For Peer Review Only Journal: BMJ Open

BMJ Open BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

Repeat infusion of autologous bone marrow cells in multiple sclerosis – protocol for a phase I extension study (SIAMMS- II)

For peer review only Journal: BMJ Open

Manuscript ID: bmjopen-2015-009090

Article Type: Protocol

Date Submitted by the Author: 15-Jun-2015

Complete List of Authors: Rice, Claire; University of Bristol, School of Clinical Sciences Marks, David; Bristol Royal Hospital for Children, Adult BMT Unit; University of Bristol, School of Clinical Sciences Walsh, Peter; North Bristol NHS Trust, Neurosciences Kane, Nick; North Bristol NHS Trust, Neurosciences Guttridge, Martin; NHS Blood and Transplant, Redondo, Juliana; University of Bristol, School of Clinical Sciences Sarkar, Pamela; University of Bristol, School of Clinical Sciences; North Bristol NHS Trust, Neurosciences Owen, Denise; North Bristol NHS Trust, Neurosciences Wilkins, Alastair; University of Bristol, School of Clinical Sciences; North Bristol NHS Trust, Neurosciences Scolding, Neil; University of Bristol Institute of Clinical Neurosciences,

Neurology http://bmjopen.bmj.com/

Primary Subject Neurology Heading:

Secondary Subject Heading: Haematology (incl blood transfusion)

Multiple sclerosis < NEUROLOGY, IMMUNOLOGY, Bone marrow Keywords: transplantation < HAEMATOLOGY

on September 30, 2021 by guest. Protected copyright.

For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 1 of 11 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 Repeat infusion of autologous bone marrow cells in multiple sclerosis – a 4 phase I extension study (SIAMMS-II) 5 6 7 8 Claire M. Rice1,2*, David I. Marks3, Peter Walsh2, Nick M. Kane2, Martin G. 9 4 1 1,2 2 1,2 10 Guttridge , Juliana Redondo , Pamela Sarkar , Denise Owen , Alastair Wilkins , 11 and Neil J. Scolding1,2 12 13 14 15 For peer review only 16 1School of Clinical 3Adult BMT Unit 4NHS Blood and 17 Sciences, Bristol Royal Hospital Transplant 18 19 University of Bristol, for Children Filton, 20 Southmead Hospital, University Hospitals North Bristol Park 21 Bristol, BS10 5NB, UK Bristol NHS Foundation Northway, Filton, 22 Trust & University of Bristol, BS34 7QH, UK 23 2Bristol Institute of Bristol, 24 Clinical Neurosciences, St Michael's Hill, 25 Southmead Hospital, Bristol, 26 Bristol, BS16 1JB, UK BS2 8BJ, UK 27 28 29 30 31 32 33

34 http://bmjopen.bmj.com/ 35 * Correspondence: [email protected] 36 37 38 39 Keywords: cell therapy 40 bone marrow 41 multiple sclerosis 42 reparative therapy 43 on September 30, 2021 by guest. Protected copyright. 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 11 BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 Abstract 4 5 Introduction: The ‘Study of Intravenous Autologous Marrow in Multiple Sclerosis 6 (SIAMMS)’ trial was a safety and feasibility study which examined the effect of 7 8 intravenous infusion of autologous bone marrow without myeloablative therapy. This 9 trial was welltolerated and improvement was noted in the global evoked potential 10 (GEP) a neurophysiological secondary outcome measure recording speed of 11 conduction in central nervous system pathways. The efficacy of intravenous delivery 12 of autologous marrow in progressive MS will be examined in the phase II study the 13 ‘Assessment of Bone MarrowDerived Cellular Therapy in Progressive Multiple 14 Sclerosis (ACTiMuS; NCT01815632)’. In parallel with the ‘ACTiMuS’ study, the 15 current studyFor ‘SIAMMSII’ peer will explore review the feasibility of repeated,only nonmyeloablative 16 autologous bone marrowderived cell therapy in progressive MS. Furthermore, 17 18 information will be obtained regarding the persistence or otherwise of improvements 19 in conduction in central nervous system pathways observed in the original ‘SIAMMS’ 20 study and whether these can be reproduced or augmented by a second infusion of 21 autologous bone marrowderived cells. 22 23 24 Methods and analysis: An open, prospective, single centre phase I extension study. 25 The 6 patients with progressive MS who participated in the ‘SIAMMS’ study will be 26 invited to undergo repeat bone marrow harvest and receive an intravenous infusion 27 28 of autologous, unfractionated bone marrow as a daycase procedure. The primary 29 outcome measure is the number of adverse events and secondary outcome 30 measures will include change in clinical rating scales of disability, global evoked 31 potential and cranial MRI. 32 33

