and conduct of the study; 21 22 data collection and management; drafting, review, or approval of the manuscript; and 23 decision to submit the manuscript for publication. 24 25 26 Competing interests 27 28 We declare to have no conflicts of interest. 29 30 31 32 33 34 http://bmjopen.bmj.com/ 35 36 37 38 39 40 41
42 on October 2, 2021 by guest. Protected copyright. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 24 Page 25 of 40 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 References 4 5 6 1. Hurlbert RJ, Hadley MN, Walters BC, et al. Pharmacological therapy for acute spinal cord 7 injury. Neurosurgery 2013;72 Suppl 2:93 105. 8 2. Siddiqui AM, Khazaei M, Fehlings MG. Translating mechanisms of neuroprotection, 9 regeneration, and repair to treatment of spinal cord injury. Prog Brain Res 10 2015;218:15 54. 11 3. Dietz V, Curt A. Translating preclinical approaches into human application. Handb Clin 12 Neurol 2012;109:399 409. 13 4. Thuret S, Moon LD, Gage FH. Therapeutic interventions after spinal cord injury. Nat Rev 14 Neurosci 2006;7(8):628 43. 15 5. Yiu G, HeFor Z. Glial inhibition peer of CNS axonreview regeneration. Nat only Rev Neurosci 2006;7(8):617 16 27. 17 6. Xie F, Zheng B. White matter inhibitors in CNS axon regeneration failure. Exp Neurol 18 2008;209(2):302 12. 19 7. Kopp MA, Liebscher T, Niedeggen A, et al. Small molecule induced Rho inhibition: 20 NSAIDs after spinal cord injury. Cell Tissue Res 2012;349(1):119 32. 21 8. Schmandke A, Schmandke A, Strittmatter SM. ROCK and Rho: biochemistry and neuronal 22 functions of Rho associated protein kinases. Neuroscientist 2007;13(5):454 69. 23 9. Baer AS, Syed YA, Kang SU, et al. Myelin mediated inhibition of oligodendrocyte 24 precursor differentiation can be overcome by pharmacological modulation of Fyn 25 RhoA and protein kinase C signalling. Brain 2009;132(Pt 2):465 81. 26 10. Tanaka H, Yamashita T, Yachi K, et al. Cytoplasmic p21(Cip1/WAF1) enhances axonal 27 regeneration and functional recovery after spinal cord injury in rats. Neuroscience 28 2004;127(1):155 64. 29 11. Wang X, Budel S, Baughman K, et al. Ibuprofen Enhances Recovery from Spinal Cord 30 Injury by Limiting Tissue Loss and Stimulating Axonal Growth. Journal of 31 Neurotrauma 2009;26(1):81 95. 32 12. Wu D, Yang P, Zhang X, et al. Targeting a dominant negative rho kinase to neurons 33
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75. Metz GA, Curt A, van de Meent H, et al. Validation of the weight drop contusion model in http://bmjopen.bmj.com/ 34 rats: a comparative study of human spinal cord injury. J Neurotrauma 2000;17(1):1 35 17. 36 76. Lammertse D, Tuszynski MH, Steeves JD, et al. Guidelines for the conduct of clinical 37 trials for spinal cord injury as developed by the ICCP panel: clinical trial design. 38 Spinal Cord 2007;45(3):232 42. 39 40 77. Steeves JD, Lammertse D, Curt A, et al. Guidelines for the conduct of clinical trials for 41 spinal cord injury (SCI) as developed by the ICCP panel: clinical trial outcome measures. Spinal Cord 2007;45(3):206 21. 42 on October 2, 2021 by guest. Protected copyright. 43 78. Geisslinger G, Dietzel K, Bezler H, et al. Therapeutically relevant differences in the 44 pharmacokinetical and pharmaceutical behavior of ibuprofen lysinate as compared to 45 ibuprofen acid. Int J Clin Pharmacol Ther Toxicol 1989;27(7):324 8. 46 79. Seidl RO, Nusser Muller Busch R, Kurzweil M, et al. Dysphagia in acute tetraplegics: a 47 retrospective study. Spinal Cord 2010;48(3):197 201. 48 80. Center for Drug Evaluation and Research. Guidance for industry, estimating the 49 maximum safe starting dose in initial clinical trials for therapeutics in adult health 50 volunteers 2005 [Available from: 51 http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Gui 52 dances/ucm078932.pdf 53 81. Mandula H, Parepally JM, Feng R, et al. Role of site specific binding to plasma albumin 54 in drug availability to brain. J Pharmacol Exp Ther 2006;317(2):667 75. 55 82. Mills RF, Adams SS, Cliffe EE, et al. The metabolism of ibuprofen. Xenobiotica 56 1973;3(9):589 98. 