Ketamine for Depression Relapse Prevention Following ECT: a Randomised Pilot Trial With Blood Biomarker Evaluation
Phase: EARLY_PHASE1
Conditions: Depression
Sponsor: St Patrick's Hospital, Ireland

Cover Page 
NCT02414932    
Unique Protocol ID: SPUH 05/14    
Brief Title: Ketamine for Depression Relapse 
Prevention Following ECT (KEEP-WELL) 
Date of this document: 24.3.14 
Please see the attached Study Protocol and 
Statistical Analysis Plan for this study 

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1. INFORMATION ON CLINICAL TRIAL PROTOCOL 
2. STUDY TITLE 
 
Ketamine for depression relapse prevention following ECT: a randomised pilot trial with blood 
biomarker evaluation (The KEEP WELL Study) 
 
 
3. STUDY SPONSOR 
 
St Patrick’s Mental Health Services.  
 
 
Clinical Trial Protocol 

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4.  DETAILS 
4.1 Study title 
 
Ketamine for depression relapse prevention following ECT: a randomised pilot trial with blood 
biomarker evaluation (The KEEP WELL Study)  
 
4.2 Reference numbers 
Protocol identification (code or reference number):  
SPMHS REC Reference 05/14 
EudraCT number:  
 
 
                          2014-000339-18 
 
 
Date and version number:  
 
 
 
24.3.14, Version 1.0 
4.3 Applicant details 
 
Chief investigator/ Co-ordinating investigator 
Name/title: Prof. Declan McLoughlin 
 
Contact details:  
Postal: Dept. of Psychiatry, St. Patrick’s University Hospital, James’ St., Dublin 8.  
Email: d.mcloughlin@tcd.ie  
Telephone: 01 2493385 
 
 
Sponsor 
Name: St Patrick’s Mental Health Services 
 
Contact details: The contact person for the sponsor is: Professor J Lucey,  
Postal: Office of the Medical Director, St Patrick’s University Hospital, James’ St., Dublin 8 
Email: (Secretary) jbraddock@stpatsmail.com 
Telephone: (Secretary) 01 2493345 
 
 
Funder: Not yet allocated, as of 23.3.14. Contact details to be updated when funding secured.  
 
4.4 Signatures 
 
 
 
 
1. Sponsor: Prof. J. Lucey, Medical Director, St. Patrick’s University Hospital 
(on behalf of St Patrick’s Mental Health Services)  
 
 
 
 
 

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2. Principal Investigator: Prof. Declan McLoughlin, Dept. of Psychiatry. St. Patrick’s University 
Hospital.  
 
 
 
 
 
 
Date:25.08.14 
4.5 Other relevant information 
 
Collaborator Contact Information 
 
Dr Enda Shanahan 
Postal:  ECT Clinic, St Patrick’s University Hospital, James’ St, Dublin 8 
Email: enda.shanahan@upcmail.ie 
Telephone: 087 8308152 
 
Prof. Leslie Daly 
Postal:  CSTAR (Centre for Support and Training in Analysis and Research), The School of Public 
Health, Physiotherapy and Population Science, Woodview House, University College Dublin, Belfield 
Dublin 4 
Email: leslie.daly@ucd.ie 
Telephone: T + 353 1 716 3451 
 
 
 
5. CONFIDENTIALITY STATEMENT 
 
This document contains confidential information that must not be disclosed to anyone other 
than the sponsor, the investigative team, regulatory authorities, and members of the Research 
Ethics Committee. 

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6. TABLE OF CONTENTS   
 
1. 
INFORMATION ON CLINICAL TRIAL PROTOCOL TEMPLATE 
1 
2. 
STUDY TITLE 
1 
3. 
STUDY SPONSOR 
1 
4. 
APPLICATION DETAILS 
2 
5. 
CONFIDENTIALITY STATEMENT 
3 
6. 
TABLE OF CONTENTS 
4 
7. 
DOCUMENT HISTORY 
5 
8. 
SYNOPSIS 
5 
9. 
ABBREVIATIONS 
9 
10. 
INTRODUCTION 
11 
11. 
STUDY OBJECTIVE 
15 
12. 
TRIAL DESIGN 
18 
13. 
TREATMENT OF TRIAL SUBJECTS 
23 
14. 
SAFETY REPORTING 
36 
15. 
STATISTICS 
40 
16. 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
43 
17. 
DATA HANDLING AND RECORD KEEPING 
43 
18. 
RETENTION OF ESSENTIAL DOCUMENTS 
44 
19. 
QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES 
44 
20. 
AUDITS AND INSPECTIONS 
45 
21. 
ETHICS 
45 
22. 
FINANCING AND INSURANCE/INDEMNITY 
47 
23. 
CLINICAL STUDY REPORT AND PUBLICATION POLICY 
47 
24. 
REFERENCES 
48 
 
APPENDICES 
1. Appendix 1 - Investigator’s Brochures, ketamine and midazolam. 
2. Appendix 2 - Advertisement Material for Recruitment  
3. Appendix 3 - Validated Questionnaires for use in this trial 
4. Appendix 4 - Information Leaflet (Control and Patient), Letter of Invitation 
5. Appendix 5 - Trial-Specific Consent Form 
6. Appendix 6 - Copy of Indemnity, St Patrick’s University Hospital 
 
 

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7. DOCUMENT HISTORY 
 
Document 
Date of Issue 
Summary of Change 
Protocol version no. 
Version 1.0, 24.3.14 
Version 2.0, 10.6.14 
 
 
 
Version 3.0 
Not applicable 
Consent for both phases to be 
obtained pre-ECT, data retained 
5-10 years only, effects of 
ketamine can be unpleasant.  
 
SPMHS as sponsor 
 
Original protocol 
Version 1.0, 24.3.14 
Not applicable 
 
8. SYNOPSIS  
Title of study 
Ketamine for depression relapse prevention following ECT: a randomised 
pilot trial with blood biomarker evaluation (The KEEP WELL Study).  
Name of 
sponsor/company 
St Patrick’s Mental Health Services.  
Contact person: Prof. J. Lucey, Medical Director, St Patrick’s University Hospital 
Phase of 
development 
Pilot Trial 
Objectives 
Primary Objective 
This pilot trial has two phases. The primary objective is to conduct a randomised, 
controlled, patient- and rater-blinded pilot study of ketamine vs. an active 
comparator (midazolam) for four weeks following successful ECT, to assess trial 
process to inform a future definitive trial.  
Secondary Objectives  
1. To calculate a 95% confidence interval for an unadjusted hazard ratio 
that will allow interpretation of statistical difference between ketamine 
and midazolam groups to assess ketamine for reducing six-month relapse 
rates following successful ECT - Phase II (Trial Phase)  
2. To evaluate changes in blood levels of neuroplasticity-associated proteins 
as biomarkers to predict the effect of ketamine in six-month relapse rates 
in ECT responders - Phase II (Trial Phase) - this is an exploratory 
objective  
3.  To conduct an open prospective cohort study of patients with severe 
depression being treated with ECT vs. a healthy control group to 
investigate telomerase activity in depression and response to ECT - Phase 
I (Open Phase)  
4. To investigate epigenetic modulation of depression/stress-related genes 
in depression and response to ECT in the prospective cohort study of 
patients with severe depression being treated with ECT vs. a healthy 
control group above - Phase I (Open Phase)  
5. To measure the association between stressful early life experiences and 
epigenetic modification of gene expression in the prospective cohort 
study of patients with severe depression being treated with ECT - Phase I 

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(Open Phase)  
6.  To assess for association between scores on a brief personality 
assessment and response to ECT in the prospective cohort study of 
patients with severe depression being treated with ECT  - Phase I (Open 
Phase)  
  
Trial design 
This pilot trial has two phases.  
Phase I - a prospective open cohort study involving patients with major 
depressive disorder referred for ECT and a healthy control comparison group for 
investigation of: telomerase activity in both depression and response to ECT, 
epigenetic modulation of depression/stress related genes in depressed and non-
depressed people, epigenetic modulation of depression/stress-related genes in 
depression and response to ECT, the association between stressful early life 
experiences and epigenetic modification of gene expression in depression,  and 
personality factors and response to ECT.  
Peripheral blood telomerase activity will be measured at pre-ECT/Baseline and 
on completion of the ECT course or at one month for controls. Whole blood 
samples to examine epigenetic modulation of depression/stress related genes, 
including DNA methylation, chromatin activation, and microRNAs will also be 
collected from patients and controls at Baseline and 4 weeks/ post ECT. 
Demographic, clinical and cognitive data will be collected at Baseline and at four 
weeks/end of ECT. Participants having ECT will also complete a brief 
personality screening test and a questionnaire on early life events.  
Phase II – a two-group parallel-design randomised controlled pilot trial 
involving Phase I participants who responded to ECT. Participants will be 
randomly allocated in a 1:1 ratio to a four-week course of either once-weekly 
ketamine or midazolam infusions. Patients and raters will be blind to treatment 
allocation (assessed at six months). The anaesthetist administering ketamine/ 
midazolam infusions will not be blinded but will not be involved in assessments 
or data analysis. Computerised random allocation, using randomly permuted 
blocks and stratified for family history of alcohol dependence, will be done 
independently. Both groups will continue usual care. Participants will be 
followed-up over six-months following ECT to identify if and when relapse 
occurs, using repeated clinical and cognitive measures at weeks 6, 8, 12, 20 and 
26.  
Phase II participants will also be involved in a study to assess biomarkers to 
predict effect of ketamine on six-month relapse rates in ECT responders. Changes 
in blood levels of neuroplasticity-associated proteins will be assessed at four 
points before, during and after the first infusion of either ketamine or midazolam.  
Key inclusion 
criteria 
Inclusion Criteria for Phase I: 
(i) Patients ≥ 18 years of age referred for ECT 
(ii) Unipolar major depressive disorder (DSM-IV) 
(iii) 24-item Hamilton Rating Scale for Depression (HRSD-24) score of ≥21 
(iv) Can provide valid informed consent 
 
Inclusion Criteria for Phase II: 
(i) Received a course of ECT ≥ 5 sessions in Phase I 
(ii) Achieved response criteria - ≥ 60% decrease from Baseline HRSD-24 score, 
and score ≥ 16 on two consecutive weekly ratings 
(iii) Have a nominated adult who can stay with them for 24hours on outpatient 
treatment days 

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(iv) Mini-Mental State Examination (MMSE) score of ≥ 24 
(v) Can provide valid informed consent 
Key exclusion 
criteria 
Exclusion Criteria Phase I and II: 
(i) Any condition rendering patient medically unfit for ECT; general anaesthesia, 
ketamine or midazolam – assessed by physical examination, routine haematology 
and biochemistry investigations prior to enrolment in Phase I (routine care) 
(ii) Active suicidal intention 
(iii) Dementia, intellectual disability, or MMSE <24  
(iv) Lifetime history of bipolar affective disorder 
(v) Current history of post-traumatic stress disorder 
(vi) Other Axis I diagnosis (DSM-IV) 
(vii) ECT in the six months prior to recruitment 
(viii) Alcohol dependence or substance misuse in the six months prior to 
recruitment  
(ix) Pregnancy or breast-feeding 
(x) Residing in a nursing home 
(xi) Prisoner 
(xii) Diagnosis of terminal illness 
(xiii) Inability or refusal to provide valid informed consent 
Number of 
subjects 
Phase I (Open Phase) 
Patients referred for ECT, n=78, Healthy controls, n=78 
Total, n=156 
Phase II (Trial Phase) 
ECT responders randomised to ketamine (n=20) or midazolam (n=20) 
Total,  n=40 (from Phase I cohort) 
Total number of participants, n=156 
Test product, 
dose and mode of 
administration 
Investigative Medicinal Product - Ketamine Hydrochloride (Pfizer Healthcare 
Ireland) 10 mg/ml infusion at 0.5mg/kg made up as 50ml colourless saline 
solution and administered intravenously over 40 minutes via syringe driver pump.  
 
Active Comparator - Midazolam Hydrochloride 10mg/5ml solution at 
0.045mg/kg (Roche Products Ireland Ltd). Made up as 50ml colourless saline 
solution and administered intravenously over 40 minutes via syringe driver pump. 
Duration of 
treatment 
 
Phase I participants,  duration of involvement – 4-6 weeks 
Phase I and II Participants, duration of involvement – 7.5 months. 
Duration of treatment with IMP in Phase II – 4 weeks.  
 
Statistical 
methods 
Pilot trial data will be analysed on an intention-to-treat basis for all patients who 
complete at least one infusion and one follow-up assessment. Data analyses will 
be performed blinded to allocation, by Prof Leslie Daly in CSTAR.  
Descriptive statistics will be used to report rates of: recruitment (Phases 1 and 2); 
willingness to be randomised; willingness to complete assessments (Phases 1 and 
2); medical/ cognitive/ psychotomimetic/ general adverse events between groups 
in Phase 2; adherence to allocated treatments; adherence to follow-up between 
groups; and reasons for drop-outs between groups.  
Relapse-free survival times will be compared between groups using Kaplan-
Meier survival curves and log-rank test. Cox proportional hazard regression 
analysis will provide a 95% confidence interval for a hazard ratio for ketamine 
versus midazolam groups, accounting for the stratification factor “alcohol family 
history”.  Secondary exploratory analyses (Cox proportional hazards regression 

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models) will be used to identify pre-randomisation clinical factors or covariates 
(e.g. family history of alcohol dependence; gender; age; presence of psychosis 
and extent of treatment-resistance in index depressive episode; ECT remission 
status) that might influence response and help guide randomisation stratification 
in a future definitive trial.    
In the telomerase study, Baseline demographic and clinical data will be compared 
between groups using independent t-tests and chi-square tests as appropriate.  
Between groups data will be analysed by ANCOVA, with age and gender as 
covariates. In the depressed group, data will be analysed with linear models to 
test relationships between both Baseline telomerase activity and pre/post ECT 
change in activity and both clinical (e.g. HRSD-24) and cognitive measures.   
Linear regression will be used to investigate the association between epigenetic 
modulation of depression/stress-related genes and childhood trauma in depressed 
patients and the healthy control group. 
For all projects, continuous data will be tested for normality and transformed to 
enable linear modelling techniques. Where multiple hypotheses are applied to one 
dataset they will be corrected using Bonferroni or similarly conservative 
techniques. 
Sample size 
Phase I  (Open Phase) 
In this prospective cohort study, we aim to recruit 78 ECT patients and 78 healthy 
volunteers, n=156 in total. Data collected in this cohort study will be used in 
studies of : 
(i) 
Telomerase activity in depression – limited data available to inform 
sample size however sample of n=156 will be adequate to detect 
meaningful differences at a 0.05 significance level with 80% power.  
(ii) 
Studies of epigenetic modulation of depression/stress related genes, 
and the association between stressful early life experiences with 
epigenetic modification of gene expression - based on previous 
studies, sample sizes were estimated to detect meaningful differences 
at a 0.05 significance level with 80% power. 
(iii) 
Personality factors affecting ECT – no formal sample size calculation 
as this is an exploratory objective.  
Phase II (Trial Phase) 
A formal sample size calculation has not been performed for this pilot study; 
numbers are based on previous trial recruitment and completion rates.  
Please see section 15.2 for further details regarding sample size.  