34 Ethics and dissemination: The study has UK National Research Ethics Committee http://bmjopen.bmj.com/ 35 approval (13/SW/0255). Study results will be disseminated via peerreviewed 36 publications and conference presentations. 37 38 39 40 Trial Registration: ClinicalTrials.gov registry NCT01932593 41 42 on September 30, 2021 by guest. Protected copyright. 43 44 Strengths and limitations of this study 45 46 47 Strengths: 48 Regulated clinical trial of cell therapy for progressive MS 49 Extension data for phase I clinical trial 50 51 Limitations: 52 Open label trial 53 Small sample size 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 3 of 11 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 Introduction 4 5 Although effective treatments for relapsing remitting multiple sclerosis (MS) are 6 available, there are no proven therapies available to halt or reverse the progressive 7 8 phase of the disease which ultimately affects the majority of people with MS. There is 9 preclinical evidence to support a reparative role for bone marrowderived cells in 10 demyelinating disease and, following on from this, we have begun to explore the 11 potential of autologous, unselected bone marrow cells for repair in progressive MS. 12 ‘SIAMMS’ was a safety and feasibility study of intravenous autologous bone marrow 13 (BM) infusion in patients with progressive MS 1. This study was well tolerated and 14 also raised the possibility of partial repair; conduction times in multiple central 15 nervous systemFor (CNS) peer pathways collated review as a composite onlyscore (GEP) 2 3 improved in 16 all patients studied (n=6) 1. A randomised, placebocontrolled trial will determine 17 whether autologous bone marrow infusion exerts genuine reparative effects in 18 4 19 progressive MS (‘ACTiMuS’; NCT01815632) but the purpose of ‘SIAMMSII’ is to 20 explore whether the improvements observed in the initial study performed over 5 21 years ago have persisted and whether these can be repeated or augmented. 22 23 24 Methods and design 25 26 27 Objective and hypothesis 28 29 Our hypothesis is that intravenouslydelivered autologous bone marrow cell therapy 30 in chronic MS has reparative properties. We postulate that bone marrowderived cells 31 contribute to repair within the CNS via a multiplicity of mechanisms including 32 immunomodulation and reparative and/or neuroprotective effects. Furthering our 33 understanding of these processes will enable development and refinement of cell

34 therapy for progressive MS. http://bmjopen.bmj.com/ 35 36 37 The phase II ‘ACTiMuS’ trial will explore the efficacy of intravenous infusion of 38 autologous bone marrowderived cell therapy in progressive MS and its laboratory 39 arm will explore the underlying mechanisms of any observed effect. ‘SIAMMSII’ will 40 run in parallel with ‘ACTiMuS’ and will investigate whether the previously observed 41 effects can be replicated and/or augmented. 42

on September 30, 2021 by guest. Protected copyright. 43 Trial design 44 45 46 ‘SIAMMSII’ is an open, prospective, single centre, safety and feasibility extension 47 study. The study schema is presented in figure 1. The study has UK National 48 Research Ethics Committee approval (13/SW/0255). 49 50 Sample size, eligibility & enrolment 51 52 The study is limited to the 6 people who participated in the original ‘SIAMMS’ study, 53 all of whom are under active follow up at the Bristol and Avon Multiple Sclerosis 54 (BrAMS) Unit, North Bristol NHS Trust, Bristol, UK. All participants have progressive 55 56 MS and must fulfil the inclusion and exclusion criteria as detailed in table 1. 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 4 of 11 BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 Trial interventions 4 5 Participants will have a bone marrow harvest and reinfusion of autologous marrow 6 as a daycase procedure. A short general anaesthesic will be given for the bone 7 marrow harvest which will be taken from the posterior iliac crests. Approximately 8 600ml marrow will be collected together with a single bone marrow trephine. The 9 10 marrow aspirate will be processed by NHSBT (filtered, bagged and labelled) prior to 11 intravenous infusion. 12 13 Assuming specific written informed consent is granted, a bone marrow trephine and a 14 small sample of the bone marrow aspirate will be retained for research. Additional 15 blood samplesFor for research peer purposes review may be requested only throughout the duration of 16 the study. 17 18 Outcome Measures 19 20 21 Primary outcome measure 22 23 The primary outcome measure is the number of adverse events (AE). For the 24 purposes of the study, an AE is defined as any unfavourable and unintended sign, 25 symptom or illness that develops or worsens during the period of the study. This is 26 irrespective of the likelihood that the AE is related to study interventions. AEs may be 27 expected or unexpected and include unwanted side effects, toxicity or sensitivity 28 reactions, as well as abnormal laboratory results, injury or intercurrent illnesses. 29 30 31 A serious adverse event (SAE) is defined as an AE which results in death, is life 32 threatening or requires hospitalisation or prolongation of inpatient stay or which 33 results in persistent or significant disability or incapacity. Any congenital anomaly or

34 birth defect or any event considered to be a medical event of importance will be also http://bmjopen.bmj.com/ 35 be classified as a SAE. All SAEs must be reported to the trial coordinating centre as 36 soon as possible. Those hospital admissions that were planned prior to trial entry will 37 not be recorded as SAEs. 38

39 4 40 As per the ‘ACTiMuS’ trial, expected AEs include: 41 42 Local bruising and discomfort following bone marrow harvest 43 Increase in lower limb spasticity following bone marrow harvest on September 30, 2021 by guest. Protected copyright. 44 Acute urinary retention following bone marrow harvest 45 Temporary exacerbation of MS following general anaesthesia 46 Hypovolaemia or anaemia following blood and marrow donation 47 Exacerbation of MS due to sepsis e.g. urinary tract infection or chest infection 48 Assessment at or admission to hospital following fall 49 50 51 Bloods taken for safety analyses will be screened as follows: urea and electrolytes, 52 liver function tests, full blood count with differential white cell count, coagulation, 53 group and save, Creactive protein, glucose, calcium, magnesium, chloride, 54 bicarbonate, phosphate, viral serology (including cytomegalovirus, EpsteinBarr virus, 55 herpes simplex virus, varicella zoster virus, toxoplasmosis, Hepatitis B&C, human 56 immunodeficiency virus, human T cell lymphoma virus and syphilis screening. 57 Urinalysis (microscopy and culture) will also be performed. 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 5 of 11 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 4 Secondary outcome measures 5 6 Change in clinical measures of disability, GEP and cranial MRI findings are included 7 as secondary outcome measures. 8

9 Clinical outcomes will be assessed at entry and at 6 months and 1 year. The clinical 10 5 11 rating scales will include the widely used Expanded Disability Status Scale (EDSS) 6 12 together with the Multiple Sclerosis Functional Composite (MSFC) . The latter is a 13 threepart quantitative assessment including a timed walk, ninehole peg test and 14 Paced Auditory Serial Addition Test (PASAT). In addition, participants will be asked 15 to completeFor the MS peer Impact Scale review (MSIS29) which isonly a wellvalidated patient 16 completed rating scale 710. The measures taken for clinical secondary outcome 17 measures will be: 18

