statistical analysis plan version 2.0 date: 06/06/2018...poppi version date: 06/06/2018 version...

Poppi: A blinded randomised placebo-controlled trial investigating the efficacy of morphine analgesia for procedural pain in infants. Funded by the Wellcome Trust and National Institute for Health ISRCTN Registry ID: 82342359 eudraCT number: 2014-003237-25

NPEU Clinical Trials Unit Version 2.0 Effective Date: 21 March 2017

Form ID: ST105-A of 24

Statistical Analysis Plan Version 2.0

Date: 06/06/2018

Protocol version: 6.0

A blinded randomised placebo-controlled trial investigating the

efficacy of morphine analgesia for procedural pain in infants.

Authors: Jennifer Bell (Trial Statistician, NPEU CTU) Louise Linsell (Senior Trial Statistician, 2017 to present,

NPEU CTU) Reviewers: Rebeccah Slater (Principal Investigator) Caroline Hartley (Poppi Postdoctoral Research Fellow)

Poppi Version date: 06/06/2018 Version number: 2.0

Poppi Statistical Analysis Plan v2.0 18_06_06.docx of 24

TABLE OF CONTENTS List of abbreviations ................................................................................................................... 4

1 Introduction ....................................................................................................................... 5

2 Deviation from analysis described in protocol .................................................................. 6

3 Background information .................................................................................................... 7

3.1 Rationale .................................................................................................................... 7

3.2 Objectives of the trial ................................................................................................. 7

3.3 Trial design ................................................................................................................. 7

3.4 Eligibility ..................................................................................................................... 8

3.5 Interventions .............................................................................................................. 8

3.6 Definition of primary and secondary outcomes ........................................................ 8

3.6.1 Primary outcomes .................................................................................................. 8

3.6.2 Secondary outcomes .............................................................................................. 9

3.7 Hypothesis framework ............................................................................................... 9

3.8 Sample size & power .................................................................................................. 9

3.9 Intervention allocation ............................................................................................ 10

3.10 Data collection schedule .......................................................................................... 10

3.11 Interim analyses and stopping rules ........................................................................ 11

3.11.1 Trial stopping rules........................................................................................... 11

3.12 Trial reporting .......................................................................................................... 12

4 Protocol non-compliances ............................................................................................... 12

4.1 Major ........................................................................................................................ 12

4.2 Minor ........................................................................................................................ 12

5 Adherence to the intervention ........................................................................................ 13

6 Analysis populations ........................................................................................................ 13

6.1 Post-randomisation exclusions ................................................................................ 13

6.2 Population definitions .............................................................................................. 14

6.2.1 Per-protocol population ...................................................................................... 14

6.2.2 Safety analysis population ................................................................................... 14

7 Descriptive analyses ......................................................................................................... 14

7.1 Representativeness of trial population and participant throughput ...................... 14

7.2 Baseline comparability of randomised groups ........................................................ 15

7.3 Other descriptive analysis ........................................................................................ 16

7.4 Losses to follow-up .................................................................................................. 16

8 Comparative analyses ...................................................................................................... 16

8.1 Detailed definition of outcomes .............................................................................. 17



8.2 Primary analysis ....................................................................................................... 17

8.2.1 Analysis of the primary and co-primary outcomes.............................................. 17

8.2.2 Analysis of secondary outcomes .......................................................................... 17

8.2.3 Safety analysis ...................................................................................................... 18

8.3 Secondary analyses .................................................................................................. 18

8.4 Pre-specified subgroup analyses ............................................................................. 18

8.5 Sensitivity analyses .................................................................................................. 18

8.6 Significance levels .................................................................................................... 18

8.7 Missing data ............................................................................................................. 19

8.8 Statistical software employed ................................................................................. 19

9 Safety data analysis .......................................................................................................... 19

10 Additional analysis ........................................................................................................... 19

10.1 Additional objectives ............................................................................................... 19

10.2 Analysis of additional objectives.............................................................................. 20

11 References ....................................................................................................................... 21

11.1 Trial documents ....................................................................................................... 21

11.2 Other references ...................................................................................................... 21

12 Approval ........................................................................................................................... 22

13 Document history ............................................................................................................ 22



LIST OF ABBREVIATIONS AE Adverse event CI Chief Investigator CRF Case Report Form DMC Data Monitoring Committee eCRF Electronic Case Report Form EEG Electroencephalography EME Efficacy Mechanisms & Evaluation HDU High-dependency unit IMP Investigational Medicinal Product ISRCTN International Standard Randomised Controlled Trial

Number ITU Intensive treatment unit IVF In vitro fertilisation IVH Intraventricular haemorrhage kg Kilogram LDU Low-dependency unit ml Millilitre NIHR National Institute for Health Research NPEU CTU National Perinatal Epidemiology Unit Clinical Trials