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9. ABBREVIATIONS 
 
ACE-R              Addenbrooke’s Cognitive Examination, Revised 
AE 
 
Adverse event 
Akt                    Ak-Thyoma/Protein Kinase B 
AR 
 
Adverse reaction 
BDNF                Brain Derived Neurotrophic Factor 
BPRS                 Brief Psychiatric Rating Scale 
CA 
 
Competent authority 
CADSS             Clinician-Administered Dissociative States Scale 
AMI                   Autobiographical Memory Interview 
CRA 
 
Clinical research associate  
CRF 
 
Case report form 
CRO 
 
Contract research organisation 
CSTAR 
Centre for Support and Training in Analysis and Research, University College Dublin 
CT 
 
Clinical trial 
CTA 
 
Clinical trial authorisation 
CXR   
Chest x-ray 
DNA                  Deoxyribonucleic Acid 
DSM-IV            Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition 
ECG   
Electrocardiogram 
ECT 
 
Electroconvulsive therapy 
eEF-2                Eukaryotic elongation factor 2 
EHQ                  Edinburgh Handedness Questionnaire 
EU  
 
European Union 
e-CRF  
Electronic case report form 
FBDS                Forward and Backward Digit Spans  
FCSRT              Free and Cued Selective Recall Test 
GCP 
 
Good Clinical Practice 
GP 
 
General Practitioner 
GSK-3               Glycogen Synthase Kinase 3 
HRSD-24          Hamilton Depression Rating Scale, 24-item version 
HSE 
 
Health Service Executive 
IB 
 
Investigator’s brochure 
ICF 
 
Informed consent form 
ICH 
 
International Conference on Harmonisation 
IEC 
 
Independent Ethics Committee 
IMB 
 
Irish Medicines Board 
IMP 
 
Investigational medicinal products 
IMPD  
Investigational medicinal product dossier 
LMP                  (Date of) Last Menstrual Period 
MDD              Major depressive disorder 
MMSE              Mini Mental State Exam 
MSTRD            Maudsley Staging for Treatment-Resistant Depression  

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mTOR               Mammalian target of Rapamycin 
NART               National Adult Reading Test 
NMDA  
N-methyl-D-aspartate 
NOK                  Next of kin 
PBMC               Peripheral Blood Mononuclear Cells  
PBS                   Phosphate Buffered Saline 
PI 
 
Principal Investigator (Refers to Prof. Declan McLoughlin) 
PIL 
 
Patient/subject information leaflet 
PRISE               Patient-Rated Inventory of Side Effects 
QIDS-SR 16     Quick Inventory of Depressive Symptoms, Self-Report, 16-item 
RA                     Nominated Responsible Adult 
REC 
 
Research ethics committee  
ROI 
 
Republic of Ireland 
SAE 
 
Serious adverse event 
SAR 
 
Serious adverse reaction 
SCID                 Structured Clinical Interview for DSM-IV Axis I Disorders  
SmPC  
Summary of product characteristics 
SOP 
 
Standard operating procedure 
SPUH                St. Patrick’s University Hospital 
SUSAR 
Suspected unexpected serious adverse reaction 
TCIN                 Trinity College Institute of Neuroscience 
YMRS               Young Mania Rating Scale 

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10. INTRODUCTION 
 
10.1 
Background information    
 
DEPRESSION, ECT AND RELAPSE 
Major depressive disorder (MDD) is a debilitating mental illness with a lifetime prevalence of 
12-20%. In Ireland depressive disorders account for 6,000 of 20,000 annual psychiatry admissions 
(http://www.dohc.ie/statistics/pdf/stats11_psyc.pdf).  It’s the most costly brain disorder in Europe, 
accounting for 1% (€118 billion annually) of the total European economy 1.  Indeed, depression is 
currently the second largest cause globally for years lived with disability.2 .  It is thus a public health 
priority, additionally so in Ireland with worryingly high suicide rates (http://www.3ts.ie/wp-
content/uploads/2013/05/Suicide-in-Ireland-Survey-2003-2008-Report.pdf ). 
About 30% of patients don’t respond to antidepressants even after multiple trials with/without 
psychotherapies 3.  However, electroconvulsive therapy (ECT) offers up to 70% of such treatment-
resistant patients complete remission 4, 5. ECT is a medically safe procedure and is more acutely 
effective than psychotherapy or antidepressants for severe, often treatment-resistant, depression4. The 
major concerns are cognitive side-effects but for most people these are transient and many cognitive 
functions improve 6.   Treatments involve passing small electrical charges through the brain to induce 
a seizure lasting ~30 seconds under anaesthesia with muscle-relaxant. 6-10 treatments are 
administered in a course, 2-3 times weekly. One million people worldwide annually receive ECT, 
including nearly 250 people in Ireland (http://www.mhcirl.ie/File/The-Administration-of-ECT-in-
Approved-Centres-Activity-Report-2012.pdf). 
However, we reported in a recent meta-analysis that relapse rates following successful ECT 
are high: 27.1% after three months and 37.7% after six months 7.  Even continuation ECT (C-ECT), 
albeit mostly at unadjustable fixed schedules, did not improve six-month relapse rates (37.2%). 
Notably, these rates are similar to patients who respond only after ≥3 antidepressant steps and most 
likely reflect the recurrent nature of treatment-resistant depression3. We also found that, compared to 
placebo, taking antidepressants after ECT halved the risk (risk ratio=0.49, p<0.0001, NNT=3.3) for 
relapse at six months from nearly 80%7. Some form of continuation therapy is therefore essential. 
To date, the few reported randomised controlled trials for relapse prevention following ECT 
have focused on antidepressants and/or fixed-schedule C-ECT 7.   Searching www.clinicaltrials.gov, 
there are just five on-going or yet-to-be-reported trials of relapse prevention following ECT: three 
comparing various combinations of C-ECT and/or antidepressants; one assessing triiodothyronine; and 
one studying antidepressants/C-ECT/cognitive behavioural therapy.  There is a need for other, 
potentially better, methods for relapse prevention.  One possibility is the anaesthetic/analgesic 
ketamine. 
 
KETAMINE AS AN ANTIDEPRESSANT 
 
Ketamine is a competitive glutamate N-methyl-D-aspartate receptor (NMDAR) antagonist 
with half-life of 2-3 hours.  Ketamine has a remarkably rapid antidepressant effect, targeting core 
symptoms, in treatment-resistant depression when given as single sub anaesthetic doses, usually a 40 
minute 0.5 mg/kg intravenous infusion 8. Ketamine is psychotomimetic (with abuse potential) but at 
low dosage it’s safe, with patients and healthy controls experiencing mild dissociative and psychotic 
symptoms that resolve soon after finishing infusions 8.  To control for these effects, and also avoid 
“carry-over” effects in crossover studies while improving blinding, midazolam is now being used as 
control in parallel-group design trials rather than inactive placebo saline 9. Thereafter robust 
antidepressant effects (~70% responder rates) occur within 2-4 hours and persist for a few days, i.e. 
beyond immediate NMDAR blockade.  Chronic, mostly recreational, high-dose ketamine use can 
cause uropathy and dependency 8. However, repeated (e.g. 2-3/week for two weeks) infusions of sub 
anaesthetic ketamine are safe with more sustained antidepressant effects 8, 10.  

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These findings have led to the most exciting development in treating and understanding 
depression in over 50 years and represent a paradigm shift away from conventional slow-acting 
monaminergic antidepressants 11. Preclinical studies have shown that within just two hours ketamine 
increases synaptogenesis and spine formation in rodent prefrontal cortex 12 and rapidly reverses 
chronic stress-induced depressive behaviours and prefrontal neuronal atrophy 13. These effects are 
mediated, at least in part, via Akt/GSK-3/mammalian target of rapamycin (mTOR) signalling and 
increased dendritic translation of synaptic proteins 12, as well as deactivation of eukaryotic elongation 
factor 2 (eEF2) kinase, resulting in de-suppression of brain-derived neurotrophic factor (BDNF) 
translation 14.  BDNF mediates synaptic plasticity and is implicated in mechanisms of antidepressants 
and ECT 11. Interestingly, a lesser response to ketamine was found in low-activity Met BDNF 
Val66Met polymorphism carriers 15 while increased plasma BDNF was detectable in responders 
compared to non-responders four hours post-infusion 16, though this hasn’t been found in all studies. 
Changes in blood mononuclear cell levels of phosphorylated mTOR, eEF2 and GSK-3beta have also 
been associated with response to ketamine17, suggesting potential as biomarkers for response. 
Ketamine has been used for ECT anaesthesia and is associated with earlier improvement and 
possibly fewer cognitive side-effects but no overall better response 8.  However, no trials have yet 
been reported, or registered, for using ketamine as an adjunctive treatment to reduce relapse rates 
following successful depression treatment - an important potential use of ketamine that this proposal 
addresses.   
 
NEUROPLASTICITY BIOMARKERS 
Identifying a simple peripheral blood-based biomarker for diagnosis of depression and 
treatment response would be a major step. Based upon the above findings, the most appropriate 
biomarkers for initial evaluation are neuroplasticity markers, i.e. BDNF 11, 14, 16 as well as 
phosphorylated species of mTOR, eEF2 and GSK-3beta 12, 14, 17  Additionally, several meta–analyses 
have established that both serum and plasma BDNF are reduced in major depression and rise to 
normal levels with effective antidepressant treatment 18. Another neuroplasticity peripheral biomarker 
for predicting treatment response may be the enzyme telomerase which maintains the length of 
telomeres, regions at chromosome ends that protect them from degradation or fusion.  Telomere length 
provides a measure of biological, rather than chronological, age and reduced leukocyte telomere 
length occurs in depression and other stress-related conditions 19. Telomerase functions in 
neuroplasticity and is critical for adult neurogenesis and antidepressant-like behaviours in a mouse 
depression model 20.  Interestingly, telomerase activity is elevated in un-medicated depressed patients. 
Following sertraline treatment, those with lower baseline activity showed a greater activity increase 
which was associated with superior antidepressant response 21. Moreover, in a study of dementia 
caregivers with depressive symptoms, improvement was associated with increased telomerase 
activity22.  
The purpose of this research programme is to conduct a randomised pilot trial of ketamine for 
relapse prevention following ECT and develop molecular biomarkers for ketamine response and 
depression, one of the major health challenges for the 21st Century.  Outcomes will include knowledge 
gained from the pilot trial to inform a future randomised controlled trial, and from analyses of 
preselected blood molecules.  The latter will aid development of clinical biomarkers to help severely 
depressed patients.   
 
EPIGENETICS, DEPRESSION AND EARLY LIFE STRESS 
 
Epigenetics refers to control mechanisms that work alongside DNA sequences, altering their 
activity without changing the sequence itself 23 24 Recent research shows that depressed patients have a 
different epigenetic profile compared with control subjects 23, 24We will compare epigenetic 
modulation of depression/stress-related genes (e.g. BDNF, FKBP5) in depressed patients and healthy 
controls. Studies also suggest that reversing or bringing about compensatory epigenetic alterations in 
gene expression may be important in the mechanism of action of antidepressant therapies. We will test 

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the hypothesis that antidepressant therapy with ECT reverses or brings about compensatory epigenetic 
changes by examining epigenetic modulation of depression/stress-related genes in patients pre- and 
post-ECT. Epigenetics provides a potential mechanism by which adverse external stimuli such as early 
life stress can result in lasting changes in expression of depression/stress-related genes thus increasing 
the risk of depression in adulthood (23, 24. To gain insight into the pathogenesis of depression, we will 
measure the association between early life trauma and epigenetic modification of depression/stress-
related genes in depressed patients and the healthy control group 25, 26. Epigenetic modifications that 
will be examined may include DNA methylation, which involves the addition of a methyl group at the 
promoter region of a gene’s DNA sequence. This modification usually suppresses gene expression and 
may be measured by pyrosequencing 27 a DNA sequencing technique that identifies methylated DNA 
nucleotide bases. A second type of epigenetic modification that may be measured is chromatin 
activation status. To allow the storage of condensed information within the nucleus, DNA is stored in 
the form of chromatin. Active, open chromatin promotes gene expression while chromatin in its closed 
state suppresses gene expression. Chromatin activation status can be analysed using a technique called 
chromatin immunoprecipition 28 whereby genomic DNA associated with chromatin in its open or 
closed state can be precipitated with targeted antibodies and analysed by quantitative PCR. Small non-
protein coding RNA molecules known as microRNAs (miRNA)that also act as epigenetic modulators 
of gene expression, by suppressing messenger RNA translation to protein, may be measured using 
reverse transcription PCR 29. The downstream changes modulated by these epigenetic changes can be 
examined by measuring messenger RNA (mRNA) and protein products of the genes involved. 
Examining such epigenetic modulations may provide insight into the pathogenesis of depression.  
 
PERSONALITY AND RESPONSE TO ECT 
 
Personality disorder is a recognised psychiatric diagnosis characterised by enduring maladaptive 
patterns of behaviour, cognition and inner experience (DSM-IV).  Concomitant diagnoses of 
depression and personality disorder result in a doubling of the risk of a poor outcome for depression 
compared with no personality disorder 30. There is evidence that a diagnosis of personality disorder 
with depression can affect response rates to ECT 31, 32 and also relapse rates following successful ECT 
33. There is also evidence that specific personality traits can affect response to ECT 34. It has been 
suggested that clinicians under-prescribe ECT for depressed patients with a personality disorder 35. 
Personality disorder or traits can be diagnosed clinically following repeated assessments and collateral 
history, or using a structured diagnostic interview for personality disorder e.g. SCID-II. Patients 
referred for ECT are often severely depressed and the extensive patient input required to make a 
formal diagnosis of personality disorder may be impracticable. Therefore a study assessing for 
association of a brief personality assessment and response to ECT may be useful. The validated 
screening tool SAPAS (Standardised Assessment of Personality – Abbreviated Scale) 36 is an eight-
item dichotomously-rated screening tool for personality disorder.  A score of 3 on the SAPAS 
correctly identified the presence of DSM IV personality disorder in 90% of participants in one study 
with sensitivity and specificity of 0.94 and 0.85 respectively.  It has been shown that the majority of 
self-report personality traits are stable pre- and post-ECT treatment 37. We propose to assess for 
association between scores on a brief personality screening questionnaire and response to ECT by 
administering SAPAS to participants in Phase I.  
 
 
Rationale for the study 
As described above, there is evidence of ketamine’s antidepressant effect and a need for treatments to 
prevent relapse of depression following successful ECT. There are also identified needs for 
biomarkers for depression, response to ECT and response to ketamine, to further the understanding of 
the biology of depression and mechanism of action of ECT and ketamine in depression.   
Based on the background as described above, we hypothesise the following: 
 
1. Ketamine will reduce six month relapse rates following successful ECT 

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2. Blood molecular changes evident within hours after the first ketamine infusion (e.g. increase in 
levels of BDNF, phosphorylated mTOR, etc.) will predict lower six-month relapse rates 
3. Telomerase levels will be lower in depressed patients than controls but will normalise in response to 
ECT 
4. Depressed patients will have a different epigenetic profile compared with healthy controls and 
response to ECT will be associated with reversal of these differences or compensatory epigenetic 
changes in depression/stress-related genes 
5. There will be an association between early life trauma and epigenetic modification of 
depression/stress-related genes in depressed patients and the healthy control group 
6. Scores on a brief personality assessment will be associated with response to ECT 
 
Investigative Medicinal Product - Ketamine Hydrochloride 10 mg/ml infusion at 0.5mg/kg (Pfizer 
Healthcare Ireland), made up as 50ml colourless saline solution and administered intravenously over 
40 minutes via syringe driver pump. 
 