19 20 1. Physicianbased EDSS: time to EDSS progression of at least one point 21 from a baseline EDSS of 4.0, 4.5 or 5.0 or at least 0.5 points from a 22 baseline EDSS ≥5.5. 23 2. Patientbased MSIS29 physical impact scale version 2: overall mean 24 change from baseline to end of study 25 3. MSFC: overall mean change of zscores, from baseline to final visit 26 27 Multimodal evoked potentials will be examined at 0, 6 and 12 months. Evoked 28 potential abnormalities will be quantified according to a 4point graded ordinal score 29 modified from Leocani et al (0=normal; 1=increased latency; 2=increased latency and 30 3 31 abnormal amplitude; 3=absent) and the composite GEP score calculated . 32 33 The recording of the evoked potentials shall be in accordance with the Guidelines of 11

34 the International Federation of Clinical Neurophysiology and analysis will be http://bmjopen.bmj.com/ 35 performed using standard methods 12 (box 1). Electrophysiological responses shall 36 be considered abnormal if they exceed 2.5 standard deviations of the normal values 37 or cannot be detected. 38

39 40 Participants will undergo cranial MRI at entry and at 6 months after bone marrow 41 infusion. The secondary MRI outcome measures will relate to lesion load and atrophy 42 measures of the brain. 43 on September 30, 2021 by guest. Protected copyright. 44 Annual subjective patient and treating physician assessments of efficacy will also be 45 recorded. 46 47 Trial status 48

49 50 ‘SIAMMSII’ opened to recruitment in March 2014 and is ongoing. 51 52 53 Analysis 54 55 A full statistical analysis plan will be written prior to data collection. The null 56 hypothesis is that there will be no significant difference in the primary and secondary 57 58 outcomes between intervention and control arms at 12 months. 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 6 of 11 BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 4 Secondary outcomes will be scored according to standard methodology but the 5 limitations of the small sample size are acknowledged. 6 7 8 9 Conclusion 10 11 On the background of extensive preclinical studies and anticipated low risk of 12 significant harm, we commenced a phase I trial intravenous delivery of filtered but 13 otherwise unmodified autologous bone marrow in 2006. The successful completion of 14 this early trial and the suggestion that electrophysiological improvement may have 15 occurred, 1 Formade further peer exploration reviewof the reparative potential only of autologous marrow 16 in MS mandatory. We have begun to assess the efficacy of this approach in the 17 4 18 randomised, doubleblind ‘ACTiMuS’ trial. However. ‘SIAMMSII’ will give some 19 preliminary information about the value of retreating progressive MS with repeat 20 infusion of autologous bone marrow. 21 22 A number of clinical trials are now beginning to explore the safety and therapeutic 23 effectiveness of bone marrowderived cell therapy for MS using specific sub 24 populations of bone marrow cells such as multipotent mesenchymal stromal cells 25 (reviewed in 13). We have set out our rationale for using unfractionated marrow in an 26 accompanying manuscript 4 but, in essence, our approach utilises the potential 27 28 reparative effects of multiple cell populations and has not be shown to be associated 29 with increased clinical risk. 30 31 There is now a wealth of preclinical data which supports a clear scientific rationale 32 for bone marrowderived cell therapy in MS. This, together with the extensive clinical 33 experience of bone marrow transplantation which has been acquired over several

34 decades, justifies the examination of the putative clinical benefit of bone marrow http://bmjopen.bmj.com/ 35 derived cell therapy for MS in clinical trials. 36 37 13 38 ‘SIAMMSII’, the ‘ACTiMuS’ trial and other ongoing studies will determine whether 39 bone marrowderived cell therapy genuinely effects neurological repair in MS and will 40 further understanding of the potential multiplicity of reparative mechanisms. 41 Optimisation of treatment is likely to be an iterative process which is dependent on 42 efficient backtranslation of information gained from carefully designed clinical trials on September 30, 2021 by guest. Protected copyright. 43 and it is hoped that future refinements will exploit more efficiently the therapeutic 44 potential of bone marrow cell therapy for the treatment of progressive MS. 45 46 47 48 Acknowledgements 49 50 We are very grateful to all those providing financial support for the ‘SIAMMSII’ trial 51 as listed above. 52 53 Contributors 54 55 CMR and NJS were responsible for overall study design. NMK and PW are involved 56 in the neurophysiological outcome measures. GM has coordinated NHSBT 57 58 involvement. AW has contributed to protocol review and refinement. PS, DO, CMR, 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 7 of 11 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 AW and NJS are involved in the clinical trial processes. JR, PS, CMR, AW and NJS 4 are responsible for the laboratory analyses. CMR drafted the manuscript, the final 5 version of which all authors read and approved. 6 7 Funding 8

9 10 Funding for the clinical aspects of the trial is supplied by The Sir Halley Stewart 11 Trust. Support for the laboratory studies which will run in parallel with the clinical 12 study is provided by the Medical Research Council (UK). CMR and PS are supported 13 by the Burden Neurological Institute. 14 15 CompetingFor interests peer review only 16 17 The authors declare that they have no competing interests. 18

19 20 21 Abbreviations 22 23 ‘ACTiMuS’ Assessment of Cellular Therapy in progressive Multiple Sclerosis 24 AE adverse event 25 BM bone marrow 26 BSAEP brainstem auditory evoked potential 27 CNS central nervous system 28 CXR chest Xray 29 30 EDSS expanded disability status scale 31 GEP global evoked potential 32 GMP good manufacturing practice 33 Hb haemoglobin