Unit PI Principal Investigator PIPP Premature Infant Pain Profile PIPP-R Premature Infant Pain Profile – Revised PMG Project Management Group RCT Randomised controlled trial ROP Retinopathy of Prematurity RMS Root mean squared SAE Serious adverse event SD Standard deviation SUSAR Suspected unexpected serious adverse reaction TSC Trial Steering Committee μg Microgram

http://www.ct-toolkit.ac.uk/glossary/suspected-unexpected-serious-adverse-reactions-susar



1 INTRODUCTION This document details the proposed presentation and analysis for the main paper(s) reporting results from the Wellcome Trust and National Institute for Health Research (NIHR) Efficacy Mechanisms & Evaluation (EME) programme funded blinded randomised placebo-controlled trial Poppi. Recruitment to the trial was stopped on 13 December 2017 following recommendation by the Data Monitoring Committee (DMC) and a decision by the Trial Steering Committee (TSC). The DMC reviewed data collected in the first 25 trial participants on 4 December 2017, followed by a further review of the first 31 trial participants on 7 December 2017, and recommended that recruitment to the trial should be stopped due to safety concerns relating to the side effects of oral morphine. The TSC met on 13 December 2017 to review the same data on the first 31 trial participants and to discuss this recommendation. They unanimously agreed that the trial should not continue in its present form, and recommended that it should be paused. The TSC advised that data collected so far should be fully analysed by the investigators and, in light of the analysis, a revised protocol submitted to the appropriate regulatory bodies. The NIHR supported the TSC recommendation initially, but have since (5 March 2018) decided to withdraw funding for the Poppi trial, and invite a new application to address these issues. This statistical analysis plan therefore describes the proposed analysis of the data collected in the first 31 participants in the Poppi trial. The analysis plan will be available on request when the principal papers are submitted for publication in a journal. Suggestions for subsequent analyses by journal editors or referees, will be considered carefully, and carried out as far as possible in line with the principles of this analysis plan; if reported, the source of the suggestion will be acknowledged. The statistical analysis will be carried out by an identified, appropriately qualified and experienced statistician, who will ensure the integrity of the data during their processing. Examples of such procedures include quality control and evaluation procedures. Some additional analyses will be carried out by an appropriately qualified postdoctoral research fellow, with the relevant experience and expertise in analysis in this field.



2 DEVIATION FROM ANALYSIS DESCRIBED IN PROTOCOL Due to final analysis taking place earlier and with a smaller sample size than anticipated, the following deviations from the analysis described in the protocol have been made:

Significance levels – 95% confidence intervals will be reported for all comparisons of outcomes, instead of reporting 99% confidence intervals for secondary outcomes

Models will not adjust for minimisation factors due to the small sample size

Clinical stability analysis will not use Poisson or linear regression for count outcomes (number of episodes of bradycardia, tachycardia, desaturation and apnoea). Instead, the Wilcoxon rank-sum test will be performed on the standardised difference in the number of episodes of bradycardia, tachycardia and desaturation before and after the clinical intervention. Median differences between the groups with confidence intervals will be calculated for these standardised differences

Increased respiratory support will be analysed using crude risk ratios, instead of logistic regression

Episodes of apnoea will be treated as a binary composite of new-onset apnoeas or an increase in the number of apnoeas, analysed using crude risk ratios

The safety analysis will not use logistic regression, but will instead calculate risk differences with confidence intervals

Additional analyses (section 10).



3 BACKGROUND INFORMATION 3.1 Rationale Infant pain has both immediate and long-term negative consequences, yet in clinical practice it is often under-treated. To date, very few pain-relieving drugs have been tested in infants, mainly because of difficulties in assessing response to treatment. Morphine is a potent analgesic that provides effective pain relief in adults, but there is inconclusive evidence for its effectiveness in infants. The purpose of this trial is to establish whether oral morphine provides effective analgesia for procedural pain in infants. Retinopathy of prematurity (ROP) screening and clinical heel lancing are painful medical procedures that are routinely performed on premature infants. The trial will investigate whether morphine is an effective analgesic for these procedures. 3.2 Objectives of the trial

Primary Objective (i) To test whether administration of morphine reduces clinical pain scores (PIPP-R)

during the 30 second period after ROP screening compared with a placebo

(inactive solution).

Co-primary Objective (ii) To test whether administration of morphine reduces nociceptive-specific brain

activity following a clinically-essential heel lance compared with a placebo

(inactive solution).

Secondary Objectives (iii) To test whether administration of morphine improves clinical stability in the 6-

hour and 24-hour period following the start of the clinical intervention. The

clinical intervention is defined as the heel lance followed by ROP screening.