Active Comparator - Midazolam Hydrochloride 10mg/5ml solution at 0.045mg/kg (Roche Products 
Ireland Ltd). Made up as 50ml colourless saline solution and administered intravenously over 40 
minutes via syringe driver. 
 
A recent review of trials of ketamine for use as an antidepressant showed the most commonly used 
dosage in the past was as described above, i.e. 40 minute infusion of 0.5mg/kg 8. Bioavailability of 
ketamine is highest when administered intravenously. There have been insufficient studies of 
intramuscular, oral or intranasal ketamine for depression to warrant studying these preparations for 
relapse prevention. Studies comparing ketamine with midazolam have used the sub anaesthetic dose of 
0.045mg/kg as this dose mimics some of the effects of ketamine at 0.5mg/kg 8.  The regimen of four 
weekly infusions was chosen to facilitate subjects travelling for appointments, and because ketamine 
has proven effects as a rapid-acting antidepressant but has not yet been studied as a series of infusions 
for relapse prevention. While the half-life of ketamine is 3 hours, in previous studies the 
antidepressant effect was maintained to up to seven days 10. It is proposed that by administering a 
series of four weekly infusions, a cumulative effect may be observed that could be protective during 
the vulnerable first month post ECT and could affect relapse rates.  
 
Risks and Hazards 
Ketamine has a rapid, robust antidepressant effect when administered at low-dosage (0.5mg/kg) 
intravenously 8. Ketamine is psychotomimetic (with abuse potential) but is safe at low dosage, with 
patients and healthy controls experiencing mild dissociative and psychotic symptoms that resolve soon 
after finishing infusions 8. Chronic, mostly recreational, high-dose ketamine use can cause uropathy 
and dependency. However, repeated (e.g. 2-3/week for two weeks) infusions of sub anaesthetic 
ketamine are safe with more sustained antidepressant effects 8, 10. In sub anaesthetic doses, ketamine is 
a safe drug but can cause transient rises in pulse and blood pressure during infusion and for up to 80 
minutes afterward 8. Thus monitoring procedures have been out in place and will be followed as per 
the clinical trial protocol. A recent review of ketamine in depression concluded that outside 
recreational usage, there have been no reports of persistent adverse effects with sub anaesthetic uses of 
ketamine 8.  
Midazolam has recently been used as an active comparator to ketamine in parallel-group design trials 
as it mimics some of the effects of ketamine and may improve blinding over inactive placebo saline. 
At sub anaesthetic doses there have been no reported serious adverse events. However transient 
physical symptoms can occur during infusions, including minor lowering of blood pressure. As above, 
strict monitoring procedures will be followed before, during and after infusions. Please see the 
monitoring procedures as described in section 15.6 of this protocol for more detail.  
 

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11 STUDY OBJECTIVE 
 
11.1 
Primary objective 
This pilot trial has two phases.  
The primary objective is to conduct a randomised, controlled, patient- and rater-blinded pilot study 
of ketamine vs. an active comparator (midazolam) for four weeks following successful ECT, to 
assess trial process to inform a future definitive trial. This primary objective will be fulfilled in 
Phase II (Trial Phase), Project 1. Several secondary objectives will be fulfilled in both Phase I 
(Open Phase) and Phase II (Trial Phase).  
 
11.2 
Secondary objective/s 
(i) 
To calculate a 95% confidence interval for an unadjusted hazard ratio that will allow 
interpretation of statistical difference between ketamine and midazolam groups to 
assess ketamine for reducing six-month relapse rates following successful ECT - 
Phase II (Trial Phase)  
(ii) 
To conduct an open prospective cohort study of patients with severe depression being 
treated with ECT vs. a healthy control group to investigate telomerase activity in 
depression and response to ECT - Phase I (Open Phase)  
(iii) 
To investigate epigenetic modulation of depression/stress-related genes in depression 
and response to ECT in the prospective cohort study of patients with severe 
depression being treated with ECT vs. a healthy control group above - Phase I (Open 
Phase)  
(iv) 
To measure the association between stressful early life experiences and epigenetic 
modification of gene expression in the prospective cohort study of patients with 
severe depression being treated with ECT - Phase I (Open Phase)  
(v) 
To assess for association between scores on a brief personality assessment and 
response to ECT in the prospective cohort study of patients with severe depression 
being treated with ECT - Phase I (Open Phase)  
 
11.3 
Exploratory objectives 
 
(i) 
To evaluate changes in blood levels of neuroplasticity-associated proteins as 
biomarkers to predict the effect of ketamine in six-month relapse rates in ECT 
responders - Phase II (Trial Phase)  
 
11.4 
Primary and secondary/ exploratory endpoints/outcome measures 
 
Phase I 
(i) 
Telomerase activity in depression and response to ECT 
The outcome in this project is peripheral blood telomerase activity, measured at pre-ECT/baseline and 
after completing the ECT course or one month for controls, using the commercially available 
TRAPeze® Kit RT Telomerase Detection Kit (Chemicon International, Millipore). Response to ECT 
will be assessed by scores on the Hamilton Depression Rating Scale, 24-item (HRSD-24). HRSD 
scores are assessed at baseline, weekly during ECT, and at end-of-ECT. To enter Phase 1 patients 
must score ≥21 on HRDS-24. Response to ECT is defined as achieving ≥60% decrease from baseline 
HRSD-24 and score ≤16 on two consecutive weekly ratings. 
 

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(ii) 
Epigenetic modulation of depression/stress-related genes in depression and response 
to ECT 
Outcomes for this project include markers of epigenetic modulation of depression/stress related genes, 
including microRNA and DNA methylation, chromatin activation status and mRNA expression for e.g. 
FKBP5 and BDNF. RNA and DNA will be extracted from whole blood samples from patients and 
controls at Baseline and 4 weeks/ post ECT. Blood samples for plasma protein levels of the relevant 
gene products will also be examined.  Response to ECT will be assessed by scores on the Hamilton 
Depression Rating Scale, 24-item (HRSD-24) as detailed above.  
 
(iii) 
The association between stressful early life experiences and epigenetic modification 
of gene expression in depression vs. healthy controls  
Outcomes for this project include presence of stressful early life experiences assessed by baseline 
scores on the validated, 28-item self-rated Childhood Trauma Questionnaire, short form (CTQ-SF)38, 
and measures of epigenetic modification of depression/stress-related genes e.g. DNA methylation, 
chromatin activation status and mRNA expression for FKBP5 and BDNF. These will be measured via 
whole blood samples from patients and controls at Baseline and 4 weeks/ post ECT.  
(iv) 
Personality factors and response to ECT 
The outcomes for this project are baseline scores on the brief personality assessment measure the 
Standardised Assessment of Personality- Abbreviated Scale (SAPAS)36, and 8-item self-rated 
validated scale (ref), and response to ECT assessed by scores on the Hamilton Depression Rating 
Scale, 24-item (HRSD-24)39. HRSD scores are assessed at baseline, weekly during ECT, and at end-
of-ECT.  
 
Phase II 
(i) 
Pilot trial of ketamine vs. midazolam 
The focus of this pilot trial is on trial process with assessment of the primary clinical outcome being 
secondary because the pilot itself is not designed to measure efficacy 40. 
Process outcomes that will inform a future definitive ketamine relapse prevention trial include 
information on the following: 
x 
recruitment methods and rate 
x 
willingness of participants to be randomised 
x 
willingness of participants to complete assessments 
x 
randomisation 
x 
success of blinding 
x 
ability to administer a course of ketamine infusions 
x 
medical safety and acceptability of ketamine infusions in an ECT responder population 
x 
rates of adverse dissociative and psychiatric events 
x 
adherence to allocated treatment 
x 
adherence to follow-up  
x 
reasons for drop-out from treatment 
x 
reasons for drop-out from follow-up 
x 
a 95% confidence interval for the difference between the ketamine and midazolam groups in 
six-month relapse rates to help with power calculations for a future definitive trial  
 
Clinical outcomes:   

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These measures will be assessed at baseline, intra-treatment, end-of-treatment and follow-up time 
points illustrated in Figure 2 (Schedule of events).   
(i) Diagnosis and treatment history: Diagnosis of major depressive disorder will be confirmed using 
the mood episodes module of the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID)41.  
The Maudsley Staging Method for Treatment Resistant Depression (MSTRD)42 will be used to 
provide a measure of treatment-resistance. Handedness will be recorded with the Edinburgh 
Handedness Questionnaire43.  The National Adult Reading Test (NART) 44 will measure premorbid 
ability.   
Additional baseline data from patient interview and case-note review will include age, sex, weight, 
height, occupation, educational attainment, duration of index depressive episode, number of previous 
depressive episodes, previous ECT, history of medical illness and surgical treatments, personal and 
family history of alcohol/substance dependency, presence of psychotic symptoms (detected by SCID), 
and current medications and other therapies.  Changes in medications during Phases 1 and 2 will be 
documented at follow-up interviews (see Figure 2, Schedule of events). 
(ii) Depression outcomes: The primary clinical outcome measure is the relapse rate at six months as 
measured using the objectively-rated 24-item Hamilton Rating Scale for Depression (HRSD-24).  To 
enter Phase 1 patients must score ≥21.  Response to ECT is defined as achieving ≥60% decrease from 
baseline HRSD-24 and score ≤16 on two consecutive weekly ratings.  Remission criteria are ≥60% 
decrease in HRSD from baseline and score ≤10 on two consecutive weekly ratings.  Criteria for 
relapse are ≥10 point increase in HRSD-24 compared to baseline Phase 2 score plus HRSD ≥16; in 
addition, increase in the HRSD should be maintained one week later (if indicated, additional follow-
ups will be arranged).  Hospital admission, further ECT, and deliberate self-harm/suicide also 
constitute relapse.  Timing of these events will be recorded. 
Patients must achieve at least response to participate in the Phase 2 pilot trial.  Baseline and weekly 
intra-treatment course HRSD-24 scores will be obtained during the treatment periods of Phases 1 and 
2.  During the infusion sessions in Phase 2 HRSD-24 scores will be obtained 60 minutes before the 
infusion begins and at +120 and +240 minutes afterwards. Baseline scores on sleep and appetite items 
will be maintained for repeated measures within one day.  The +240 HRSD-24 scores will serve as the 
weekly post-ECT scores up to follow-up-week 4.  Depression measures will be repeated at weeks 6, 8, 
12, 20 and 26 during the six-month follow-up.  Subjective mood ratings will be measured at the above 
time points using the Quick Inventory of Depressive Symptoms, self-report version (QIDS-SR16)44.   
(iii) Cognitive outcomes: Cognitive testing can be challenging for severely depressed patients.  It is 
vital that patients be encouraged to remain in the trial throughout the whole study period and a great 
deal of tact is required of raters.  Priority will be given to obtaining scores on the HRSD-24. In a 
recent meta-analysis we identified that ECT has the most pronounced subacute (i.e. 0-3 days post 
course) adverse effects upon verbal memory (delayed word list recall) and frontal executive function 6. 
Sub anaesthetic doses of ketamine can cause problems with orientation, concentration, working and 
episodic memory but these resolve within two hours of beginning an infusion8. There are no published 
data on effects of ketamine on cognition in ECT responders. We will use the following battery in 
Phase 1 (pre and post ECT course; the latter will serve as baseline for Phase 2) and Phase 2 (one day 
after the first and fourth infusions and at six-months; see Tables 1 and 2).  Parallel versions will be 
used to reduce practice effects. 
a) Global cognition will be assessed with the revised Addenbrooke’s Cognitive Examination 
(ACE-R; three parallel versions 45) which also generates Mini-Mental State Examination 
(MMSE 46) and verbal fluency scores.  The ACE-R provides a total score (maximum=100) 
plus subscale scores for different aspects of cognition and has been used to study cognition in 
depression and by ourselves during ECT.   

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b) Forward and Backward Digit Spans(Wechsler, 1989) - immediate short-term memory, 
attention and working memory 
c) Trail Making Test (Part A Wechsler, 1989)- motor and psychomotor speed  
d) Frontal-executive function will be rated by Trail Making Test (Part B, Wechsler, 1989) plus 
letter and category verbal fluencies. 
e) Anterograde verbal memory will be tested using the Free and Cued Selective Reminding 
Test variant (immediate and delayed recall) of the Buschke Selective Reminding Test 47. 
f) To measure retrograde amnesia for autobiographical information we will use the Kopelman 
Autobiographical memory Interview (AMI48). This will be administered pre and post ECT, 
after the fourth ketamine infusion, and at six months. 
(iv) Ketamine psychotomimetic effects and adverse events: Outcomes will include scores on the 
following scales, assessed before, during (+35-40 mins) and after (+240 mins) infusions of ketamine 
or midazolam: 
- 
Clinician-Administered Dissociative States Scale (CADSS)49 
- 
 Brief Psychiatric Rating Scale (BPRS; four-item positive symptom subscale)50 
- 
Young Mania Rating Scale (YMRS; mood item)51 
- 
Patient-Rated Inventory of Side Effects52 
 
(ii) 
Neuroplasticity biomarkers for predicting effect of adjunctive ketamine on relapse 
prevention 
Outcomes for this project include changes in blood levels of neuroplasticity biomarkers including 
BDNF, pMTOR, pGSK3beta and peEF2 in response to the first infusion of ketamine/midazolam, 
compared at time points -60, +40, +120 and +240 minutes. Telomerase activity will also be analysed 
at -60 and +240 minutes. Relapse will be assessed using HRDS-24. Criteria for relapse are ≥10 point 
increase in HRSD-24 compared to baseline Phase II score plus HRSD ≥16; in addition, increase in the 
HRSD should be maintained one week later (if indicated, additional follow-ups will be arranged).  
Hospital admission, further ECT, and deliberate self-harm/suicide also constitute relapse.  Timing of 
these events will be recorded. 
 
12 TRIAL DESIGN 
 
12.1 
General considerations  
 
This single-site study will take place in St. Patrick’s University Hospital, Dublin.  
This pilot trial consists of two phases.  
The study design is presented graphically in the form of a CONSORT flow diagram below. 
 

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Phase I  
A prospective open study that involves recruiting patients with major depressive disorder (DSM-IV 
criteria) referred for ECT and a healthy control comparison group (recruited using the enclosed 
advertisement material, Appendix 2) for participation in several concurrent prospective cohort studies 
described separately below. Trial duration for participants in Phase I is one month.  
 