34 MEP motor evoked potential http://bmjopen.bmj.com/ 35 MMEP multimodal evoked potential 36 MRI magnetic resonance imaging 37 MS multiple sclerosis 38 MSFC multiple sclerosis functional composite 39 40 MSIS29 multiple sclerosis impact scale29 41 NHSBT National Health Service Blood and Transplant 42 OCT optical coherence tomography 43 PPMS primary progressive multiple sclerosis on September 30, 2021 by guest. Protected copyright. 44 PASAT paced auditory serial addition 45 SAE serious adverse event 46 SEP sensory evoked potential 47 ‘SIAMMS’ Study of Intravenous Autologous Marrow in Multiple Sclerosis 48 ‘SIAMMSII’ Repeat infusion of autologous bone marrow cells in multiple sclerosis – 49 50 a phase I extension study 51 SPMS secondary progressive multiple sclerosis 52 VEP visual evoked potential 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 8 of 11 BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 References 4 5 1. Rice CM, Mallam EA, Whone AL, et al. Safety and feasibility of autologous bone 6 marrow cellular therapy in relapsingprogressive multiple sclerosis. Clin 7 Pharmacol Ther 2010;87(6):67985. 8 2. Leocani L, Medaglini S, Comi G. Evoked potentials in monitoring multiple 9 10 sclerosis. NeurolSci 2000;21(4 Suppl 2):S889S91. 11 3. Leocani L, Rovaris M, Boneschi FM, et al. Multimodal evoked potentials to assess 12 the evolution of multiple sclerosis: a longitudinal study. J 13 NeurolNeurosurgPsychiatry 2006;77(9):103035. 14 4. Rice C, Marks D, BenShlomo Y, et al. Assessment of bone marrowderived 15 cellularFor therapy peerin progressive reviewmultiple sclerosis (ACTiMuS): only study protocol for 16 a randomised, placebocontrolled, stepped wedge study: Submitted to Trials. 17 5. Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded 18 disability status scale (EDSS). Neurology 1983;33(11):144452. 19 20 6. Fischer JS, Rudick RA, Cutter GR, et al. The Multiple Sclerosis Functional 21 Composite Measure (MSFC): an integrated approach to MS clinical outcome 22 assessment. National MS Society Clinical Outcomes Assessment Task Force. 23 MultScler 1999;5(4):24450. 24 7. Costelloe L, O'Rourke K, McGuigan C, et al. The longitudinal relationship between 25 the patientreported Multiple Sclerosis Impact Scale and the clinician 26 assessed Multiple Sclerosis Functional Composite. Mult Scler 2008;14(2):255 27 8. 28 8. Hobart J, Lamping D, Fitzpatrick R, et al. The Multiple Sclerosis Impact Scale 29 30 (MSIS29): a new patientbased outcome measure. Brain 2001;124(Pt 5):962 31 73. 32 9. Hobart JC, Riazi A, Lamping DL, et al. How responsive is the Multiple Sclerosis 33 Impact Scale (MSIS29)? A comparison with some other self report scales.

34 JNeurolNeurosurgPsychiatry 2005;76(11):153943. http://bmjopen.bmj.com/ 35 10. Hoogervorst EL, Zwemmer JN, Jelles B, et al. Multiple Sclerosis Impact Scale 36 (MSIS29): relation to established measures of impairment and disability. Mult 37 Scler 2004;10(5):56974. 38 11. Recommendations for the Practice of Clinical Neurophysiology: Guidelines of the 39 40 International Federation of Clinical Neurophysiology: Elsevier, 1999. 41 12. Walsh P, Kane N, Butler S. The clinical role of evoked potentials. 42 JNeurolNeurosurgPsychiatry 2005;76 Suppl 2:ii16ii22. 43 13. Rice CM, Kemp K, Wilkins A, et al. Cell therapy for multiple sclerosis: an evolving on September 30, 2021 by guest. Protected copyright. 44 concept with implications for other neurodegenerative diseases. Lancet 2013; 45 382(9899):120413. 46 47 48 Figure legends 49 50 Table 1 Eligibility criteria for the ‘SIAMMS’ trial 51 52 53 Figure 1 Study schema for the ‘SIAMMSII’ trial 54 55 Box 1 Method for recording of multimodal evoked potentials 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 9 of 11 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 Figure 1 Study schema for the ‘SIAMMS-II’ trial 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 40 41 42 43 on September 30, 2021 by guest. Protected copyright. 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 10 of 11 BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 4 Table 1 Eligibility criteria for the ‘SIAMMS’ trial 5 6 7 8 Inclusion criteria Exclusion critieria 9

10 Participation in the phase I safety and feasibility Pregnancy, breastfeeding or lactation 11 ‘Study of Intravenous Autologous Marrow in History of autologous/allogeneic bone marrow 12 Multiple Sclerosis’ (SIAMMS) (REC reference transplantation or peripheral blood stem cell 13 number number 05/Q1704/137)[1] transplant other than in SIAMMS 14 Bone marrow insufficiency 15 For peer reviewHistory of lymphoproliferative only disease or previous 16 total lymphoid irradiation 17 Immune deficiency 18 History of current or recent (<5 years) malignancy 19 Chronic or frequent drug-resistant bacterial 20 infections or presence of active infection requiring antimicrobial treatment 21 Frequent and/or serious viral infection 22 Systemic or invasive fungal disease within 2 23 years of entry to study 24 Significant renal, hepatic, cardiac or respiratory 25 dysfunction 26 Contraindication to anaesthesia 27 Bleeding or clotting diathesis 28 Current or recent (within preceding 12 months) 29 immunomodulatory therapy other than 30 corticosteroid therapy 31 Treatment with corticosteroids within the 32 preceding 3 months 33 Radiation exposure in the past year other than chest / dental x-rays