57 83. International Conference on Harmonisation of Technical Requirements for Registration of 58 Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline, Clinical 59 Safety Data Management: D efinitions And Standards For Expedited Reporting E2A 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 28 Page 29 of 40 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 1994 [Available from: 4 https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E2 5 A/Step4/E2A_Guideline.pdf. 6 84. Galer BS, Jensen MP. Development and preliminary validation of a pain measure 7 specific to neuropathic pain: the Neuropathic Pain Scale. Neurology 1997;48(2):332 8 8. 9 85. Jensen MP, Dworkin RH, Gammaitoni AR, et al. Assessment of pain quality in chronic 10 neuropathic and nociceptive pain clinical trials with the Neuropathic Pain Scale. J 11 Pain 2005;6(2):98 106. 12 86. Haas BM, Bergstrom E, Jamous A, et al. The inter rater reliability of the original and of 13 the modified Ashworth scale for the assessment of spasticity in patients with spinal 14 cord injury. Spinal Cord 1996;34(9):560 4. 15 87. BannwarthFor B, Lapicque peer F, Pehourcq review F, et al. Stereoselective only disposition of ibuprofen 16 enantiomers in human cerebrospinal fluid. Br J Clin Pharmacol 1995;40(3):266 9. 17 88. Kirshblum SC, Waring W, Biering Sorensen F, et al. Reference for the 2011 revision of 18 the International Standards for Neurological Classification of Spinal Cord Injury. J 19 Spinal Cord Med 2011;34(6):547 54. 20 89. Krauss H, Maier D, Buhren V, et al. Development of heterotopic ossifications, blood 21 markers and outcome after radiation therapy in spinal cord injured patients. Spinal 22 Cord 2015;53(5):345 8. 23 90. Thaller J, Walker M, Kline AJ, et al. The effect of nonsteroidal anti inflammatory agents 24 on spinal fusion. Orthopedics 2005;28(3):299 303; quiz 04 5. 25 91. Sucato DJ, Lovejoy JF, Agrawal S, et al. Postoperative ketorolac does not predispose to 26 pseudoarthrosis following posterior spinal fusion and instrumentation for adolescent 27 idiopathic scoliosis. Spine (Phila Pa 1976) 2008;33(10):1119 24. 28 92. Lumawig JM, Yamazaki A, Watanabe K. Dose dependent inhibition of diclofenac sodium 29 on posterior lumbar interbody fusion rates. Spine J 2009;9(5):343 9. 30 93. Tuszynski MH, Steeves JD, Fawcett JW, et al. Guidelines for the conduct of clinical trials 31 for spinal cord injury as developed by the ICCP Panel: clinical trial inclusion/exclusion 32 criteria and ethics. Spinal Cord 2007;45(3):222 31. 33
94. Previnaire JG, Soler JM, El Masri W, et al. Assessment of the sympathetic level of lesion http://bmjopen.bmj.com/ 34 in patients with spinal cord injury. Spinal Cord 2009;47(2):122 7. 35 95. European Medicines Agency (EMA). Updated advice on use of high dose ibuprofen 2015 36 [updated 22 May 2015 Available from: 37 http://www.ema.europa.eu/docs/en_GB/document_library/Press_release/2015/05/WC 38 500187108.pdf. 39 40 96. Paulus HE. FDA arthritis advisory committee meeting: risks of agranulocytosis/aplastic 41 anemia, flank pain, and adverse gastrointestinal effects with the use of nonsteroidal antiinflammatory drugs. Arthritis Rheum 1987;30:593 95. 42 on October 2, 2021 by guest. Protected copyright. 43 97. Singh G, Rosen Ramey D. NSAID induced gastrointestinal complications: the ARAMIS 44 perspective 1997. Arthritis, Rheumatism, and Aging Medical Information System. J 45 Rheumatol Suppl 1998;51:8 16. 46 98. Lands LC, Stanojevic S. Oral non steroidal anti inflammatory drug therapy for cystic 47 fibrosis. Cochrane Database Syst Rev 2007(4):CD001505. 48 99. Konstan MW, Hoppel CL, Chai BL, et al. Ibuprofen in children with cystic fibrosis: 49 pharmacokinetics and adverse effects. J Pediatr 1991;118(6):956 64. 50 100. Pannu N, Nadim MK. An overview of drug induced acute kidney injury. Crit Care Med 51 2008;36(4 Suppl):S216 23. 52 101. Whelton A. Nephrotoxicity of nonsteroidal anti inflammatory drugs: physiologic 53 foundations and clinical implications. Am J Med 1999;106(5B):13S 24S. 54 102. Navarro Xavier RA, Newson J, Silveira VL, et al. A new strategy for the identification of 55 novel molecules with targeted proresolution of inflammation properties. J Immunol 56 2010;184(3):1516 25. 57 103. Kramer JL, Lammertse DP, Schubert M, et al. Relationship between motor recovery and 58 independence after sensorimotor complete cervical spinal cord injury. Neurorehabil 59 Neural Repair 2012;26(9):1064 71. 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 29 BMJ Open Page 30 of 40