Phase I, Telomerase in depression and response to ECT  
This prospective cohort study will assess telomerase activity in depressed patients referred for ECT 
compared with a healthy control comparison group. Baseline peripheral blood telomerase activity will 
be measured via whole blood samples collected from fasting and resting (45 minutes) participants and 
analysis will take place in the laboratory at Trinity College Institute of Neuroscience, according to the 
method described in section 0. Telomerase activity will be measured again following completion of 
ECT or at one month for controls. Response to ECT will be assessed using scores on the Hamilton 
Depression Rating Scale, 24-item (HRSD-24) as outlined in section 0 and telomerase activity will be 
compared between responders and non-responders to ECT. Baseline and follow-up demographic, 
clinical and cognitive assessments will also be performed comprising –  
x 
Clinical: Structured Clinical Interview for DSM-IV Axis I Disorders (SCID), Hamilton 
Depression Rating Scale, 24-item (HRSD-24), Quick Inventory of Depressive Symptoms, 
self-rated (QIDS-SR) 
x 
Background:  Demographic data, Edinburgh Handedness Questionnaire, the National Adult 
Reading Test (NART), Childhood Trauma Questionnaire (CTQ), Standardised Assessment of 
Personality – abbreviated scale (SAPAS), 
x 
Cognitive: Addenbrooke’s Cognitive Assessment, Revised (ACE-R), digit spans, Trails A and 
B, Full and Cued Selective Recall Test (FCSRT), and the Autobiographical Memory Interview. 
Please see the detailed schedule and description of all assessments in section 12.3 
 
Phase I, Epigenetic modulation of depression/stress-related genes in depression and response to 
ECT 
This study will examine epigenetic modulation of depression/stress related genes in the cohort of 
depressed patients referred for ECT and a healthy control comparison group, as above. Whole blood 
samples will be collected at baseline and on completion of ECT (or at four weeks for controls) and 
markers of epigenetic modulation will be examined using the method described in section 12.3and 
compared between responders and non-responders to ECT, defined by scores on the Hamilton 
Depression Rating Scale, 24-item (HRSD-24) as outlined in section 12.3.  
 
Phase I, The association between stressful early life experiences and epigenetic modification of 
gene expression in depression  
This case-control study will compare baseline scores on the Childhood Trauma Questionnaire, short 
form (CTQ-SF), and measures of epigenetic modification of depression/stress-related genes between 
depressed patients referred for ECT and a healthy control comparison group, as above. Whole blood 
samples will be obtained at baseline and on completion of ECT or at four weeks for controls and 
markers of epigenetic modulation will be examined using the method described in section 12.3. 
Presence of depression will be assessed by diagnosis of unipolar major depressive disorder on SCID at 
screening and scores of ≥21 on HRSD-24 at screening and baseline.  
 
Phase I, Personality factors and ECT 
This prospective cohort study of depressed patients referred for ECT will assess for association 
between baseline scores on the brief personality assessment the Standardised Assessment of 
Personality- Abbreviated Scale (SAPAS), and response to ECT assessed by scores on the Hamilton 
Depression Rating Scale, 24-item (HRSD-24).  

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Phase II 
Following Phase I, participants identified as being ECT responders will be invited to participate in 
Phase II, which consists of a two-group parallel-design randomised controlled pilot trial, and a 
prospective cohort study of biomarkers for prediction of response to ketamine, both described 
separately below.  
 
Phase II, Pilot trial of Ketamine vs. Midazolam for relapse prevention following successful ECT 
 
Phase I participants who are successfully treated with ECT and continue to meet inclusion criteria will 
be randomly allocated in a 1:1 ratio to a four-week course of either once-weekly ketamine at 
0.5mg/Kg or the active comparator midazolam at 0.045mg/Kg. The trial will take place under “real 
world” conditions with both groups continuing usual care (e.g. regular medications, psychological and 
other therapies, and out-patient review) during the randomised treatment phase and thereafter.  
Participants will be followed-up over six-months following ECT to identify if and when relapse occurs.  
 
A baseline interview will comprise treatment review, HRSD-24, QIDS-SR, and cognitive outcomes 
(ACE-R, digit spans, Trails A and B, FCSRT, and AMI). Patients and raters will be blind to treatment. 
Computerised random allocation, using randomly permuted blocks and stratified for family history of 
alcohol dependence (may be associated with antidepressant response to ketamine), will be done 
independently at CSTAR.  Allocation information will be provided in two sets of opaque envelopes 
which will be stored in the ECT department where infusions are to be administered. To ensure patient 
safety during infusions and the post infusion period, the anaesthetist administering ketamine/ 
midazolam infusions will not be blinded but will not be involved in assessments or data analysis. 
Success of blinding for patients and raters will be assessed after the first and final treatments and at the 
end of the six month follow-up.  
 
The first infusion will be administered within two weeks of completing ECT and may be administered 
as an inpatient or outpatient; further infusions will take place as an outpatient. Each infusion will take 
40 minutes, monitoring will take place for 200 minutes post-infusion. During each treatment session, 
participants will be monitored for heart rate, blood pressure, pulse oximetry, and electrocardiogram 
changes. Adverse or psychotomimetic effects of either agent will be monitored using the Clinician-
Administered Dissociative States Scale (CADSS), Brief Psychiatric Rating Scale (BPRS; four-item 
positive symptom subscale), Young Mania Rating Scale (YMRS; mood item) and the Patient-Rated 
Inventory of Side Effects administered before, during and after infusions. Cognitive outcomes will be 
repeated in week 4. All other assessment measures will be repeated weekly; including treatment 
review/MSTRD, HRSD-24, and QIDS-SR. 
 
Participants will be advised not to drive or operate heavy machinery for 24 hours post-infusions and 
will be asked to ensure they have a nominated adult who can stay with them for 24 hours on outpatient 
treatment days. Participants will be contacted by a researcher 24 hours after each session to enquire 
about side-effects.  
 
Phase II participants will be followed up for six months following ECT with repeated questionnaires 
comprising treatment review, HRSD-24 and QIDS-SR at weeks 6, 8, 12 and 20 post ECT. A final 
follow-up session in week 26 will comprise the above listed measures as well as cognitive outcomes 
(ACE-R, digit spans, Trails A and B, FCSRT, and AMI).  
 
Phase II, Neuroplasticity biomarkers for predicting effect of adjunctive ketamine on relapse 
prevention 

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Consented ECT responders who participate in the randomised controlled trial above will be assessed 
for changes in blood levels of neuroplasticity-associated proteins during the first ketamine infusion. 
Blood samples for plasma will be collected at four time points before, during and after the first 
ketamine/midazolam infusion (-60, +40, +120, and +240 minutes) and changes in biomarkers 
including BDNF, pMTOR, pGSK3beta and peEF2 will be compared between ketamine and 
midazolam groups. Six-month relapse rates (assessed as described in section 11.4) between groups 
will be compared with changes in biomarkers to assess these proteins as predictors of the effect of 
ketamine on relapse rates in ECT responders.  
 
A detailed description of all assessments and interventions is available in section 12.3.  
 
12.2 
Selection of Study Population 
 
12.2.1 Overall description of trial subjects 
 
Participants will include healthy controls of ages 18 and above, who are not being treated for a mental 
health problem, and current inpatients in St Patrick’s University Hospital, who have a diagnosis of 
unipolar MDD and are referred for ECT. Participants may be male or female, aged 18+, and from a 
variety of geographical (within Ireland) and socioeconomic backgrounds. Participants will not have 
any medical condition that would preclude treatment with ECT or ketamine/ midazolam.  
 
12.2.2 Inclusion criteria 
 
To be eligible for inclusion, each subject must meet each of the following criteria at Screening (Visit 
1) and must continue to fulfil these criteria at Baseline (Visit 2).  
 
- 
Subjects must be able and willing to give written informed consent and to comply with the 
requirements of this study protocol  
- 
Subjects must be male or female, aged 18 years or above at Baseline 
- 
If in the ECT patient group – must be diagnosed with unipolar major depressive disorder (DSM-
IV) and with a HRDS-24 of   ≥21, and referred for ECT.  
- 
Participants in the ECT patient group must not have any finding on physical examination, routine 
haematology and biochemistry investigations, and an ECG at Screening that might affect ability to 
be treated with ECT or ketamine.  
- 
If healthy volunteers, subjects who are judged to be in generally good health by the investigator 
based upon the results of the medical history and clinical assessments at Baseline (SCID).  
- 
Female subjects of child bearing potential and male subjects whose partner is of child bearing 
potential must be willing to ensure that they or their partner use effective contraception during the 
study and for 5 weeks thereafter. 
- 
Female subjects are requested to alert researchers to any possibility that they may be pregnant 
when asked at Screening, Baseline and all subsequent assessments.  
- 
For inclusion in randomised Phase II, patients must have  
o Received a significant course of ECT (i.e. at least 5 sessions) 
o Achieved at least response criteria (i.e. ≥60% decrease from Baseline HRSD-24 score 
and score ≤16 on two consecutive weekly ratings) 
o Have a nominated adult who can stay with them for 24-hours on out-patient treatment 
days 
o Have a Mini-Mental State Examination (MMSE) score of ≥24 

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o Not currently taking any of the contraindicated medications listed in section 13.7.2 of 
this protocol 
 
 
12.2.3 Exclusion criteria 
 
Subjects are excluded from the study if any of the following criteria are met at Screening or at 
Baseline: 
 
- 
Allergy/sensitivity to study medications or their ingredients 
- 
Female subjects who are pregnant or breast-feeding or considering becoming pregnant during the 
study.  
- 
Subjects who have participated in another study and received any other investigational agent 
within six months.  
- 
Subjects unable to provide valid informed consent  
- 
Any condition rendering patient medically unfit for ECT; general anaesthesia; ketamine or 
midazolam – assessed by physical examination, routine haematology and biochemistry 
investigations prior to enrolment in Phase I.  
- 
Subjects who have a history of drug or alcohol use that, in the opinion of the investigator, would 
interfere with adherence to study requirements.  
- 
Known history of, or documented positive hepatitis B or C or HIV infection. 
- 
Diagnosis of current advanced malignancy or terminal illness.  
- 
Scheduled for non-trial procedures requiring general anaesthesia during the study. 
- 
 Active suicidal intention 
- 
Dementia, intellectual disability, or MMSE <24  
- 
Lifetime history of bipolar affective disorder 
Current history of post-traumatic stress disorder 
- 
Other Axis I diagnosis (DSM-IV) 
- 
ECT in the six months prior to recruitment 
- 
Residing in a nursing home 
- 
Current prisoner 
 
12.3 
 Study assessments and procedures 
 
Participants in the healthy control group in Phase I will undergo: 
- Phlebotomy for plasma and whole blood performed according to WHO “Best Practice in 
Phlebotomy”  
- An interview comprising Structured Clinical Interview for DSM-IV Axis I Disorders (SCID), 
National Adult Reading Test (NART), background and demographic data including the 
Childhood Trauma Questionnaire (CTQ) and Standardised Assessment of Personality- 
abbreviated Scale (SAPAS), Hamilton Depression Rating Scale, 24-item (HRSD-24), Quick 
Inventory of Depressive Symptoms, self-report version (QIDS-SR), and cognitive measures -  
- Cognitive measures - Addenbrooke’s Cognitive Examination- Revised (ACE-R); Digit Spans; 
Trails A and B; Full and Cued Selective Recall Test (FCSRT) and the Autobiographical 
Memory Interview (AMI) 
- These measures including phlebotomy for plasma and whole blood will be repeated at 4 weeks. 
Trial duration for healthy controls in Phase I is five weeks.  
 

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Participants in the ECT treatment group in Phase I will undergo: 
- The above listed measures 
- Treatment review/Maudsley Staging method for Treatment Resistant Depression (MSTRD) at 
Baseline (prior to first ECT session), and at the end of the course of ECT.  
- Weekly clinical outcome measures (HRSD-24, QIDS-SR).  
- Trial duration for Phase I participants undergoing ECT is maximum eight weeks.  
 
Phase I participants who are successfully treated with ECT and suitable to progress to Phase II will 
undergo: 
- Baseline Interview (following completion of ECT) comprising treatment review/MSTRD, 
HRSD-24, QIDS-SR 
- Baseline cognitive measures (ACE-R, Digit spans, Trails A and B; FCSRT and AMI) 
 
Phase II participants will be randomized to receive four weekly intravenous infusions of either 
ketamine at 0.5mg/Kg or active comparator midazolam at 0.045mg/Kg. The first infusion will be 
administered as an inpatient within one week of completing ECT; further infusions will take place as 
an outpatient. Each infusion will take 40 minutes, monitoring will take place for 200 minutes post-
infusion.  
During each treatment session, participants will be monitored for: 
- Heart rate, blood pressure, pulse oximetry, and ECG changes 
- Adverse or psychotomimetic effects of either agent will be monitored using Clinician-
Administered Dissociative States Scale (CADSS), Brief Psychiatric Rating Scale (BPRS), 
Young Mania Rating Scale, and Patient-Rated Inventory of Side Effects (PRISE), administered 
before, during and after infusions 
- Cognitive outcomes will be repeated in week 4 
- All other assessment measures will be repeated weekly, including treatment review/MSTRD, 
HRSD-24, and QIDS-SR 
 
At the first infusion session, phlebotomy for plasma and whole blood will be performed on four 
occasions (at time points 60 mins pre-infusion and 40, 120 and 240 mins post-infusion). Participants 
will be advised not to drive or operate heavy machinery for 24 hours post-infusions and will be 
contacted by a researcher 24 hours after each session to enquire about side-effects. Participants in 
Phase II will be asked to ensure they have a nominated adult who can stay with them for 24 hours on 
outpatient treatment days.  
Phase II participants will be followed up for six months with repeated questionnaires comprising 
treatment review/MSTRD, HRSD-24 and QIDS-SR at weeks 6, 8, 12 and 20 post infusions. A final 
follow-up session in week 26 will comprise the above listed measures as well as cognitive outcomes 
(ACE-R, digit spans, Trails A and B, FCSRT, and AMI). No phlebotomy will be performed on follow-
up. Total trial duration for Phase II participants is nine months.  
 
Informed consent will be obtained prior to any study-related procedures being undertaken. 
 
 
 
 
 
 
 
 
 

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Figure 2. Schedule of events. Abbreviations are as in main text and key on pages 8 and 9.   
 