34 http://bmjopen.bmj.com/ Previous claustrophobia 35 The presence of any implanted metal or other 36 contraindication to MRI 37 Participation in another experimental study or 38 treatment within previous 24 months 39 40 41 42 43 on September 30, 2021 by guest. Protected copyright. 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 Page 11 of 11 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 Box 1 Method for recording of multimodal evoked potentials 4 5 6 7 8 VEPs will be evoked with a rear-projected checkerboard pattern using an opto- 9 mechanical device subtending 30 degrees at the retina, check-size 1 degree, white 10 -2 11 brightness of 150cdm and contrast 87.5%. 12 13 Monaural stimulation will be delivered via earphones to each side with rarefaction 14 click stimuli of 0.1ms duration at an intensity of 75dB above the subjective hearing 15 threshold whilstFor the contralateral peer ear reviewwas masked with white only noise. 16 17 SEPs will be obtained by delivering electrical stimulation with square wave pulses of 18 0.2ms duration to the median and the posterior tibial nerves, at the wrist and ankle 19 respectively. 20 21 22 Motor evoked potentials (MEPs) will be recorded from electrodes situated over the 23 abductor pollicis brevis muscle in the hand and the abductor hallucis in the foot using 24 a 9cm circular coil held over the vertex. The central motor conduction time (CMCT) 25 was calculated by subtracting ½(M+F+1) from the MEP latency where M is the distal 26 motor latency and F is the minimum F wave latency. 27 28 The GEP score will then be calculated as the sum of left and right BSAEP and VEP 29 scores (0-12) and left and right upper and lower SEPs (0-12) and CMCTs (0-12). 30 31 32 33

34 http://bmjopen.bmj.com/ 35 36 37 38 39 40 41 42 43 on September 30, 2021 by guest. Protected copyright. 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-2015-009090 on 11 September 2015. Downloaded from

Repeat infusion of autologous bone marrow cells in multiple sclerosis – protocol for a phase I extension study (SIAMMS- II)

For peer review only Journal: BMJ Open

Manuscript ID: bmjopen-2015-009090.R1

Article Type: Protocol

Date Submitted by the Author: 21-Jul-2015

Neurology http://bmjopen.bmj.com/

Primary Subject Neurology Heading:

Secondary Subject Heading: Haematology (incl blood transfusion)

Multiple sclerosis < NEUROLOGY, IMMUNOLOGY, Bone marrow Keywords: transplantation < HAEMATOLOGY

on September 30, 2021 by guest. Protected copyright.

For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 1 of 13 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 Repeat infusion of autologous bone marrow cells in multiple sclerosis - 4 protocol for a phase I extension study (SIAMMS-II) 5 6 7 8 Claire M. Rice1,2*, David I. Marks3, Peter Walsh2, Nick M. Kane2, Martin G. 9 4 1 1,2 2 1,2 10 Guttridge , Juliana Redondo , Pamela Sarkar , Denise Owen , Alastair Wilkins , 11 and Neil J. Scolding1,2 12 13 14 15 For peer review only 16 1School of Clinical 3Adult BMT Unit 4NHS Blood and 17 Sciences, Bristol Royal Hospital Transplant 18 19 University of Bristol, for Children Filton, 20 Southmead Hospital, University Hospitals North Bristol Park 21 Bristol, BS10 5NB, UK Bristol NHS Foundation Northway, Filton, 22 Trust & University of Bristol, BS34 7QH, UK 23 2Bristol Institute of Bristol, 24 Clinical Neurosciences, St Michael's Hill, 25 Southmead Hospital, Bristol, 26 Bristol, BS16 1JB, UK BS2 8BJ, UK 27 28 29 30 31 32 33

34 http://bmjopen.bmj.com/ 35 * Correspondence: [email protected] 36 37 38 39 Keywords: cell therapy 40 bone marrow 41 multiple sclerosis 42 reparative therapy 43 on September 30, 2021 by guest. Protected copyright. 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 13 BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

34 Ethics and dissemination: The study has UK National Research Ethics Committee http://bmjopen.bmj.com/ 35 approval (13/SW/0255). Study results will be disseminated via peerreviewed 36 publications and conference presentations. 37 38 39 40 Trial Registration: ClinicalTrials.gov registry NCT01932593 41 42 on September 30, 2021 by guest. Protected copyright. 43 44 Strengths and limitations of this study 45 46 47 Strengths: 48 Regulated clinical trial of cell therapy for progressive MS 49 Extension data for phase I clinical trial 50 51 Limitations: 52 Open label trial 53 Small sample size 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 3 of 13 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

on September 30, 2021 by guest. Protected copyright. 43 Trial design 44 45 46 ‘SIAMMSII’ is an open, prospective, single centre, safety and feasibility extension 47 study. The study schema is presented in figure 1. The study has UK National 48 Research Ethics Committee approval (13/SW/0255). 49 50 Sample size, eligibility & enrolment 51 52 The study is limited to the 6 people who participated in the original ‘SIAMMS’ study, 53 all of whom are under active follow up at the Bristol and Avon Multiple Sclerosis 54 (BrAMS) Unit, North Bristol NHS Trust, Bristol, UK. All participants have progressive 55 56 MS and must fulfil the inclusion and exclusion criteria as detailed in table 1. The prior clinical history of the participants is detailed in the manuscript documenting the 57 1 58 results of the original SIAMMS study . At the time of entry to the ‘SIAMMS’ trial, 4 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 4 of 13 BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 participants had had no exposure to disease modifying treatment. One had received 4 prior treatment with azathioprine and methotrexate and another participant had been 5 previously been treated with glatiramer and Avonex. In the intervening period since 6 receiving the first infusion of autologous bone marrow, none of the 6 participants 7 have received additional disease modifying therapy. 8