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 104. Shavelle RM, DeVivo MJ, Brooks JC, et al. Improvements in long term survival after 4 spinal cord injury? Arch Phys Med Rehabil 2015;96(4):645 51. 5 6 7 8 9 10 11 12 13 14 15 For peer review only 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 http://bmjopen.bmj.com/ 35 36 37 38 39 40 41
42 on October 2, 2021 by guest. Protected copyright. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 30 Page 31 of 40 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 Figure Legends 4 5 6 Figure 1: Pharmacological targets of ibuprofen. Intracellular signaling cascades converge 7 8 at the GTPase RhoA, which is activated after SCI by myelin and scar associated proteins (for 9 5 7 29 10 review, ). Downstream to Rho, the activated ROCK inhibits axonal re growth, promotes 11 neurodegeneration, contributes to the development of neuropathic pain and tissue loss, and 12 28 13 impedes neurorestoration and functional recovery (reviewed by Watzlawick et al. ). This 14 pathway can be blocked by the ROCK inhibitors Y 27632 and Fasudil or the specific Rho 15 ForCIP1/WAF1 peer28 review only 11 16 inhibitors P21 , C3 transferase , and by the R( ) and S( ) enantiomers of ibuprofen, 17 17 19 21 31 32, as the most convincing Rho inhibitor among individual drugs from the group of 18 17 19 NSAIDs. Ibuprofen mediated Rho inhibition depends on the upregulation of PPARγ. 20 Treatment with PPARγ agonists was demonstrated to have anti inflammatory effects39 40 and 21 22 to protect tissue and thereby motor function in other CNS injury conditions (reviewed by 23 McTigue,41). It is not yet clear whether the inhibition of NF κB as a further target of R( )/S(+) 24 38 25 ibuprofen, is independent of PPARγ. Notably, PPARγ inhibits gene expression by 26 antagonizing the activities of the pro inflammatory transcription factors NF κB.39 Another 27 28 pathway, mainly operated by the S enantiomer of ibuprofen, is the inhibition of COX 1/2 and 29 consequently of the prostaglandin E production, which activates NF κB or counter regulates 30 2 31 it at very high concentrations.54 COX 1/2 or NF κB are associated with inflammation induced 32 neuropathic pain,53 54, neurodegeneration,50 sickness behavior,43 and the systemic 33 34 inflammatory response syndrome.44 52 Systemic inflammation contributes to neurogenic http://bmjopen.bmj.com/ 35 59 36 heterotopic ossificastions. 37 Taken together, Rho blocking NSAIDs have the potential to decrease the systemic and 38 39 acute CNS inflammatory response by targeting at least two separate pathways, PPARγ and 40 COX 1/2. The suspected side effect of neuroprotective anti inflammatory therapy, that is, that 41 62
42 it further limits the regeneration capacity of spared axons, is suggested to be abrogated by on October 2, 2021 by guest. Protected copyright. 43 Rho Inhibition. Abbreviations: CNS = central nervous system; COX = cyclooxygenase; 44 45 NSAIDs = non steroid anti inflammatory drugs; PPARγ = peroxisome proliferator activated 46 receptor γ; NF κB = nuclear factor κB; ROCK = Rho associated coiled kinase; SCI = spinal 47 48 cord injury. 49 50 51 52 Figure 2: Systematic review preclinical study selection chart. To identify animal 53 54 studies reporting the effect of ibuprofen or indometacin treatment for neurobehavioral 55 recovery after SCI the following search term was used for PubMed, EMBASE, and ISI Web 56 57 of science (search conducted May 18, 2015): (Ibuprofen OR Indometacin OR NSAID OR 58 nonsteroidal anti inflammatory drugs) AND (spinal cord injury OR hemisection OR contusion 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 31 BMJ Open Page 32 of 40
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 OR dorsal column injury OR transection OR corticospinal tract injury OR compression OR 4 spinal cord lesion). Search results were limited to animals. Studies were included if they 5 6 reported the effects of ibuprofen or indometacin in animal models after various types of SCI. 7 We included SCI experiments comparing functional motor outcome between a group of 8 9 animals receiving treatment and a control group receiving no treatment (sham group). Non 10 traumatic models of SCI were excluded, as well as studies reporting only combined 11 12 treatments. Studies had to report the number of animals for each group, the mean effect size 13 14 and its variance. Studies were excluded due to inappropriate outcome scales, combination of 15 treatments andFor statistical peer inconsistencies. review only 16 17 18 19 20 Figure 3: Meta analysis of preclinical effects on motor recovery. Improvement in 21 neurobehavioral score ranked by effect size. The overall number of included animals was 22 23 n=255 (median n=12, range: 8 – 73). Black dots represent studies using Ibuprofen; white 24 dots show Indometacin studies. The horizontal bar represents the 95% CI of the effect size 25 26 (ES). Details on the design of the included studies are summarized in Table 1. 