 
Phase 1: ECT patients and 
healthy controls 
Phase 2: ECT responders randomised to 
ketamine or midazolam 
Assessment 
Baseline 
(pre 
ECT) 
Weekly 
during 
ECT 
course 
(patients 
only) 
End of 
ECT 
course 
( 4 
weeks 
for 
controls) 
Pre-
infusions 
Infusions #1-4; 
weeks 1-4 
Follow-
ups: 
weeks 
6, 8, 12, 
20 
Final 
follow-
up 
week 
26 
DIAGNOSIS AND TREATMENT 
Background, 
SCID, 
NART 
 
 
 
 
 
 
 
Treatment  
review/ 
MSTRD 
 
 
 
 
 (1-4) 
 (6-20) 
 
CLINICAL OUTCOMES 
HRSD-24 
 
  
 
 (1-
4) 
 (6-20) 
 
QIDS-SR 
 
  
 
  (1-
4) 
 (6-20) 
 
COGNITIVE OUTCOMES 
ACE-R 
 
 
 
 
1st  + 4th  
 
Digit spans 
 
 
 
 
1st  + 4th  
 
Trails A + B 
 
 
 
 
1st  + 4th  
 
FCSRT 
 
 
 
 
1st  + 4th  
 
AMI 
  
 
  
 
           4th 
 
 
KETAMINE EFFECTS 
CADSS 
 
 
 
 
  (1-
4) 
 
 
BPRS 
 
 
 
 
  (1-
4) 
 
 
YMRS 
 
 
 
 
  (1-
4) 
 
 
PRISE 
 
 
 
 
  (1-
4) 
 
 
BLOOD SAMPLES 
Phlebotomy 
 
 
 
 
1st 
 
 
CONSENT 
Signed 
Consent 
 
 
 

 
 
 
Verbal 
Assent 




 (1-
4)
 (6-20)

ELIGIBILTY  
Eligibility 
check 




 (1-
4)
 (6-20)

RANDOMISATION 
Allocation 
 
 
 
 
 
 
 
 
 

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The following circumstances will constitute a protocol violation and will be reported to the Sponsor 
and discussed at the next Trial Steering Committee meeting. Multiple protocol violations may result in 
premature termination of the trial, at the decision of the Sponsor or investigators.  
(i) 
Eligibility cannot be determined prior to referral for ECT, thus any assessment or 
intervention performed prior to referral for ECT constitutes a protocol violation 
(ii) 
Baseline phlebotomy performed after ECT session 1 
(iii) 
Phase I follow-up phlebotomy performed more than one week after the end of ECT 
treatment 
(iv) 
Follow-up questionnaires repeated on more than the five specified occasions in Figure 2 
(v) 
Questionnaires repeated more than seven months after the end of ECT treatment 
(vi) 
Administration of more than four separate infusions of either ketamine or midazolam 
(vii) 
Administration of ketamine or midazolam within six days of the last infusion 
(viii) 
Administration of the first infusion of ketamine or midazolam more than two weeks after 
the end of ECT treatment 
 
 
12.3.1 Description of Study Assessments 
 
Medical and Surgical History 
Details of current and previous diagnoses and treatments will be recorded.  
 
Demographics 
The date of birth, gender and race will be recorded. 
 
Vital Signs 
Vital signs will not be recorded for the purposes of the trial for participants in Phase I, but monitoring 
of vital signs for patients undergoing ECT is part of standard care.  
In Phase II, participants’ heart rate, pulse oximetry and blood pressure will be monitored before and 
during infusions and for a further 200 minutes.  
 
ECG Test 
A 12-lead ECG will be examined by the investigator at screening for Phase I. ECG monitoring 
(rhythm strip) will take place during ECT sessions as per usual care. ECG monitoring will be 
performed for all Phase II participants at infusion sessions, before and after administration of the IMP.  
Abnormal findings will be noted for clinical significance, and the report will be signed by the 
investigator. 
 
Clinical Laboratory Tests 
Clinical laboratory tests will not be performed as part of this trial. Results of clinical laboratory tests of 
potential participants in the ECT treatment group of Phase I (who may progress to Phase II) will be 
reviewed by the anaesthetist as part of routine clinical screening prior to ECT.  
 
Pregnancy Tests 
Women of child-bearing potential who participate in the study are requested to inform researchers if 
there is any possibility they may be pregnant. At this point, urine pregnancy test may be performed 
with consent. Date of last menstrual period (LMP) will be documented at the start of a course of ECT 
(Phase I, Open Phase), and at the first ketamine infusion (Phase II, Trial Phase). Information relating 
to the importance of contraception during the trial is provided in the Participant Information Sheet.  
 

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Concomitant Medication  
For all participants in Phase I, all over-the-counter or prescription medication, vitamins, and/or herbal 
supplements will be recorded on CRFs.  The indication for treatments will be recorded. Please see 
section 13.7 for further details. 
The Maudsley Staging Method for Treatment-Resistant Depression (MSTRD) will be used as a 
measure of treatment resistance in participants in the ECT treatment group in Phase I and will be 
performed at baseline Phase I assessment and at end-of-ECT. For Phase II participants, the MSTRD 
will be repeated at Phase II baseline and again at each infusion and follow-up session.  
 
Cognitive Assessments 
Cognitive measures including Addenbrooke’s Cognitive Examination- Revised (ACE-R); Digit Spans; 
Trails A and B; Full and Cued Selective Recall Test (FCSRT) and the Autobiographical Memory 
Interview (AMI) will be performed at baseline and end-of-ECT or four weeks in Phase I, and at 
baseline and final infusion session in Phase II, as well as at final Phase II follow-up at week 26. These 
validated cognitive measures will be performed and scored by trained researchers, and have been 
chosen as each assesses an important aspect of cognition.  
 
Psychiatric Assessments  
Structured Clinical Interview for DSM-IV Axis I Disorders (SCID) will be performed once by trained 
researchers at baseline assessment in Phase I. SCID is a structured interview which is diagnostic for 
disorders in DSM-IV and will be used in this study to confirm a diagnosis of major depressive 
disorder in participants undergoing ECT in Phase I, and to out rule any psychiatric diagnosis in 
participants in the healthy control group in Phase I.   
Hamilton Depression Rating Scale, 24-item (HRSD-24) is a validated depression rating measure 
which will be performed on a repeated basis by trained researchers in both Phases I and II as a 
measure of depressive symptomatology and response to treatment.  
Quick Inventory of Depressive Symptoms, self-report version (QIDS-SR) is a validated self-report 
measure of depressive symptoms which will be used repeatedly in both Phases I and II. It is best 
practice to include both self- and clinician-rated measures of depressive symptoms to assess treatment 
response in depression.  
 
National Adult Reading Test 
National Adult Reading Test (NART) is a measure of premorbid intelligence relative to expected 
intelligence (i.e. NART assesses IQ) and will be performed once at baseline assessment in Phase I.   
 
Childhood Trauma Questionnaire 
Childhood Trauma Questionnaire (CTQ) is a validated measure to assess for adverse early life events 
and will be completed once at baseline assessment in Phase I.  
 
Standardised Assessment of Personality- abbreviated Scale (SAPAS) 
SAPAS is a validated brief (8-item) personality assessment questionnaire which will be completed 
once at baseline assessment in Phase I. It is not diagnostic of DSM-V personality disorder.  
 
Side Effect Measures 
During infusion sessions in Phase II, adverse or psychotomimetic effects of either ketamine or 
midazolam will be monitored using validated scales comprising: Clinician-Administered Dissociative 
States Scale (CADSS), Brief Psychiatric Rating Scale (BPRS), Young Mania Rating Scale, and 
Patient-Rated Inventory of Side Effects (PRISE), administered before, during and after infusions in 
order to capture the range of possible subjective and objective side effects of either agent.  

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Telomerase Assay 
Peripheral blood telomerase activity will be measured at baseline assessment in Phase I and after 
completing the ECT course or one month for controls. Blood will be collected from fasting and resting 
(45 minutes) participants into heparinised tubes containing a Ficoll separation gradient. Peripheral 
Blood Mononuclear Cells (PBMCs) will be extracted, pelleted, washed once with phosphate buffered 
saline (PBS) and repelleted. All traces of PBS will be removed and pellets will be stored at -80°C. The 
commercially available TRAPeze® Kit RT Telomerase Detection Kit (Chemicon International, 
Millipore) will be used to assess telomerase activity according to manufacturer’s instructions and as 
used by others. This kit allows fluorometric detection of telomerase activity through using 
Amplifluor® primers.  
 
Neuroplasticity Biomarkers 
At the first infusion session in Phase II, changes in blood levels of BDNF, pMTOR, pGSK3beta and 
peEF2 in response to the ketamine/midazolam will be compared at time points -60, +40, +120 and 
+240 minutes. Blood samples for plasma will be collected using EDTA vacutainer tubes and 
centrifuged to generate plasma that will be aliquoted and stored at -80oC.  BDNF levels will be 
determined using ELISA (ChemiKine, USA) following the manufacturer’s instructions. PBMCs will 
be collected, pelleted and stored at -80oC as described above.  Semi-quantitative immunoblotting of 
PBMC lysates will be used to measure changes in levels of the other ketamine-induced  proteins using 
antibodies against activated mTOR phosphorylated at serine residue 2448 (AbCam, USA), inhibitory 
serine-9 phosphorylated GSK-3beta (Cell Signalling Technology, USA), and phospho-eEF2 (Thr56; 
Cell Signalling Technology, USA) with appropriate secondary antibodies and controlling respectively 
for total levels of mTOR, GSK-3beta and eEF2 measured using relevant antibodies.  Protein bands 
will be identified using standard chemiluminescent techniques (Millipore) visualised in a darkbox 
imager (LAS 3000, Fujifilm) and analysed using ImageJ (Image Processing and Analysis in Java) 
software.  PBMCs will be collected and analysed for telomerase activity as described in Project 2 
above at -60 and +240 minutes. One whole blood sample in a PAXgene tube will also be collected at 
time points -60 & +120 minutes for extraction of total RNA to examine RNA species such as mRNA 
or miRNA using reverse transcription PCR.  
 
Epigenetics  
In Phase I, whole blood samples will be collected using one PAXgene tube and one EDTA tube at 
baseline and post-ECT/four weeks for controls, to analyse epigenetic modulation of depression/stress-
related gene expression. Epigenetic modifications that will be examined may include DNA 
methylation, measured by pyrosequencing 27, a DNA sequencing technique that identifies methylated 
DNA nucleotide bases. A second type of epigenetic modification that may be measured is chromatin 
activation status, using chromatin immunoprecipition 28 whereby genomic DNA associated with 
chromatin in its open or closed state can be precipitated with targeted antibodies and analysed by 
quantitative PCR. Small non-protein coding RNA molecules known as microRNAs that also act as 
epigenetic modulators of gene expression, by suppressing messenger RNA translation to protein, may 
be measured using reverse transcription PCR 29.   
 
12.3.2 Endpoints assessments 
 
Please see detailed information on outcomes and assessment of endpoints in section 12.3. 
 
Efficacy Assessment 
 

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This pilot trial is not designed to assess efficacy 40. The focus is on trial process with assessment of the 
primary clinical outcome being secondary. However, efficacy data will be collected in the course of 
the trial and will be reported as part of the study findings. 
The primary outcome relating to efficacy (the assessment of which is not a primary objective) is the 
relapse rate at six months as measured by HRSD-24. Criteria for relapse are ≥10 point increase in 
HRSD-24 compared to baseline Phase 2 score plus HRSD ≥16; in addition, increase in the HRSD 
should be maintained one week later (if indicated, additional follow-ups will be arranged).  Hospital 
admission, further ECT, and deliberate self-harm/suicide also constitute relapse.  Timing of these 
events will be recorded. 
 
Secondary efficacy endpoints 
(i) 
Subjective mood ratings as measured by scores on QIDS-SR. 
(ii) 
Tolerability of ketamine vs. midazolam in terms of cognitive outcomes as measured by scores 
on ACE-R, digit spans, Trails A and B, FCSRT, and AMI.  
(iii) 
Tolerability of ketamine vs. midazolam in terms of psychotomimetic effects as measured by 
scores on CADSS, BPRS, YMRS, and PRISE.  
(iv) 
Number of adverse effects in ketamine vs. midazolam groups.  
 
Safety Assessment 
 
The following safety evaluations will be performed during the study: adverse event monitoring, vital 
signs, cognitive and clinical assessments. Regular laboratory investigations and physical examinations 
are not indicated. Blinded data will be presented to the DSMB for safety evaluation every four months. 
Minutes of the DSMB meetings including safety evaluations will be presented to the TSC at every 
meeting.  
 
12.3.3 Screening procedure  
 
Screening 
Screening will take place prior to recruitment to Phase I, Open Phase 
Screening for both healthy volunteers and patients referred for ECT will involve the following: 
- 
HRDS-24 
- 
MMSE 
- 
Brief medical history  
- 
Brief psychiatric history – to out rule bipolar affective disorder, alcohol or substance 
dependence, post traumatic stress disorder 
- 
Review of clinical notes to out rule exclusion criteria e.g. resident in a nursing home 
Date of screening, subject age, gender and reason for ineligibility (if subject is not eligible) will be 
recorded. The results of the screening evaluation must meet the inclusion/exclusion criteria for the 
subject to continue in the study. There will be one decision point regarding eligibility, according to the 
inclusion criteria specified in section 12.2.2. There are no laboratory screening tests or physical 
examinations. The maximum duration between screening and randomisation is one month. Informed 
consent will be obtained prior to any study related procedures being undertaken.  
 
Recruitment  
Phase I, Open Phase 
Healthy volunteers will be recruited by advertising in volunteer organisations with an interest in 
mental health, using the advertisement material in Appendix 2. 
Participants for the ECT treatment cohort will be recruited by verbal approach on the ward by a 
researcher, following their referral for ECT treatment.  

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Phase II, Trial Phase 
Phase II participants will be recruited from Phase I participants who have received ECT. ECT 
responders who consent to randomisation will be allocated to one of two treatment groups. 
12.3.4 Baseline assessments 
 
The following pre-treatment Baseline assessments will be performed prior to enrolling in Phase I:  
 
- Confirmation of eligibility (review inclusion/exclusion criteria)  
o In the case of Phase I participants having ECT, this involves examining the 
documented results of physical examination, laboratory investigations, ECG and chest 
X-ray which are routinely performed by the team referring the participant for ECT  
- Recording of demographics, medical history and concomitant medications  
- For women of child-bearing potential the LMP will be recorded 
- Phlebotomy for plasma and whole blood performed according to WHO “Best Practice in 
Phlebotomy”  
- An interview comprising: 
o  Structured Clinical Interview for DSM-IV Axis I Disorders (SCID), National Adult 
Reading Test (NART), Childhood Trauma Questionnaire (CTQ) and Standardised 
Assessment of Personality- abbreviated Scale (SAPAS), Hamilton Depression Rating 
Scale, 24-item (HRSD-24), Quick Inventory of Depressive Symptoms, self-report 
version (QIDS-SR)  
o Cognitive measures - Addenbrooke’s Cognitive Examination- Revised (ACE-R); 
Digit Spans; Trails A and B; Full and Cued Selective Recall Test (FCSRT) and the 
Autobiographical Memory Interview (AMI) 
- Phase I Participants undergoing ECT will also undergo a treatment review/Maudsley Staging 
Method for Treatment Resistant Depression (MSTRD)  
 
Phase I Participants who respond to ECT will be invited to participate in Phase II and upon 
clarification of their assent, will undergo the following Phase II baseline assessment: 
 
- Baseline Interview (following completion of ECT) comprising treatment review/MSTRD, 
HRSD-24, QIDS-SR and eligibility review 
- Baseline cognitive measures (ACE-R, Digit spans, Trails A and B; FCSRT and AMI) 
 
12.3.5 Subsequent study visits and procedures 
 
Phase I, Open Phase 
Subsequent visits for Phase I participants in the healthy control group will involve: 
- One follow-up visit with repeat of baseline assessments (above) including phlebotomy and 
eligibility review at four weeks 
o SAPAS and CTQ will not be repeated  
Subsequent visits for participants in the ECT treatment group will involve: 
- Weekly repeat of HRSD and QIDS-SR during course of ECT 
- End-of-ECT assessment with repeat of baseline measures (above) including phlebotomy, 
eligibility check and treatment review 
o SAPAS and CTQ will not be repeated 
 
Phase II, Trial Phase  

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Following randomisation, participants in Phase II will attend on four occasions for infusions of 
ketamine or midazolam and will also have follow-up assessments over a period of six months.  
1. The first infusion session will take place within one week of completing ECT and will 
comprise: 
- Phlebotomy for plasma and whole blood on four occasions (at time points 60mins pre-infusion 
and 40, 120 and 240 mins post-infusion) 
- Eligibility check and treatment review/MSTRD  
- HRSD-24 and QIDS-SR 
- 40-minute infusion of either ketamine or midazolam 
- Regular monitoring of heart rate, blood pressure, pulse oximetry, and ECG changes before, 
during and for 200 minutes after infusion  
- Clinician-Administered Dissociative States Scale (CADSS), Brief Psychiatric Rating Scale 
(BPRS), Young Mania Rating Scale, and Patient-Rated Inventory of Side Effects (PRISE), 
administered before, during and after infusions 
- Telephone contact by a researcher in 24 hours to enquire about side-effects  
 
2. Three subsequent infusion sessions will involve the above measures except phlebotomy.  
3. The final infusion session will also include repeat of cognitive assessments including 
Addenbrooke’s Cognitive Examination- Revised (ACE-R); Digit Spans; Trails A and B; Full 
and Cued Selective Recall Test (FCSRT) and the Autobiographical Memory Interview (AMI) 
 
Phase II participants will be followed up for six months with repeated questionnaires comprising 
eligibility review, treatment review/MSTRD, HRSD-24 and QIDS-SR at weeks 6, 8, 12 and 20 post 
infusions. A final follow-up session in week 26 will comprise the above listed measures as well as 
cognitive outcomes (ACE-R, digit spans, Trails A and B, FCSRT, and AMI). No phlebotomy will be 
performed on follow-up.  
 