9 10 Trial interventions 11 12 Participants will have a bone marrow harvest and reinfusion of autologous marrow 13 as a daycase procedure. A short general anaesthesic will be given for the bone 14 marrow harvest which will be taken from the posterior iliac crests. Approximately 15 600ml marrowFor will be peer collected together review with a single boneonly marrow trephine. The 16 marrow aspirate will be processed by NHSBT (filtered, bagged and labelled) prior to 17 intravenous infusion. 18

19 20 Assuming specific written informed consent is granted, a bone marrow trephine and a 21 small sample of the bone marrow aspirate will be retained for research. Additional 22 blood samples for research purposes may be requested throughout the duration of 23 the study. 24 25 Outcome Measures 26 27 Primary outcome measure 28

29 30 The primary outcome measure is the number of adverse events (AE). For the 31 purposes of the study, an AE is defined as any unfavourable and unintended sign, 32 symptom or illness that develops or worsens during the period of the study. This is 33 irrespective of the likelihood that the AE is related to study interventions. AEs may be

34 expected or unexpected and include unwanted side effects, toxicity or sensitivity http://bmjopen.bmj.com/ 35 reactions, as well as abnormal laboratory results, injury or intercurrent illnesses. 36 37 A serious adverse event (SAE) is defined as an AE which results in death, is life 38 threatening or requires hospitalisation or prolongation of inpatient stay or which 39 40 results in persistent or significant disability or incapacity. Any congenital anomaly or 41 birth defect or any event considered to be a medical event of importance will be also 42 be classified as a SAE. All SAEs must be reported to the trial coordinating centre as 43 soon as possible. Those hospital admissions that were planned prior to trial entry will on September 30, 2021 by guest. Protected copyright. 44 not be recorded as SAEs. 45 46 As per the ‘ACTiMuS’ trial, 4 expected AEs include: 47 48 Local bruising and discomfort following bone marrow harvest 49 50 Increase in lower limb spasticity following bone marrow harvest 51 Acute urinary retention following bone marrow harvest 52 Temporary exacerbation of MS following general anaesthesia 53 Hypovolaemia or anaemia following blood and marrow donation 54 Exacerbation of MS due to sepsis e.g. urinary tract infection or chest infection 55 Assessment at or admission to hospital following fall 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 5 of 13 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 Bloods taken for safety analyses will be screened as follows: urea and electrolytes, 4 liver function tests, full blood count with differential white cell count, coagulation, 5 group and save, Creactive protein, glucose, calcium, magnesium, chloride, 6 bicarbonate, phosphate, viral serology (including cytomegalovirus, EpsteinBarr virus, 7 herpes simplex virus, varicella zoster virus, toxoplasmosis, Hepatitis B&C, human 8 immunodeficiency virus, human T cell lymphoma virus and syphilis screening. 9 10 Urinalysis (microscopy and culture) will also be performed. 11 12 Secondary outcome measures 13 14 Change in clinical measures of disability, GEP and cranial MRI findings are included 15 as secondaryFor outcome peer measures. review only 16 17 Clinical outcomes will be assessed at entry and at 6 months and 1 year. The clinical 18 rating scales will include the widely used Expanded Disability Status Scale (EDSS) 5 19 6 20 together with the Multiple Sclerosis Functional Composite (MSFC) . The latter is a 21 threepart quantitative assessment including a timed walk, ninehole peg test and 22 Paced Auditory Serial Addition Test (PASAT). In addition, participants will be asked 23 to complete the MS Impact Scale (MSIS29) which is a wellvalidated patient 24 completed rating scale 710. The measures taken for clinical secondary outcome 25 measures will be: 26 27 1. Physicianbased EDSS: time to EDSS progression of at least one point 28 from a baseline EDSS of 4.0, 4.5 or 5.0 or at least 0.5 points from a 29 30 baseline EDSS ≥5.5. 31 2. Patientbased MSIS29 physical impact scale version 2: overall mean 32 change from baseline to end of study 33 3. MSFC: overall mean change of zscores, from baseline to final visit

34 http://bmjopen.bmj.com/ 35 Multimodal evoked potentials will be examined at 0, 6 and 12 months. Evoked 36 potential abnormalities will be quantified according to a 4point graded ordinal score 37 modified from Leocani et al (0=normal; 1=increased latency; 2=increased latency and 38 abnormal amplitude; 3=absent) and the composite GEP score calculated 3. 39 40 41 The recording of the evoked potentials shall be in accordance with the Guidelines of 11 42 the International Federation of Clinical Neurophysiology and analysis will be 43 performed using standard methods 12 (box 1). Electrophysiological responses shall on September 30, 2021 by guest. Protected copyright. 44 be considered abnormal if they exceed 2.5 standard deviations of the normal values 45 or cannot be detected. 46 47 Participants will undergo cranial MRI at entry and at 6 months after bone marrow 48 infusion. The secondary MRI outcome measures will relate to lesion load and atrophy 49 50 measures of the brain. 51 52 Annual subjective patient and treating physician assessments of efficacy will also be 53 recorded. 54 55 Trial status 56 57 ‘SIAMMSII’ opened to recruitment in March 2014 and is ongoing. 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 6 of 13 BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 4 5 Analysis 6 7 8 A full statistical analysis plan will be written prior to data collection. The null 9 hypothesis is that there will be no significant difference in the primary and secondary 10 outcomes between intervention and control arms at 12 months. 11 12 Secondary outcomes will be scored according to standard methodology but the 13 limitations of the small sample size are acknowledged. 14 15 For peer review only 16 17 Conclusion 18 19 On the background of extensive preclinical studies and anticipated low risk of 20 significant harm, we commenced a phase I trial intravenous delivery of filtered but 21 otherwise unmodified autologous bone marrow in 2006. The successful completion of 22 this early trial and the suggestion that electrophysiological improvement may have 23 occurred, 1 made further exploration of the reparative potential of autologous marrow 24 in MS mandatory. We have begun to assess the efficacy of this approach in the 25 4 26 randomised, doubleblind ‘ACTiMuS’ trial. However. ‘SIAMMSII’ will give some 27 preliminary information about the value of retreating progressive MS with repeat 28 infusion of autologous bone marrow. 29 30 There is now a wealth of preclinical data which supports a clear scientific rationale 31 for bone marrowderived cell therapy in MS. This, together with the extensive clinical 32 experience of bone marrow transplantation which has been acquired over several 33 decades, justifies the examination of the putative clinical benefit of bone marrow