27 28 29 30 Figure 4: Longitudinal clinical trial design. Diagram of frequency and scope of trial 31 32 procedures. The evaluation for eligibility should start as early as possible after acute SCI. 33
The baseline will be obtained at the day of the inclusion from day 4 and latest at day 21 post http://bmjopen.bmj.com/ 34 35 trauma, in any case as early as possible. Start of the study medication is directly after the 36 baseline assessment. The duration of the intervention is 4 weeks for cohort I, and 12 weeks 37 38 for cohort II. Frequent safety laboratory measurements are performed. Samples for 39 40 pharmacokinetic measurements are collected two times in cohort I and three times in cohort 41 II. The follow up visits for determination of secondary endpoints are performed at week 4 (± 3
42 on October 2, 2021 by guest. Protected copyright. 43 days) and after the end of intervention at week 24 (± 14 days). Final safety laboratory 44 measurements will be performed 4 weeks after the end of the study medication. 45 46 Abbreviations: SCI = spinal cord injury. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml 32 Page 33 of 40 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 4 5 6 7 8 9 10 11 12 13 14 15 For peer review only 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Figure 1: Pharmacological targets of ibuprofen. Intracellular signaling cascades converge at the GTPase 30 RhoA, which is activated after SCI by myelin and scar associated proteins (for review,5 7 29). Downstream 31 to Rho, the activated ROCK inhibits axonal re growth, promotes neurodegeneration, contributes to the 32 development of neuropathic pain and tissue loss, and impedes neurorestoration and functional recovery 33 (reviewed by Watzlawick et al.28). This pathway can be blocked by the ROCK inhibitors Y 27632 and Fasudil 34 or the specific Rho inhibitors P21CIP1/WAF1, C3 transferase28, and by the R( ) and S( ) enantiomers of http://bmjopen.bmj.com/ 35 ibuprofen,11 17 19 21 31 32, as the most convincing Rho inhibitor among individual drugs from the group of NSAIDs. Ibuprofen mediated Rho inhibition depends on the upregulation of PPARγ.17 Treatment with 36 PPARγ agonists was demonstrated to have anti inflammatory effects39 40 and to protect tissue and thereby 37 motor function in other CNS injury conditions (reviewed by McTigue,41). It is not yet clear whether the 38 inhibition of NF κB as a further target of R( )/S(+) ibuprofen,38 is independent of PPARγ. Notably, PPARγ 39 inhibits gene expression by antagonizing the activities of the pro inflammatory transcription factors NF 40 κ�B.39 Another pathway, mainly operated by the S enantiomer of ibuprofen, is the inhibition of COX 1/2 41 and consequently of the prostaglandin E2 production, which activates NF κB or counter regulates it at very high concentrations.54 COX 1/2 or NF κB are associated with inflammation induced neuropathic pain,53 54, 42 on October 2, 2021 by guest. Protected copyright. 43 neurodegeneration,50 sickness behavior,43 and the systemic inflammatory response syndrome.44 52 Systemic inflammation contributes to neurogenic heterotopic ossificastions.59 44 Taken together, Rho blocking NSAIDs have the potential to decrease the systemic and acute CNS 45 inflammatory response by targeting at least two separate pathways, PPARγ and COX 1/2. The suspected 46 side effect of neuroprotective anti inflammatory therapy, that is, that it further limits the regeneration 47 capacity of spared axons,62 is suggested to be abrogated by Rho Inhibition. Abbreviations: CNS = central 48 nervous system; COX = cyclooxygenase; NSAIDs = non steroid anti inflammatory drugs; PPARγ = 49 peroxisome proliferator activated receptor γ; NF κB = nuclear factor κB; ROCK = Rho associated coiled 50 kinase; SCI = spinal cord injury.