A detailed description of each of the assessments is available in section 12.3. 
 
10.4.6 Method of assigning Subjects to treatment groups  
 
This is a two-phase pilot trial. There are no treatment groups in Phase I, Open Phase.  
In Phase II, Trial Phase, ECT responders will be randomised after Baseline data collection. Subjects 
will be randomly assigned to one of two treatment groups in a 1:1 ratio.  
 
Randomisation 
Computerised random allocation, using randomly permuted blocks will be done independently by the 
Centre for Support and Training in Analysis and Research (CSTAR, University College Dublin, 
www.cstar.ie). Blocks will be stratified for family history of alcohol dependence as this has been 
associated with antidepressant response to ketamine 53. Allocation information will be provided in a 
set of sealed numbered envelopes prepared by CSTAR.  The information in these will be made 
available only to the anaesthetist to ensure allocation concealment. A second identical set of envelopes 
will also be provided by CSTAR and these may be accessed for emergency purposes.  
 
Blinding  
Study treatment assignment will be blinded for both the raters and the participants. Two sets of opaque 
envelopes containing allocation information will be provided by CSTAR following randomisation as 
above. To ensure patient safety during infusions and in the post-infusion period, the anaesthetist 
administering the ketamine/midazolam infusions will not be blinded but he will not be involved in 
assessments or data analysis. Infusions will be prepared by the anaesthetist in a location separate to the 
infusion area and labelled as “trial infusion” prior to transfer to the infusion area. Success of blinding 

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for patients and raters will be assessed after the first and final treatments and at the end of the six-
month follow up.  
The second set of envelopes containing allocation information will remain unopened but may be used 
where emergency unblinding is indicated. Unblinding for one or all participants will take place if it is 
in the best interests of the participants.  In the case of an emergency, when knowledge of the treatment 
assignment is essential for the clinical management of the subject, any investigator may unblind a 
single subject. Please see further details in section 13.7.2.  
Circumstances in which unblinding for multiple or all subjects may take place include – multiple 
SAEs or SUSARs, new information regarding safety of the investigative medicinal products, and 
unsatisfactory progression of the trial.  
Any breaking of the blind, whether intentional or unintentional, will be recorded and reported to the 
sponsor as soon as possible. Unblinding for multiple or all subjects will be discussed by the trial 
steering committee at the next meeting. Unblinding will be recorded and justified in the final report.   
 
12.4 
Definition of end-of-trial 
 
End-of-trial is defined as the final follow-up visit/ home visit/telephone assessment of the last Phase II 
participant. End-of-trial will be reported to the REC and IMB within 90 days, or 15 days if the study is 
terminated prematurely. A summary report of the study will be provided to the joint REC of St. James’ 
and Tallaght Hospitals, the REC of St Patrick’s Mental Health services, and IMB within 1 year of end-
of-trial.  
The end-of-study visit form will include: 
- Assessment of endpoints i.e. clinical (HRSD-24, QIDS-SR) and cognitive (ACE-R, digit spans, 
Trails A and B, FCSRT, AMI) outcomes  
- Assessment of safety  - check for any adverse effects 
- Recording of concomitant medication  
 
12.4.1 Premature termination of the study 
 
The Sponsor and/or the Trial Steering Committee have the right at any time to terminate the study for 
clinical or administrative reasons. The DSMB may request that the trial be prematurely terminated and 
this request will be discussed in a timely manner by the Trial Steering Committee.  
 
Premature termination of the trial may take place in the event of the following: 
(i) 
New information regarding safety of investigative medicinal products 
(ii) 
Multiple SAEs or SUSARs 
(iii) 
Unsatisfactory progression of the trial 
(iv) 
Major breach of data confidentiality 
(v) 
Any situation in which premature termination of the trial is judged by the investigators 
and/or Sponsor to be in the best interests of trial participants.  
Premature termination of the trial will be reported to the IMB and Trial Steering Committee and 
justified in the final report 
 
12.5 
Discontinuation/withdrawal of subjects from study treatment 
 
Subjects have the right to voluntarily discontinue study treatment or withdraw from the study at any 
time for any reason without any consequences. The investigator has the right to discontinue a subject 
from study treatment or withdraw a subject from the study at any time if it is in the best interest of the 
subject. 

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Subjects must discontinue the investigational medicinal product(s) and be withdrawn from the study 
for any of the following reasons: 
 
- 
withdrawal of consent by the subject  
- 
any medical condition that the investigator or sponsor determines may jeopardize the subject’s 
safety if she or he continues receiving the study treatment 
- 
pregnancy 
- 
ineligibility (either arising during the study or retrospectively having been overlooked at 
screening) 
- 
an adverse event which requires discontinuation of the study medication 
- 
treatment failure and disease progression  
- 
Lack of compliance with the study and/or study procedures (e.g., dosing instructions, study visits). 
- 
Lost to follow-up – at least three documented attempts must be made to contact any subject lost to 
follow-up.  
 
Ideally all subjects who discontinue should comply with specified follow-up procedures as detailed in 
section 11.3 of this protocol, i.e. assessment via interview comprising clinical and cognitive measures. 
The only exception to this requirement is when a subject withdraws consent for all study procedures. 
There is no mandatory physical health monitoring to be performed in the event of a subject 
withdrawing consent after a complete infusion session including 200 minutes post-infusion 
monitoring, or between infusion sessions. However, in the event that consent is withdrawn in an 
infusion session, monitoring of vital signs and mental health must be performed for 200 minutes 
following the end of the infusion, comprising the measures detailed in section 11.3.  
If a subject is withdrawn before completing the study, the reason for withdrawal must be entered on 
the appropriate case report form (CRF) page. If a subject is withdrawn due to an adverse event, the 
investigator will arrange for follow-up visits until the adverse event has resolved or stabilised. 
 
13. TREATMENT OF TRIAL SUBJECTS   
 
13.1 
Description of study treatment(s) 
 
Investigative Medicinal Product - Ketamine Hydrochloride 10 mg/ml infusion at 0.5mg/kg 
(Pfizer Healthcare Ireland) made up as 50ml colourless saline solution and administered 
intravenously over 40 minutes via syringe driver pump.  
 
Active Comparator - Midazolam Hydrochloride (Hypnovel) 10mg/5ml solution at 0.045mg/kg 
(Roche Products Ireland Ltd) made up as 50ml colourless saline solution and administered 
intravenously over 40 minutes via syringe driver pump.  
 
This dose and administration was chosen based on a previous randomised controlled trial of 
ketamine and midazolam 10 in which this dosage was well-tolerated.  
 
13.2 
Formulation, packaging, and handling 
 
Commercial labels will remain on both study treatments. These detail the component, date of expiry 
and manufacturer. Further labels will be attached to each unit of product indicating: 
(i) Use of the treatment in this trial 
(ii) Name of sponsor, and principal investigator; 
(iii) Trial reference code allowing identification of the trial site, investigator and trial subject.  

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These labels will be added by pharmacy staff to each vial on receipt of delivery of the treatment to St 
Patrick’s University Hospital Pharmacy, in accordance with Annex 13 (EU Guidelines to Good 
Manufacturing Practice, Investigational Medicinal Products).  
 
Suppliers: 
Ketamine: Pfizer Healthcare Ireland, 9 Riverwalk, Citywest Business Campus, Dublin 24. 
Midazolam: Roche Products (Ireland) Ltd., 3004 Lake Drive, Citywest, Naas Rd., Dublin 24.  
 
Pharmacy performing additional packaging:  
St Patrick’s University Hospital Pharmacy, James’ St, Dublin 8.  
 
13.3 
Storage and disposition of study treatments(s) 
 
The treatments will be stored securely in a clean dry area of the pharmacy department at St Patrick’s 
University Hospital. Ampoules will be stored in the outer carton (labelled as above) in order to protect 
from light. Products will be prepared into infusions in the clinic room of the ECT department by the 
consultant anaesthetist who will administer them. The anaesthetist will be unblinded throughout and 
patients and raters will remain in a separate area for infusions and assessments. Once made up as 
identical colourless solutions, the underlying labels will be obscured by bags and infusions will begin 
within one hour of preparation. Any unused product will be returned to pharmacy and disposed of 
according to the protocols specified by the pharmacy for destruction of unused pharmaceutical 
products.  
 
The study treatment will be stored at St Patrick’s University Hospital Pharmacy Dept. under the 
responsibility of Ms. Amanda Fitzpatrick, Chief Pharmacist, St. Patrick’s University Hospital.  
Temperatures in the storage area of the pharmacy are monitored constantly by electronic thermostat 
and a printed record is available. An alarm process is instigated if the temperature varies from the 
specified room temperature. The study treatment will be stored locked in a secure area until dispensed 
for use or returned to the sponsor. The IMP ketamine is for investigational use only and is only to be 
used within the context of this study.  
 
13.4 
Accountability of the study treatments 
 
The study medication will be supplied to the pharmacy by Pfizer Healthcare Ireland (ketamine) and 
Roche Products (Ireland) Ltd (midazolam). Standard shipment arrangements will continue. Upon 
delivery, receipt of the products will be recorded by pharmacy staff and labels applied as per section 
13.2, with products then transferred to the secure storage area. Unopened products which are unused by 
end-of-trial will be returned to the manufacturer. Opened unused products will be destroyed in the 
pharmacy following the protocol for destruction of pharmaceutical products at the end of every infusion 
session.  
The investigator is responsible for the control of the treatments under investigation. Adequate records 
for the receipt and disposition of the IMP will be maintained.  
The investigator will use a standard prescription form and the investigator/research nurse will collect 
the medication from the pharmacy no more than three hours before dosing.  
Accountability and subject compliance with study treatments will be assessed by maintaining 
dispensing and return records.  Discrepancies in these return records will be dealt with initially by re-
checking and communication with pharmacy. Should a discrepancy arise which cannot be accounted 
for, this will be recorded and discussed by the TSC at their next four-monthly meeting.  
 

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13.5 
Assessment of compliance 
 
In this study, interventions will be administered intravenously by the research team and thus there is 
no opportunity for non-compliance. The investigator is responsible for ensuring that the study 
treatment is administered in compliance with the protocol. Subject compliance will be assessed by 
maintaining dispensing records. 
 
13.6 
Overdose of study treatment 
 
Given the safeguards in place (consultant anaesthetist to prepare and administer infusions, assisted by 
members of the research team), it is deemed unlikely that an overdose of study treatment could occur. 
In the improbable event of an overdose, the study subject will be monitored for any change in 
neurological status or vital signs by the consultant anaesthetist and research team members. If there are 
signs of change, e.g. drowsiness, change in blood pressure/heart rate and a known overdose has 
occurred, the subject will be counselled and accompanied by a member of the research team to the 
local emergency department (St. James’ Hospital) for medical investigation. A letter detailing 
treatment and doses administered will be provided. Reasonable efforts will be made to contact the 
NOK/RA if the subject consents. Should the subject require medical investigation and/or treatment 
due to an overdose of study treatment, cost will be covered by the SPUH indemnity policy, unless due 
to negligence or malpractice.  
 
13.7 
Prior and concomitant therapy 
 
Any medication, other than the study medication taken during the study will be recorded in the CRF. 
Medications will be documented at the point of consent, at Baseline, and changes will be noted at 
every assessment or intervention appointment thereafter.  
 
13.7.1 Permitted medications/non- investigational medicinal products 
 
All medications aside from those listed in section 13.7.2 are permitted. Treatment-as-usual will 
continue for all participants during this study. No non-investigational medicinal products will be used 
outside authorisation for the purposes of this trial.   
 
13.7.2 Prohibited medications 
 
The following medications are contraindicated in Phase II as they may significantly alter the 
pharmacokinetics of ketamine.54. Additionally, the medication theophylline is contraindicated as 
concomitant use of ketamine and theophylline may significantly reduce the seizure threshold with 
reports of unpredictable extensor-type seizures 55.  
Contraindicated medications: 
 
Ketoconazole 
Voriconazole 
Itraconazole 
Telithromycin 
Clarithromycin 
Saquinavir 
Nefazodone 
Erythromycin 
Diltiazem 
Fluconazole 
Verapamil 
Theophylline
 
Participants taking any of these medications at Screening for Phase II will be excluded from the trial. 
Medication history will be checked at Baseline in Phase II and each subsequent assessment, and 
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interventions (i.e. infusions of ketamine or midazolam), however will be followed up according to the 
framework presented in section 12.3.5. Data collected will be included in intention-to-treat analyses if 
one infusion and one follow-up assessment have been completed.  
It is not permitted for subjects to participate in investigational treatment studies while participating in 
this study. Please see exclusion criteria in 12.2.3. 
 
14. SAFETY REPORTING 
 
Safety and tolerability will be evaluated throughout the study according to the framework presented in 
section 12.3.  
 
14.1 
Definitions 
 
14.1.1 Adverse event (AE) 
 
Any untoward medical occurrence in a patient or clinical trial subject administered a medicinal product 
and which does not necessarily have a causal relationship with this treatment. 
An adverse event can therefore be any unfavourable and unintended sign (including an abnormal 
laboratory finding, for example), symptom or disease temporally associated with the use of a medicinal 
product, whether or not considered related to the medicinal product  
 
14.1.2 Adverse reaction (AR) 
 
All untoward and unintended responses to a medicinal product related to any dose. 
The phrase ‘responses to a medicinal product’ means that a causal relationship between a study 
medication and an AE is at least a reasonable possibility, i.e., the relationship cannot be ruled out. 
All cases judged by either the reporting medically qualified professional or the sponsor as having a 
reasonable suspected causal relationship to the study medication qualify as adverse reactions.   
 