34 derived cell therapy for MS in clinical trials. Indeed, in addition to our own studies http://bmjopen.bmj.com/ 35 36 using filtered but otherwise unselected bone marrow, a number of clinical trials are 37 now exploring the safety and therapeutic effectiveness of bone marrowderived cell 38 therapy for MS using specific subpopulations of bone marrow cells. We and others 13 14 15 39 have recently reviewed the approaches being explored but, whilst candidates 40 certainly include multipotent mesenchymal stromal cells, the cell population(s) of 41 greatest therapeutic potential have not been definitively identified. The rationale for 42 1 4

our use of unfractionated marrow has been set out in detail elsewhere but in on September 30, 2021 by guest. Protected copyright. 43 essence our approach utilises the potential reparative effects of multiple cell 44 populations and has not be shown to be associated with increased clinical risk. 45 46 47 ‘SIAMMSII’, the ‘ACTiMuS’ trial and other ongoing studies will determine whether 48 bone marrowderived cell therapy genuinely effects neurological repair in MS and will 49 further understanding of the potential multiplicity of reparative mechanisms. 50 Optimisation of treatment is likely to be an iterative process dependent on efficient 51 backtranslation of information gained from carefully designed clinical trials but it is 52 hoped that future refinements will exploit more efficiently the therapeutic potential of 53 bone marrow cell therapy for the treatment of progressive MS. 54 55 56 Acknowledgements 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 7 of 13 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 We are very grateful to all those providing financial support for the ‘SIAMMSII’ trial 4 as listed in the funding section and to the participants in the original ‘SIAMMS’ study 5 for their consideration of participation in this follow up study. 6 7 Contributors 8

9 10 CMR and NJS were responsible for overall study design. NMK and PW are involved 11 in the neurophysiological outcome measures. GM has coordinated NHSBT 12 involvement. AW has contributed to protocol review and refinement. PS, DO, CMR, 13 AW and NJS are involved in the clinical trial processes. JR, PS, CMR, AW and NJS 14 are responsible for the laboratory analyses. CMR drafted the manuscript, the final 15 version of whichFor all authors peer read and reviewapproved. only 16 17 Funding 18

19 20 Funding for the clinical aspects of the trial is supplied by The Sir Halley Stewart 21 Trust. Support for the laboratory studies which will run in parallel with the clinical 22 study is provided by the Medical Research Council (UK). CMR and PS are supported 23 by the Burden Neurological Institute. 24 25 Competing interests 26 27 The authors declare that they have no competing interests. 28

29 30 Abbreviations 31 32 ‘ACTiMuS’ Assessment of Cellular Therapy in progressive Multiple Sclerosis 33 AE adverse event

34 BM bone marrow http://bmjopen.bmj.com/ 35 BSAEP brainstem auditory evoked potential 36 CNS central nervous system 37 CXR chest Xray 38 EDSS expanded disability status scale 39 40 GEP global evoked potential 41 GMP good manufacturing practice 42 Hb haemoglobin 43 MEP motor evoked potential on September 30, 2021 by guest. Protected copyright. 44 MMEP multimodal evoked potential 45 MRI magnetic resonance imaging 46 MS multiple sclerosis 47 MSFC multiple sclerosis functional composite 48 MSIS29 multiple sclerosis impact scale29 49 50 NHSBT National Health Service Blood and Transplant 51 OCT optical coherence tomography 52 PPMS primary progressive multiple sclerosis 53 PASAT paced auditory serial addition 54 SAE serious adverse event 55 SEP sensory evoked potential 56 ‘SIAMMS’ Study of Intravenous Autologous Marrow in Multiple Sclerosis 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 8 of 13 BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 ‘SIAMMSII’ Repeat infusion of autologous bone marrow cells in multiple sclerosis – 4 a phase I extension study 5 SPMS secondary progressive multiple sclerosis 6 VEP visual evoked potential 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 43 on September 30, 2021 by guest. Protected copyright. 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 Page 9 of 13 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 References 4 5 1. Rice CM, Mallam EA, Whone AL, et al. Safety and feasibility of autologous bone 6 marrow cellular therapy in relapsingprogressive multiple sclerosis. Clin 7 Pharmacol Ther 2010;87(6):67985. 8 2. Leocani L, Medaglini S, Comi G. Evoked potentials in monitoring multiple 9 10 sclerosis. NeurolSci 2000;21(4 Suppl 2):S889S91. 11 3. Leocani L, Rovaris M, Boneschi FM, et al. Multimodal evoked potentials to assess 12 the evolution of multiple sclerosis: a longitudinal study. J Neurol Neurosurg 13 Psychiatry 2006;77(9):103035. 14 4. Rice C, Marks D, BenShlomo Y, et al. Assessment of bone marrowderived 15 cellularFor therapy peerin progressive reviewmultiple sclerosis (ACTiMuS): only study protocol for 16 a randomised, placebocontrolled, stepped wedge study: Submitted to Trials. 17 5. Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded 18 disability status scale (EDSS). Neurology 1983;33(11):144452. 19 20 6. Fischer JS, Rudick RA, Cutter GR, et al. The Multiple Sclerosis Functional 21 Composite Measure (MSFC): an integrated approach to MS clinical outcome 22 assessment. National MS Society Clinical Outcomes Assessment Task Force. 23 Mult Scler 1999;5(4):24450. 24 7. Costelloe L, O'Rourke K, McGuigan C, et al. The longitudinal relationship between 25 the patientreported Multiple Sclerosis Impact Scale and the clinician 26 assessed Multiple Sclerosis Functional Composite. Mult Scler 2008;14(2):255 27 8. 28 8. Hobart J, Lamping D, Fitzpatrick R, et al. The Multiple Sclerosis Impact Scale 29 30 (MSIS29): a new patientbased outcome measure. Brain 2001;124(Pt 5):962 31 73. 32 9. Hobart JC, Riazi A, Lamping DL, et al. How responsive is the Multiple Sclerosis 33 Impact Scale (MSIS29)? A comparison with some other self report scales. J