51 345x227mm (300 x 300 DPI) 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 34 of 40
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 4 5 6 7 8 9 10 11 12 13 14 15 For peer review only 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 http://bmjopen.bmj.com/ 35 36 37 38 39 Figure 2: Systematic review preclinical study selection chart. To identify animal studies reporting the effect of ibuprofen or indometacin treatment for neurobehavioral recovery after SCI the following search term was 40 used for PubMed, EMBASE, and ISI Web of science (search conducted May 18, 2015): (Ibuprofen OR 41 Indometacin OR NSAID OR nonsteroidal anti-inflammatory drugs) AND (spinal cord injury OR hemisection 42 OR contusion OR dorsal column injury OR transection OR corticospinal tract injury OR compression OR spinal on October 2, 2021 by guest. Protected copyright. 43 cord lesion). Search results were limited to animals. Studies were included if they reported the effects of 44 ibuprofen or indometacin in animal models after various types of SCI. We included SCI experiments 45 comparing functional motor outcome between a group of animals receiving treatment and a control group 46 receiving no treatment (sham group). Non-traumatic models of SCI were excluded, as well as studies 47 reporting only combined treatments. Studies had to report the number of animals for each group, the mean effect size and its variance. Studies were excluded due to inappropriate outcome scales, combination of 48 treatments and statistical inconsistencies. 49 102x97mm (300 x 300 DPI) 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 35 of 40 BMJ Open
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 4 5 6 7 8 9 10 11 12 13 14 15 For peer review only 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Figure 3: Meta analysis of preclinical effects on motor recovery. Improvement in neurobehavioral score 33 ranked by effect size. The overall number of included animals was n=255 (median n=12, range: 8 – 73). 34 Black dots represent studies using Ibuprofen; white dots show Indometacin studies. The horizontal bar http://bmjopen.bmj.com/ 35 represents the 95% CI of the effect size (ES). Details on the design of the included studies are summarized 36 in Table 1. 420x313mm (72 x 72 DPI) 37 38 39 40 41
42 on October 2, 2021 by guest. Protected copyright. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 36 of 40
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 2 3 4 5 6 7 8 9 10 11 12 13 14 15 For peer review only 16 17 18 19 20 21 22 23 Figure 4: Longitudinal clinical trial design. Diagram of frequency and scope of trial procedures. The 24 evaluation for eligibility should start as early as possible after acute SCI. The baseline will be obtained at the 25 day of the inclusion from day 4 and latest at day 21 post-trauma, in any case as early as possible. Start of the study medication is directly after the baseline assessment. The duration of the intervention is 4 weeks 26 for cohort I, and 12 weeks for cohort II. Frequent safety laboratory measurements are performed. Samples 27 for pharmacokinetic measurements are collected two times in cohort I and three times in cohort II. The 28 follow-up visits for determination of secondary endpoints are performed at week 4 (± 3 days) and after the 29 end of intervention at week 24 (± 14 days). Final safety laboratory measurements will be performed 4 30 weeks after the end of the study medication. Abbreviations: SCI = spinal cord injury. 31 135x62mm (300 x 300 DPI) 32 33 34 http://bmjopen.bmj.com/ 35 36 37 38 39 40 41
42 on October 2, 2021 by guest. Protected copyright. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml
1 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 3______3______10______10______24______10 Addressed on on Addressed number page 19______19______24______24______24______and 2
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Protocol version version Protocol Funding 3 identifier version and Date Roles and and Roles responsibilities Title Title Trial registration registration Trial 2a registry intended of name registered, yet Ifname. not registry and identifier Trial Administrative information Administrative documents* related and protocol trial clinical a in address to items Recommended Checklist: 2013 SPIRIT Section/item Item Page 37 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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5 to 9______9______to 5 n.a.