14.1.3 Serious adverse event  
 
Any untoward medical occurrence or affect that at any dose:  
- 
results in death,  
- 
is life-threatening*,  
- 
requires hospitalisation or prolongation of existing hospitalisation,  
- 
results in persistent or significant disability or incapacity,  
- 
is a congenital anomaly or birth defect 
- 
important medical events** 
 
*Regarding a life-threatening event, this refers to an event in which the subject was at risk of death at 
the time of the event; it does not refer to an event which hypothetically might have caused death if it 
were more severe.  
 
**Some medical events may jeopardise the subject or may require an intervention to prevent one of the 
above characteristics/consequences. Such events (hereinafter referred to as ‘important medical events’) 
should also be considered as ‘serious’ in accordance with the definition  
 

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14.1.4 Severe adverse events 
 
The term ‘severity’ is used here to describe the intensity of a specific event. This has to be distinguished 
from the term ‘serious.  
 
14.1.5 Suspected unexpected serious adverse reactions 
An adverse reaction, the nature or severity of which is not consistent with the applicable product 
information (e.g. investigator’s brochure for an unauthorised investigational medicinal product or 
summary of product characteristics for an authorised medicinal product. 
 
14.2 
Evaluations of AEs and SAEs 
 
14.2.1 Assessment of seriousness 
The investigator should make an assessment of seriousness as defined in section 14.1.  
 
14.2.2 Assessment of casualty 
All adverse events judged by either the investigator or the sponsor as having a reasonable suspected 
causal relationship to an investigational medicinal product qualify as adverse reactions. 
The causality assessment given by the investigator should not be downgraded by the sponsor. 
The investigator/sponsor must make an assessment of whether the AE/SAE is likely to be related to 
treatment according to the following definitions: 
 
Unrelated 
Where an event is not considered to be related to the study medication. 
 
Possibly 
Although a relationship to the study medication cannot be completely ruled out, the nature of the event, 
the underlying disease, concomitant medication or temporal relationship make other explanations 
possible. 
 
Probably 
The temporal relationship and absence of a more likely explanation suggest the event could be related 
to the study medication. 
 
All AEs/SAEs judged as having a reasonable suspected causal relationship (e.g. possibly, probably) to 
the study medication will be considered as ARs/SARs. 
 
All AEs/SAEs judged as being related (e.g. possibly, probably) to an interaction between the study 
medication and another medication will also be considered to be ARs/SAR. 
 
Alternative causes such as natural history of the underlying disease, concomitant therapy, other risk 
factors and the temporal relationship of the event to the treatment should be considered. 
 
14.2.3 Assessment of severity 
 
The investigator will make an assessment of severity for each AE/SAE and record this on the CRF 
according to one of the following categories: 

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Mild 
An event that is easily tolerated by the subject, causing minimal discomfort and not interfering with 
every day activities. 
 
Moderate 
An event that is sufficiently discomforting to interfere with normal everyday activities. 
 
Severe 
An event that prevents normal everyday activities. 
 
13.7.1 Assessment of expectedness 
 
The exepctedness of an adverse reaction will be determined by the sponsor according to the 
investigator’s brochure.  
 
13.7.2 Emergency unblinding procedures 
 
Emergency unblinding can be performed by any investigator by opening one or all of the second set of 
envelopes containing allocation information. These will be securely stored in the ECT department 
where infusions are to be administered. Instructions for emergency unblinding will be included on 
each CRF/ e-CRF, and will also be prominently displayed in the ECT department.  
 
13.8 Reporting procedures for all adverse events 
 
All AEs occurring during the study observed by the investigator or reported by the subject, whether 
attributed to the study medication or not, will be recorded on the CRF. 
 
The following information will be recorded: description, date of onset and end date, severity, 
assessment of relatedness to the study medication, other suspect medication or device and action taken. 
Follow-up information will be provided as necessary. 
 
AEs considered related to the study medication as judged by an investigator or the sponsor will be 
followed until resolution or until the event is considered stable. All related AEs that result in a 
subject’s withdrawal from the study or are present at the end of the study, will be followed up until a 
satisfactory resolution occurs. 
 
It will be left to the investigator’s clinical judgment whether or not an AE is of sufficient severity to 
require the subject’s removal from treatment. A subject may also voluntarily withdraw from treatment 
due to what he or she perceives as an intolerable AE. If either of these occurs, the subject will undergo 
an end-of-study assessment and be given appropriate care under medical supervision until symptoms 
cease or the condition becomes stable. 
 
The severity of events will be assessed on the following scale:   mild, moderate, severe. 
The relationship of AEs to the study medication will be assessed by the investigator. 
 
13.9 Reporting procedures for serious adverse events 

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The investigator will report all serious adverse events immediately to the sponsor except for those that 
the protocol or investigator’s brochure identifies as not requiring immediate reporting. The immediate 
report will be followed by detailed, written reports. The immediate and follow-up reports will identify 
subjects by unique code numbers assigned to the latter. 
 
The immediate report will be made by the investigator within a very short period of time and under no 
circumstances should this exceed 24 hours following knowledge of the serious adverse event. 
 
All SAE information must be recorded on an SAE forms and sent expeditiously to the sponsor. 
Additional information received for a case (follow-up or corrections to the original case) need to be 
detailed on a new SAE form and sent expeditiously to the sponsor. 
 
The sponsor will keep detailed records of all adverse events which are reported to him by the 
investigator or investigators. 
 
In cases where reporting is not required immediately the investigator will report within the appropriate 
time frame, taking account of the specificities of the trial and of the serious adverse event, as well as 
possible guidance in the protocol or the IB. 
 
The sponsor will report all SUSARs to the competent authorities (IMB) and the ethics committees 
concerned (Joint REC of St James’ and Tallaght Hospitals and SPUH REC). Fatal or life-threatening 
SUSARs must be reported within 7 days. SUSARs which are not fatal and not life-threatening are to 
be reported within 15 days. The sponsor will also inform all investigators concerned of relevant 
information about SUSARs that could adversely affect the safety of subjects. 
 
If the initial report is incomplete, e.g. if the sponsor has not provided all the information/assessment 
within seven days, the sponsor  will submit a completed report based on the initial information within 
an additional eight days. 
 
If significant new information on an already reported case is received by the sponsor, the clock starts 
again at day zero, i.e. the date of receipt of new information. This information will be reported as a 
follow-up report within 15 days. 
 
In addition to the expedited reporting above, the sponsor shall submit once a year throughout the 
clinical trial or on request, a safety report to the competent authority (the IMB in Ireland) and ethics 
committees. The annual safety report will be presented in the DSUR format as per ICH guideline E2F 
- Note for guidance on development safety update reports. This is a legal requirement. 
 
13.10 Data safety monitoring board (DSMB)   
 
The DSMB will be an independent committee established prior to the commencement of the trial to 
assess progress, safety data and data security and will meet every four months during the trial. No 
member of the DSMB will have a conflict of interest with the Sponsor. Blinded data will be presented 
to the DSMB for safety evaluation every four months. Minutes of the DSMB meetings including 
safety evaluations will be presented to the TSC at every meeting.  
The DSMB may review unblinded data on request for individual or all participants. Additionally, the 
DSMB may request to terminate the trial according to section 13.9. The advice of the DSMB will be 
notified upon receipt by the Sponsor to the joint REC of St. James’ and Tallaght Hospitals, the REC of 

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St Patrick’s Mental Health Services, and the IMB.  With this notification a statement will be included 
indicating whether the advice will be followed. 
The Trial Steering Committee will comprise investigators, clinical experts not directly involved in the 
trial, and staff nominated by the Sponsor. The committee will include members who are independent 
of the investigators, SPUH, funders and the Sponsor. The TSC will consider and act, as appropriate, 
upon the recommendations of the DSMB and ultimately carries the responsibility for deciding on 
premature termination of the trial. The TSC will take responsibility for the scientific validity of the 
study protocol, assessment of study quality and conduct as well as for the scientific quality of the final 
study report. 
This section will be updated with details of the members of the DSMB and TSC once established.  
 
13.11 Pregnancy 
 
Pregnancy is not considered an AE or SAE however the investigator will collect pregnancy 
information for female trial subjects or female partners of male trial subjects who become pregnant 
while participating in the study.  
The investigator will record the information on a Pregnancy Notification Form and submit this to the 
sponsor. Any pregnancy that occurs in a trial subject or a trial subject’s partner during a trial will be 
followed to outcome. It may be necessary to monitor the development of the newborn for an 
appropriate period post delivery.  
While ketamine has been shown to be teratogenic in rats, there are no data of its use during human 
pregnancy, particularly at sub-anaesthetic doses. Insufficient data are available on Midazolam to 
assess its safety in pregnancy; however other benzodiazepines have been associated with 
teratogenicity. There have been no studies of sub-anaesthetic doses of midazolam in pregnancy. Date 
of last menstrual period (LMP) will be recorded at the start of ECT treatment (Phase I, Open Phase) 
and the first ketamine infusion (Phase II, Trial Phase).  Information regarding the importance of 
adequate contraception during the trial and informing researchers if there is any possibility of 
pregnancy is provided in the Participant Information Leaflet (Appendix 4).  
 
15. STATISTICS  
 
15.1  Description of statistical methods 
 
Descriptive statistics will be used to report: rates of recruitment (Phases I and II), willingness to be 
randomised (Phase II), willingness to complete assessments (Phases I and II), medical/ cognitive/ 
psychotomimetic/ general adverse events between groups in Phase II, adherence to allocated 
treatments, adherence to follow-up between groups, and reasons for drop-outs between groups.  
Relapse-free survival times will be compared between groups using Kaplan-Meier survival curves and 
log-rank test.  
As this is a pilot trial and insufficiently powered to achieve statistical significance 40, there will be no 
formal comparison of the two treatment groups in Phase II, Trial Phase. However, Cox proportional 
hazard regression analysis will provide a 95% confidence interval for an unadjusted hazard ratio for 
ketamine versus midazolam groups. This will be used to inform a future definitive trial.  
Secondary exploratory analyses (Cox proportional hazards regression models) will be used to identify 
pre-randomisation clinical factors or covariates (e.g. Family history of alcohol dependence; gender; 
age; presence of psychosis and extent of treatment-resistance in index depressive episode; ECT 
remission status) that might influence response and hep guide randomisation stratification in a future 
definitive trial.  
Data will be analysed using IBM PASW (SPSS) version 20, and “R” (R Foundation for Statistical 
Computing, Austria).  

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15.2  Determination of sample size 
 
Phase I, Open Phase - sample size 
 
Phase I will consist of a prospective cohort study of depressed patients receiving ECT and a healthy 
control group. We aim to recruit 78 ECT patients and 78 healthy volunteers, n=156 in total. Data 
collected in this cohort study will be used in the studies of telomerase activity in depression and 
studies of epigenetic modulation of depression/stress related genes, and the association between 
stressful early life experiences with epigenetic modification of gene expression, described in section 
12.  
(i) 
Only limited data are currently available to inform sample size calculations for the study 
of telomerase activity and so should be interpreted as being preliminary.  For example, 
Wolkowitz et al (2012) found mean (SD) baseline telomerase activity was significantly 
increased in depressed patients (10.8(5.7) units) compared to controls (7.9(5.0) units).21  A 
sample size of 47 per group will have 80% power to show a similar difference between 
pre-ECT depressed and control groups with a two-sided t-test and α=0.05. Lavretsky et al 
(2013) reported mean telomerase activity to increase from 2.7(1.3) units to 3.6(0.2) 
following yogic meditation for depressive symptoms.22  A sample size of 47 will have 
91% power to detect such a difference pre/post ECT in a two-sided t-test and α=0.05. As 
we plan to recruit above these numbers, sample size will be adequate.  
 
(ii) 
 Phase I (Open Phase) involves a prospective cohort study of epigenetic modulation of 
depression/stress related genes in depression and response to ECT, and a study examining 
the association between stressful early life experiences and epigenetic modification of 
gene expression within this cohort. There have been few studies of this nature previously. 
Similar studies examining DNA methylation profiles in depressed people vs. controls 
reported sample sizes of n=38 (Fuchikami, 2011), n=85 (D’Addario et al., 2013) and n= 
46 Cruceanu et al., (2013). Other studies of epigenetic modulation in antidepressant 
treatment have used samples of n=25 (Lopez et al., 2012), n=9 (Belzeaux et al, 2012), and 
n=10 (Bocchio-Chiavetto, 2013).  Previous studies examining associations between early 
life adversity and epigenetic modification of gene expression have reported sample sizes 
from n=215 (Perroud, 2011), n=99 (Tyrka, 2012) to n=24 (McGowan, 2009). Based on 
previous studies including those mentioned above, sample sizes were estimated to detect 
meaningful differences at a 0.05 significance level with 80% power.  
 
(iii) 
Participants in Phase I who are having ECT will also complete a brief personality 
assessment as part of a study to assess for association between response to ECT and scores 
on a brief personality assessment. Although there are studies which have examined 
diagnosis o personality disorder and response to ECT, there are no previous studies of a 
brief personality assessment such as the SAPAS and response to ECT to inform sample 
size, therefore this study is considered exploratory.  
 
Phase II, Trial Phase - sample size 
Phase II involves a pilot trial of ketamine vs. midazolam for relapse prevention following successful 
ECT and a study of biomarkers to predict response to ketamine.  
(i) 
There are no available data to inform the biomarker study in Phase II as this is an 
exploratory objective.  
 
(ii) 
For the pilot trial, in line with recommendations for pilot studies, a formal sample size 
calculation has not been performed. The sample size described (n=156 in Phase I, of 
which n=40 progress to Phase II), was determined through previous experience of this 

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research department of recruitment to a large (n=140) randomised controlled trial of 
depressed patients undergoing ECT, in which 60 patients were recruited in the trial’s first 
16 months. 80% of participants were retained after 12 months, and one participant 
withdrew during treatment. For this pilot trial we wish to recruit 20 patients per group, a 
total of 40, which is an acceptable number for the purposes of a pilot trial 40. Response 
rates to ECT are ~60%, so at least 66 patients need to be recruited to Phase I. Allowing for 
a 15% drop-out rate, we will therefore seek to recruit 78 patients in Phase I, and the same 
number of healthy volunteers as a control group, i.e. Phase I total, n=156.  As this study is 
a less intensive trial than the one described above, with the attraction of an additional 
therapy, we expect the recruitment rate to be higher than that reported above and to recruit 
78 participants to Phase I within 16 months. We expect that 47 of the ECT patient group 
of 78 will meet response criteria following ECT, and that 40 of these will additionally 
consent to be randomised and participate in the Phase II pilot trial.  
 
This section will be updated regularly to reflect recruitment rates on commencement of the trial.  
 