34 Neurol Neurosurg Psychiatry 2005;76(11):153943. http://bmjopen.bmj.com/ 35 10. Hoogervorst EL, Zwemmer JN, Jelles B, et al. Multiple Sclerosis Impact Scale 36 (MSIS29): relation to established measures of impairment and disability. Mult 37 Scler 2004;10(5):56974. 38 11. Recommendations for the Practice of Clinical Neurophysiology: Guidelines of the 39 40 International Federation of Clinical Neurophysiology: Elsevier, 1999. 41 12. Walsh P, Kane N, Butler S. The clinical role of evoked potentials. J Neurol 42 Neurosurg Psychiatry 2005;76 Suppl 2:ii16ii22. 43 13. Rice CM, Kemp K, Wilkins A, et al. Cell therapy for multiple sclerosis: an evolving on September 30, 2021 by guest. Protected copyright. 44 concept with implications for other neurodegenerative diseases, Lancet 45 2013;382(9899):120413. 46 14. Xiao J, Yang R, Biswas S, et al. Mesenchymal stem cells and induced pluripotent 47 stem cells as therapies for multiple sclerosis. International Journal of 48 Molecular Sciences 2015;16(5):9283302. 49 50 15. Holloman JP, Ho CC, Hukki A, et al. The development of hematopoietic and 51 mesenchymal stem cell transplantation as an effective treatment for multiple 52 sclerosis. American Journal of Stem Cells 2013;2(2):95107. 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 10 of 13 BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 Tables 4 5 6 Table 1 Eligibility criteria for the ‘SIAMMSII’ trial 7 8

9 Inclusion criteria Exclusion critieria 10

11 Participation in the phase I safety and feasibility Pregnancy, breastfeeding or lactation 12 ‘Study of Intravenous Autologous Marrow in History of autologous/allogeneic bone marrow 13 Multiple Sclerosis’ (SIAMMS) (REC reference transplantation or peripheral blood stem cell 14 number number 05/Q1704/137)1 transplant other than in SIAMMS 15 For peer reviewBone marrow insufficiency only 16 History of lymphoproliferative disease or previous 17 total lymphoid irradiation 18 Immune deficiency 19 History of current or recent (<5 years) malignancy 20 Chronic or frequent drugresistant bacterial 21 infections or presence of active infection requiring 22 antimicrobial treatment Frequent and/or serious viral infection 23 Systemic or invasive fungal disease within 2 24 years of entry to study 25 Significant renal, hepatic, cardiac or respiratory 26 dysfunction 27 Contraindication to anaesthesia 28 Bleeding or clotting diathesis 29 Current or recent (within preceding 12 months) 30 immunomodulatory therapy other than 31 corticosteroid therapy 32 Treatment with corticosteroids within the 33 preceding 3 months Radiation exposure in the past year other than

34 http://bmjopen.bmj.com/ chest / dental xrays 35 Previous claustrophobia 36 The presence of any implanted metal or other 37 contraindication to MRI 38 Participation in another experimental study or 39 treatment within previous 24 months 40 41 42 on September 30, 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 Page 11 of 13 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 Box 1 Method for recording of multimodal evoked potentials 4 5 6 7 VEPs will be evoked with a rearprojected checkerboard pattern using an opto 8 mechanical device subtending 30 degrees at the retina, checksize 1 degree, white 9 2 10 brightness of 150cdm and contrast 87.5%. 11 12 Monaural stimulation will be delivered via earphones to each side with rarefaction 13 click stimuli of 0.1ms duration at an intensity of 75dB above the subjective hearing 14 threshold whilst the contralateral ear was masked with white noise. 15 For peer review only 16 SEPs will be obtained by delivering electrical stimulation with square wave pulses of 17 0.2ms duration to the median and the posterior tibial nerves, at the wrist and ankle 18 19 respectively. 20 21 Motor evoked potentials (MEPs) will be recorded from electrodes situated over the 22 abductor pollicis brevis muscle in the hand and the abductor hallucis in the foot using 23 a 9cm circular coil held over the vertex. The central motor conduction time (CMCT) 24 was calculated by subtracting ½(M+F+1) from the MEP latency where M is the distal 25 motor latency and F is the minimum F wave latency. 26 27 The GEP score will then be calculated as the sum of left and right BSAEP and VEP 28 29 scores (012) and left and right upper and lower SEPs (012) and CMCTs (012). 30 31 32 33

34 http://bmjopen.bmj.com/ 35 36 37 38 39 40 41 42 43 on September 30, 2021 by guest. Protected copyright. 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 12 of 13 BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 2 3 Figure Legend 4 5 6 Figure 1 Study schema for the ‘SIAMMSII’ trial 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 43 on September 30, 2021 by guest. Protected copyright. 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 Page 13 of 13 BMJ Open BMJ Open: first published as 10.1136/bmjopen-2015-009090 on 11 September 2015. Downloaded from

1 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 145x122mm (300 x 300 DPI) 37 38 39 40 41 42 43 on September 30, 2021 by guest. Protected copyright. 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