______7______ins and washouts), assessments, and visits for visits and assessments, washouts), and ins BMJ Open http://bmjopen.bmj.com/ on October 2, 2021 by guest. Protected copyright. For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml For peer review only be collected. Reference to where list of study sites can be obtained obtained be can sites ofstudy list where to Reference collected. be allocation ratio, and framework (eg, superiority, equivalence, noninferiority, exploratory) exploratory) noninferiority, equivalence, superiority, (eg, framework and ratio, allocation participants. A schematic diagram is highly recommended (see Figure) Figure) (see recommended highly is diagram schematic A participants. Time schedule of enrolment, interventions (including any run- any (including interventions enrolment, of schedule Time efficacy and harm outcomes is strongly recommended recommended strongly is andharm outcomes efficacy median, proportion), and time point for each outcome. Explanation of the clinical relevance of chosen chosen of relevance clinical of the Explanation foroutcome. each point timeand proportion), median, pressure), analysis metric (eg, change from baseline, final value, time to event), method of aggregation (eg, aggregation methodof event), to time value, final baseline, from change (eg, metric analysis pressure), Primary, secondary, and other outcomes, including the specific measurement variable (eg, systolic blood blood (eg,systolic variable measurement specific the including outcomes, other and secondary, Primary, Relevant concomitant care and interventions that are permitted or prohibited during the trial trial the during prohibited or permitted are that interventions and care concomitant Relevant (eg, drug tablet return, laboratory tests) tests) laboratory return, tablet drug (eg, Strategies to improve adherence to intervention protocols, and any procedures for monitoring adherence adherence monitoring for procedures any and protocols, intervention to adherence improve to Strategies change in response to harms, participant request, or improving/worsening disease) disease) improving/worsening or request, participant harms, to response in change Criteria for discontinuing or modifying allocated interventions for a given trial participant (eg, drug dose drug (eg, participant trial given for a interventions allocated modifying or fordiscontinuing Criteria administered administered Interventions for each group with sufficient detail to allow replication, including how and when they will be be they will and when how including replication, allow to detail sufficient with group each for Interventions individuals who will perform the interventions (eg, surgeons, psychotherapists) psychotherapists) surgeons, (eg, interventions the perform will who individuals Inclusion and exclusion criteria for participants. If applicable, eligibility criteria for study centres and centres and criteriastudy for eligibility applicable, If for participants. criteria exclusion and Inclusion studies (published and unpublished) examining benefits and harms for each intervention intervention each for harms and benefits examining unpublished) and (published studies 13 13 12 12 11d 11d 11c 11c 11b 11b 11a 11a 10 10
6a 6a of relevant summary including trial, the forundertaking justification and question of research Description Participant timeline timeline Participant Outcomes Outcomes Interventions Interventions Eligibility criteria criteria Eligibility Study setting Study 9 will data where ofcountries list and hospital) academic clinic, community (eg, settings of study Description Methods: Participants, interventions, and outcomes outcomes and interventions, Participants, Methods: Introduction Introduction Background and Background rationale Objectives design Trial 7 8 hypotheses or 6b objectives Specific group), single factorial, crossover, group, parallel (eg, oftrial type including design of trial Description comparators of forchoice Explanation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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n.a.______n.a.______n.a.______n.a.______n.a.______n.a.______n.a.______n.a.______n.a.______n.a.______
13______13______15 to 16_____ 16_____ to 15 BMJ Open http://bmjopen.bmj.com/
on October 2, 2021 by guest. Protected copyright. For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml For peer review only collected for participants who discontinue or deviate from intervention protocols protocols from intervention deviate or discontinue who forparticipants collected Plans to promote participant retention and complete follow-up, including list of any outcome data to to be data outcome of list any including follow-up, complete and retention participant promote to Plans Reference to where data collection forms can be found, if not in the protocol protocol inthe ifnot found, be can forms collection data where to Reference study instruments (eg, questionnaires, laboratory tests) along with their reliability and validity, if known. if known. validity, and reliability their with along tests) laboratory questionnaires, (eg, instruments study processes to promote data quality (eg, duplicate measurements, training of assessors) and a description of description a and assessors) of training measurements, duplicate (eg, quality data promote to processes Plans for assessment and collection of outcome, baseline, and other trial data, including any related related any including data, other trial and baseline, ofoutcome, collection and forassessment Plans allocated intervention during the the trial during intervention allocated If blinded, circumstances under which unblinding is permissible, and procedure for revealing a participant’s participant’s a forrevealing procedure and is permissible, unblinding which under circumstances blinded, If assessors, data analysts), and how how and analysts), data assessors, Who