15.3 Analysis Sets 
 
Pilot trial data will be analysed on an intention-to-treat basis for all Phase II participants who complete 
at least one infusion and one post-infusion evaluation.  Data analyses will be performed blinded to 
allocation, by Prof. Leslie Daly in CSTAR (above).  
 
15.4 Demographic and Baseline disease characteristics 
 
Demographic and Baseline disease characteristic data will be summarized for each treatment group by 
presenting descriptive statistics. 
 
15.5 Efficacy Analysis 
 
15.5.1 Primary efficacy endpoint 
 
This pilot trial is not designed to assess efficacy 40. The focus is on trial process with assessment of the 
primary clinical outcome being secondary. However, efficacy data will be collected in the course of 
the trial and will be reported as part of the study findings. 
The primary outcome relating to efficacy (the assessment of which is not a primary objective) is the 
relapse rate at six months as measured by HRSD-24. Criteria for relapse are ≥10 point increase in 
HRSD-24 compared to baseline Phase 2 score plus HRSD ≥16; in addition, increase in the HRSD 
should be maintained one week later (if indicated, additional follow-ups will be arranged).  Hospital 
admission, further ECT, and deliberate self-harm/suicide also constitute relapse.  Timing of these 
events will be recorded. 
 
15.5.2 Secondary efficacy endpoints 
(v) 
Subjective mood ratings as measured by scores on QIDS-SR. 
(vi) 
Tolerability of ketamine vs. midazolam in terms of cognitive outcomes as measured by scores 
on ACE-R, digit spans, Trails A and B, FCSRT, and AMI.  
(vii) 
Tolerability of ketamine vs. midazolam in terms of psychotomimetic effects as measured by 
scores on CADSS, BPRS, YMRS, and PRISE.  
(viii) 
Number of adverse effects in ketamine vs. midazolam groups.  
Secondary efficacy endpoints will be analysed by descriptive methods.  
 

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15.6 Safety analysis 
 
Descriptive statistics will be used to report the results of clinical monitoring (heart rate, blood 
pressure, pulse oximetry, and presence of ECG changes), cognitive assessments (ACE-R, digit spans, 
Trails A and B, FCSRT, AMI), monitoring for psychotomimetic effects (CADSS, BPRS, YMRS, 
PRISE), and adverse effects between groups in Phase II. Blinded data will be presented to the DSMB 
for safety evaluation every four months. Minutes of the DSMB meetings including safety evaluations 
will be presented to the TSC at every meeting.  
 
15.7 The level of statistical significance 
 
As this is a pilot trial 40 there will be no formal comparison of the two groups but Cox proportional 
hazard regression analysis will provide a 95% confidence interval for an unadjusted hazard ratio that 
will allow interpretation of statistical difference between ketamine and midazolam groups. 
 
15.8 Criteria for the termination of the trial 
 
The trial will be terminated once 20 subjects have been allocated to each arm of Phase II.  
 
15.9 Procedure for accounting for missing, unused and spurious data 
 
As this is a pilot trial and small numbers of participants are involved, no missing values will be 
imputed. 
 
15.10 
Procedure for reporting any deviation(s) from the original statistical plan 
 
Deviations from the original statistical plan will be reported to the Sponsor within a timely interval 
and discussed by the Trial Steering Committee at the next meeting. These will be recorded and 
justified in the final report. Where a deviation from the original statistical plan is judged by the 
investigators or Sponsor to comprise a substantial amendment to the trial protocol, the standard 
procedure for reporting substantial amendments to the IMB will be followed.  
 
16 DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
 
Direct access will be granted to authorised representatives from the sponsor, host institution and the 
regulatory authorities to permit trial-related monitoring, audits and inspections. 
 
17 DATA HANDLING AND RECORD KEEPING 
 
Data will be entered, handled and stored at St Patrick’s University Hospital. It will be anonymised and 
then processed by members of the research team at Trinity College Institute of Neuroscience and at the 
Centre for Support and Training, University College Dublin.  
 
17.1  Data collection, source documents and case report forms (CRF):  
 
Source documents for this study include clinical notes, ECT booklets, medication records, and study-
specific data collection documents. Information will be extracted from these documents and recorded 
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in such a way that the original entry can still be read. The correct entry will then be clearly inserted, 
and the alterations will be initialled and dated by the investigator. Data reported on the CRF that are 
derived from source documents must be consistent with the source documents or the discrepancies 
must be explained.  
All documents will be stored safely in a designated locked filing cabinet in a locked office within the 
Research Building at St Patrick’s University Hospital and confidentiality will be observed at all times. 
With the exception of the informed consent form, subjects will be referred to only by their subject 
identification number on all study-specific documents, whether hard copies or electronic. Anonymised 
biological materials will be stored and processed in Professor McLoughlin’s laboratory facilities in 
Trinity College Institute of Neuroscience and the Institute for Molecular Medicine in St James’s 
Hospital. Data analysis will take place in another facility (Centre for Support and Training in Research 
and Analysis (CSTAR), University College Dublin), however data will be anonymised prior to secure 
transfer to CSTAR for analysis. 
 
17.2 Data reporting  
 
The trial Data Safety Monitoring Board will be responsible for overseeing data security. Subjects will 
be identified by a study specific subject number and/or code in the database. The name and any other 
identifying detail will not be included in any study data electronic file. 
 
18 RETENTION OF ESSENTIAL DOCUMENTS 
 
Biological material will be retained securely following the protocols in place at Trinity College Dublin, 
for a period of four years following trial termination, and disposed of by staff authorized to do so by 
Trinity College Dublin and in accordance with the institution’s policies and data protection legislation. 
Data derived from biological material and essential trial documents will be retained for a period of at 
least five years in accordance with Article 17 of EU Directive 2005/28/EC. These will be retained for 
no longer than ten years and will then be destroyed in accordance with data protection legislation at 
that time. This is included in the Control and Participant Information Leaflets and consent forms. The 
essential documents are defined as those that individually and collectively permit evaluation of the 
conduct of the trial and the quality of the data produced. These documents will be filed in an organised 
way that facilitates management of the clinical trial, audit and inspection by competent authorities and 
will be readily available on request. 
As this is an academic study, recommendations regarding retention of essential documents for EMA 
approval/ clinical development of the IMP are not of concern here.  
 
19 QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES 
This study will be conducted in accordance with the current approved protocol, ICH GCP, relevant 
regulations and standard operating procedures. The measures taken to ensure data obtained is accurate, 
complete and reliable include: 
(i) 
Researchers will attend GCP training, e.g. Martha Finnegan attended GCP training 
provided by Molecular Medicine Ireland on 11.3.14. 
(ii) 
Researchers will be trained in administration of the primary assessment tool used in this 
study, the HRSD-24. Administration will be according to specified guidelines 56 and 
training will be repeated every 6 months to ensure inter-rater reliability.  
(iii) 
Quality assurance in the laboratory is assured by adherence to protocols outlined by 
Molecular Medicine Ireland(http://www.molecularmedicineireland.ie/libraries/libgroup/8) 
and manufacturers' instructions for any laboratory assay products used. 
 
The trial site, laboratory and Sponsor’s/research team’s offices are subject to GCP inspection at any 
time. In accordance with the legislation, the trial master file comprising the essential documents which 
enable both the conduct of the trial and the quality of the data produced to be evaluated will be 

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available to provide the basis for the GCP inspection. Responses to a GCP inspection report will be 
provided within 30 working days of the date of issue.  
 
20 AUDITS AND INSPECTIONS 
 
This trial may be subject to internal or external auditing or inspections procedure to ensure adherence 
to GCP. Access to all trial-related documents will be given at that time. 
 
21 ETHICS 
 
An application for approval for this trial will be submitted to the joint REC of St. James’ and Tallaght 
Hospitals for ethical review. An application will also be submitted to the local site REC (St Patrick’s 
Mental Health Services).  
 
21.1 Declaration of Helsinki 
 
The sponsor will ensure that this study is conducted in accordance with the ethical principles that have 
their origins in the Declaration of Helsinki. 
 
21.2 Good Clinical Practice 
 
This study will be conducted in accordance with Good Clinical Practice (GCP), as defined by the 
International Conference on Harmonisation (ICH) and in accordance with the ethical principles 
underlying European Union Directive 2001/20/EC and 2005/28/EC. 
 
21.3 Approvals 
 
Required documents including the protocol, informed consent form, subject information leaflet, 
investigational medicinal product dossier, investigators brochure and any other required documents 
will be submitted to a recognised research ethics committee and the competent authority for written 
approval. The sponsor will submit and obtain approval from the above parties for substantial 
amendments to the original approved documents. 
 
21.4  Informed consent 
 
Written consent will be obtained by members of the research team using the study-specific consent 
form (Appendix 5). Potential participants will be provided with an information leaflet  and letter of 
invitation (Appendix 4) and verbal information at the first point of contact with a member of the 
research team. This process will take place following screening for Phase I, at which time informed 
consent for both Phases I and II will be completed. If invited to partake in Phase II, verbal assent will 
be confirmed after completion of ECT.  
 
Time will be provided to address questions. Every effort will be made to provide adequate time for the 
participant to consult with family, friends and their general practitioner prior to making a decision, 
however as it is common for referral for ECT to take place the day before administration of the first 
ECT treatment, in some cases, provision of information and the process of obtaining consent may take 
place on the same day. Participants will be encouraged to reflect on the information provided and ask 
questions but it is recognised that some participants may prefer to make a decision at the first point of 
contact and this will also be accomodated. Prior to any study-related screening procedures being 

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performed on the subject, the informed consent statement will be reviewed and signed and dated by 
the subject and investigator. Informed consent will be obtained verbally at each intervention e.g. 
phlebotomy, infusion, by the member of the research team who will perform the intervention.  
 
21.5 Benefits and risks assessment  
 
This study may further the understanding of the pathogenesis of depression, a major public health 
issue, and provide further information about a potential treatment to reduce relapse rates. In designing 
this study, all efforts have been made to reduce the risk to and burden for participants. It is believed 
that risk to and burden for the subject will be in proportion to the potential value of this research. 
There are no guaranteed direct benefits to participants.  However all participants may benefit 
indirectly from participation in terms of increased awareness of mental health issues. Some Phase II 
participants may benefit from the administration of ketamine in terms of reduced six-month relapse 
rates, but this is not guaranteed.  
 
This study will not include incapacitated adults or minors. Issues regarding specific vulnerable 
populations are addressed individually below: 
 
(i) This study requires inclusion of adults with mental illness to address the research question. Only 
those who have capacity to provide valid informed consent will be invited to participate. An 
assessment of capacity to make treatment decisions is made by the treating team prior to ECT 
treatment. This assessment will guide investigators in selecting those who have capacity to consent to 
enrolment in the trial. The trial is expected to benefit participants who have a mental illness indirectly 
by improving scientific knowledge of a major mental health issue. There are possible direct and 
indirect benefits to participants with a mental illness in terms of reduced relapse rate and increased 
awareness of mental health through participation, however there is no guaranteed direct benefit.  
 
(ii) Healthy volunteers will be used as a comparison group in order to investigate the role of 
telomerase in depression and response to ECT. This objective cannot accurately be achieved without a 
healthy control group. The trial is not expected to benefit healthy volunteer participants directly, there 
may be indirect benefits in increased awareness of mental health, and increased scientific knowledge 
gained. It is not anticipated that healthy volunteer participants would experience any adverse effects 
other than lifestyle inconvenience and discomfort during phlebotomy, as a result of participation. 
Risks and burden are considered minimal in this population.  
 
Women of childbearing age are defined by the Irish Central Statistics Office as women of ages 15-49 
(Census 2011 This is Ireland (Part 1) - CSO - Central Statistics Office). Women of this age group will 
not be excluded from this study as this group constitutes a significant proportion of the population of 
interest, i.e. people with treatment resistant depression. Irish women are more likely to suffer from 
depression than men and 25% of women in Ireland will require treatment for depression in their 
lifetime. Thus the primary study objective cannot be accurately achieved without inclusion of women 
of childbearing age. Previous studies of both ECT and ketamine have included women of childbearing 
age and precautions will be taken as detailed in point 12.3 to ensure adequate contraception is in place 
throughout the trial. Women of childbearing age may be in the healthy volunteer group or adults with 
mental illness group with expected benefits to these participants as listed above.  
 
21.6 Subject confidentiality 
 
Investigators will ensure that the subjects’ anonymity is maintained. The subjects will be identified 
only by initials and a subject’s identification number on the CRF and any database. All documents will 
be stored securely. The study will comply with the Data Protection Act. 

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21.7 Other ethical considerations: 
 
Use of placebo/active comparator: Participants in this pilot trial will continue usual care as 
recommended by their responsible clinical team. They may receive an additional treatment as part of 
this pilot trial but will not be denied any treatment for the purposes of this trial. Participants will be 
provided with verbal and written information regarding the possibility of being randomised to a 
placebo group. There is no evidence to suggest that subjects who are randomised to the placebo arm of 
the study will suffer poorer outcomes, as a study of this nature has not yet been performed. The DSMB 
will monitor data for safety parameters throughout the trial including the possibility of a large 
discrepancy between placebo and ketamine groups, and in the event of such a circumstance, will 
follow the specifications in this protocol.  
 
22 FINANCING AND INSURANCE/INDEMNITY 
 
Financing at the time of Version 1.0 of this protocol is through pre-existing research grants under the 
supervision of Prof. D. McLoughlin, Principal Investigator. This funding is from sources not 
connected with the design, completion or reporting of this trial. Funding is also currently being sought 
from the HRB.  Applications for further funding will continue and funding details will be updated as 
necessary.  
Insurance is provided by indemnity cover for research in place at St Patrick’s University Hospital. 
This will be in place once the trial is approved by the St Patrick’s Mental Health Services REC, an 
application for which will be submitted following approval by the authorised clinical trials REC of St. 
James’ and Tallaght Hospitals.  
Please see Appendix 6, “St Patrick’s Mental Health Services Indemnity Policy”.  
 
23 CLINICAL STUDY REPORT AND PUBLICATION POLICY 
 
The publication policy involves formal presentation of preliminary study findings at national and 
international neuroscience and psychiatry meetings. Final findings will be submitted for peer-review 
and publication in relevant high-impact scientific journals and upon publication they may be further 
publicized in national and international print and electronic media through the TCD and SPUH 
websites and public relations departments. Further knowledge dissemination will include registering 
the trial in the EudraCT database and publication of the trial protocol in a peer-reviewed journal. 
During the trial itself, a six-monthly newsletter will be sent to all participants, detailing progress in 
recruitment with lay summaries of research findings relevant to the study. Research progress and 
developments will be regularly presented at medical “grand rounds” in St. Patrick’s University 
Hospital (SPUH), and in-house research meetings and seminars in Trinity College Institute of 
Neuroscience (TCIN).  Information about the research programme and other ongoing related 
depression research will also be contributed through our group’s website 
(http://www.medicine.tcd.ie/psychiatry/research/projects/depression-neurobiology.php) available to 
the general public.   
The clinical study report will be presented to the REC and IMB within one year of the completion or 
cancellation of the trial. The format of this summary will comply with the EU Note for Structure and 
Content of Clinical Study Reports (CPMP/ICH/137/95). The clinical study report will be signed by the 
principal investigator.   
 
 
 
 

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