will be blinded after assignment to interventions (eg, trial participants, care providers, outcome outcome careproviders, participants, trial (eg, interventions to afterassignment blinded be will Who interventions interventions Who will generate the allocation sequence, who will enrol participants, and who will assign participants to to participants assign will andwho participants, enrol will who sequence, allocation the generate will Who opaque, sealed envelopes), describing any steps to conceal the sequence until interventions are assigned assigned are interventions until sequence the conceal to steps any describing envelopes), sealed opaque, Mechanism of implementing the allocation sequence (eg, central telephone; sequentially numbered, numbered, sequentially telephone; central (eg, sequence allocation the implementing of Mechanism or assign interventions interventions assign or (eg, blocking) should be provided in a separate document that is unavailable to those who enrol participants participants enrol who those to unavailable is that document separate a in provided be should blocking) (eg, factors for stratification. To reduce predictability of a random sequence, details of any planned restriction restriction planned ofany details sequence, random a of predictability reduce To forstratification. factors Method of generating the allocation sequence (eg, computer-generated random numbers), and list of any of list any and numbers), random computer-generated (eg, sequence allocation the of generating Method
Strategies for achieving adequate participant enrolment to reach target sample size size sample target reach to enrolment participant adequate forachieving Strategies clinical and statistical assumptions supporting any sample size calculations calculations samplesize any supporting assumptions statistical and clinical Estimated number of participants needed to achieve study objectives and how it was determined, including including determined, it was how and objectives study achieve to needed participants of number Estimated 18b 18b 18a 18a 17b 17b 17a 17a 16c 16c 16b 16b 16a 16a
15 15 14 14 Implementation Implementation mechanism mechanism concealment concealment generation generation Sequence Sequence Allocation Allocation
methods methods Data collection collection Data Methods: Data collection, management, and analysis and management, collection, Data Methods:
Blinding (masking) (masking) Blinding Methods: Assignment of interventions (for controlled trials) controlled (for interventions of Assignment Methods: Recruitment Recruitment Sample size size Sample
Allocation: Allocation: Page 39 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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19______19______BMJ Open http://bmjopen.bmj.com/ on October 2, 2021 by guest. Protected copyright. For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml For peer review only analyses) to relevant parties (eg, investigators, REC/IRBs, trial participants, trial registries, journals, journals, registries, trial participants, trial REC/IRBs, investigators, (eg, parties relevant to analyses) regulators) from investigators and the sponsor sponsor the and frominvestigators Frequency and procedures for auditing trial conduct, if any, and whether the process will be independent independent be will process the and whether if any, conduct, trial forauditing procedures and Frequency events and other unintended effects of trial interventions or trial conduct conduct trial or interventions trial of effects unintended other and events Plans for collecting, assessing, reporting, and managing solicited and spontaneously reported adverse reported spontaneously and solicited managing and reporting, assessing, collecting, for Plans results and make the final decision to terminate the trial trial the terminate to decision final makethe and results Description of any interim analyses and stopping guidelines, including who will have access to these interim access to have will who including guidelines, stopping and analyses interim of any Description needed needed about its charter can be found, if not in the protocol. Alternatively, an explanation of why a DMC is not is not DMC a ofwhy explanation an Alternatively, theprotocol. in ifnot found, be can itscharter about whether it is independent from the sponsor and competing interests; and reference to where further details furtherdetails where to reference and interests; competing and sponsor from the isindependent it whether Composition of data monitoring committee (DMC); summary of its role and reporting structure; statement of statement structure; reporting and role ofits summary (DMC); committee monitoring data of Composition statistical methods to handle missing data (eg, multiple imputation) imputation) multiple (eg, data missing handle to methods statistical Definition of analysis population relating to protocol non-adherence (eg, as randomised analysis), and any any and analysis), randomised as (eg, non-adherence protocol to relating population ofanalysis Definition Methods for any additional analyses (eg, subgroup and adjusted analyses) analyses) adjusted and subgroup (eg, analyses additional any for Methods statistical analysis plan can be found, if not in the protocol protocol the in iffound,not be can plan analysis statistical Statistical methods for analysing primary and secondary outcomes. Reference to where other details of the the of details other where to Reference outcomes. secondary and primary foranalysing methods Statistical procedures can be found, if not in the protocol protocol the in if not found, be can procedures (eg, double data entry; range checks for data values). Reference to where details of data management management data of details where to Reference values). data for checks range entry; data double (eg, Plans for data entry, coding, security, and storage, including any related processes to promote data quality quality data promote to processes related any including storage, and security, coding, entry, fordata Plans
23 23 22 22 21b 21b 21a 21a 20c 20c 20b 20b 20a 20a 19 19 24 24 approval (REC/IRB) board review committee/institutional ethics research seeking for Plans 25 25 outcomes, criteria, eligibility to changes (eg, modifications protocol important communicating for Plans Ethics and dissemination dissemination and Ethics Research ethics ethics Research approval Protocol Protocol amendments Harms Harms
Data monitoring monitoring Data Methods: Monitoring Methods:
Statistical methods methods Statistical Data management management Data
Auditing Auditing 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 5 BMJ Open: first published as 10.1136/bmjopen-2015-010651 on 26 July 2016. Downloaded from 21 ______21 16______16______13______13______n.a.______n.a.______16______16______separate files files separate 10 and 16 _ 16 and 10 _ _
22 to 23_____ 23_____ to 22 23______23______BMJ Open http://bmjopen.bmj.com/ ” license. license. ” on October 2, 2021 by guest. Protected copyright. For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml For peer review only how (see Item 32) 32) (seeItem how studies, if applicable if applicable studies, in order to protect confidentiality before, during, and after the trial trial afterthe and during, before, confidentiality protect to order in participation participation Provisions, if any, for ancillary and post-trial care, and for compensation to those who suffer harm from trial trial from harmsuffer who those to compensation for care,and post-trial and for ancillary ifany, Provisions, limit such access for investigators investigators for access such limit Statement of who will have access to the final trial dataset, and disclosure of contractual agreements that that agreements contractual of disclosure and dataset, trial final the to access have will of who Statement analysis in the current trial and for future use in ancillary studies, if applicable ifapplicable studies, ancillary in use future for and trial current the in analysis the public, and other relevant groups (eg, via publication, reporting in results databases, or other data data other or databases, results in reporting publication, via groups(eg, relevant other and public, the restrictions publication any including arrangements), sharing 31c 31c code andstatistical dataset, participant-level protocol, full the to access public for granting any, if Plans, n.a.______31b 31b writers professional of use intended any and guidelines eligibility Authorship 28 28 site study each and trial overall forthe investigators principal for interests competing other and Financial 24______33 33 molecular or forgenetic specimens biological of storage and evaluation, laboratory collection, for Plans
32 32 surrogates authorised and participants to given documentation related other formand consent Model as Attached 31a 31a professionals, healthcare participants, to results trial communicate sponsorto and forinvestigators Plans 30 30 29 29
Attribution-NonCommercial-NoDerivs 3.0 Unported 3.0 Attribution-NonCommercial-NoDerivs 26b ancillary in specimens and biological data ofuseparticipant and collection for provisions consent Additional Confidentiality Confidentiality 27 maintained and shared, be collected, will participants andenrolled potential about information personal How Declaration of Declaration interests Biological Biological specimens Appendices Appendices Informed consent consent Informed materials
*It is strongly recommended that this checklist be read in conjunction with the SPIRIT 2013 Explanation & Elaboration for important clarification on the items. items. the on clarification for important Elaboration & Explanation 2013 SPIRIT the with conjunction in read be thischecklist that recommended is strongly *It “ Dissemination policy Dissemination trial care trial Consent or assent assent or Consent 26a and surrogates, authorised or participants trial potential from assent or consent informed obtain will Who Amendments to the protocol should be tracked and dated. The SPIRIT checklist is copyrighted by the SPIRIT Group under the Creative Commons Commons Creative the under Group SPIRIT by the iscopyrighted checklist SPIRIT The dated. and tracked be should protocol the to Amendments Ancillary and post- and Ancillary Access to data data to Access Page 41 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60