(iv) To test whether administration of morphine reduces clinical pain scores (PIPP-R)

and reflex withdrawal activity following a clinically-essential heel lance compared

with a placebo (inactive solution).

(v) To test whether administration of morphine is safe by determining whether it

results in episodes of respiratory depression or hypotension that require

intervention.

3.3 Trial design This study is a clinical trial of an investigational medicinal product (IMP). It is a single-centre double-blind randomised placebo-controlled study investigating whether morphine sulphate provides effective analgesia compared with a placebo (inactive solution) for clinically required ROP screening and heel lancing.



A 33-month recruitment period was originally planned. Infant participants were studied on a single test occasion while they were in hospital, when they required ROP screening and a clinical heel lance on the same occasion. No extra blood tests or noxious procedures were performed for the purpose of the study. Participants were included in the study for a 48-hour period (24 hours before and after the start of the clinical intervention). The clinical intervention is defined as the heel lance followed by the ROP screening. The start of the clinical intervention is therefore defined as the time when the heel lance was performed. 3.4 Eligibility Inclusion criteria

In-patients on the neonatal unit at the John Radcliffe Hospital, Oxford

Infants born less than 32 weeks’ gestation or birth weight less than 1501 g

Infants between 34 and 42 weeks’ gestational age at the time of study. Infants were studied if they required a clinical heel lance and ROP screening on the same test occasion

Infants for whom parents/guardians gave written informed consent for inclusion in the trial.

Senior clinician considered inclusion in trial to be medically appropriate. Exclusion criteria

Intraventricular haemorrhage (IVH) > grade II

Short bowel syndrome

Receiving nil by mouth due to documented gut pathology

Received opiates in the last 72 hours

Received other analgesics or sedatives in the last 24 hours

Previously documented episode of morphine sensitivity

Congenital malformation or genetic condition known to affect neurological development

Born to mothers who regularly used opiates during pregnancy or while breastfeeding or expressing breast milk.

3.5 Interventions Treatment group: Morphine sulphate in carrier solution. 10ml of the IMP was supplied in an amber glass bottle at a concentration of 200 μg/ml. This provided a dose of 100 μg/kg which was administered orally by sterile oral/enteral syringe. Comparator: Matching placebo (carrier solution only). 3.6 Definition of primary and secondary outcomes

3.6.1 Primary outcomes

PIPP-R score during the 30 second period after ROP screening

Magnitude of nociceptive-specific brain activity evoked by heel lance



3.6.2 Secondary outcomes

Clinical stability in the 6-hour and 24-hour period following the start of the clinical

intervention. The clinical intervention is defined as the heel lance followed by ROP

screening

PIPP-R score during the 30 second period after heel lance

Amplitude of reflex withdrawal following heel lance

Drug safety: o Number of incidences of apnoea that require intervention using NeoPuff or

‘bag and mask’ o Number of incidences of hypotension that require treatment with inotropes

in the 24-hour period following the administration of the IMP or placebo. 3.7 Hypothesis framework This is a superiority trial, and all comparisons will be analysed and presented on this basis. 3.8 Sample size & power The primary objective of this study was to determine whether morphine administration reduces clinical pain scores (PIPP-R) 30 seconds after ROP screening in the infants administered morphine compared with infants administered a placebo (inactive solution). The co-primary objective was to determine whether morphine administration reduces nociceptive-specific brain activity in response to a heel lance, compared with administration of a placebo (inactive solution). A 2-point reduction in PIPP scores was considered to be a clinically-meaningful reduction in pain (1) and scores below 7 were considered to reflect minimal pain (2). Three studies have used PIPP scores to assess analgesic efficacy following ROP screening (3-5). Using the most conservative data, where the mean PIPP score post ROP screening was 8.3 (standard deviation [SD] 3.5), we would have required 66 infants per group to observe a 2-point reduction in PIPP-R scores, with a power of 90% at a two-sided significance level of 0.05. We also considered the sample size required to see a significant difference between the groups in the co-primary outcome measure (i.e. the nociceptive-specific brain activity evoked by a heel lance). In this study we assumed that a 40% reduction in the magnitude of the nociceptive-specific brain activity represents a clinically-meaningful reduction in brain activity, which in adults would be concomitant with a significant reduction in verbally reported pain scores. A 40% reduction would lead to the noxious components having a post-treatment amplitude of approximately 0.12 (SD 0.14) down from a mean of 0.20. Coincidentally, this size of reduction also required 66 infants per group for a power of 90% at a two-sided significance level of 5%. Allowing for a loss to follow-up rate of approximately 10% (i.e. due to technical difficulties during physiological monitoring), a total of 148 infants were needed to be included in the study. We anticipated the proportion of twins eligible for the trial to be around 25% based on past studies (e.g. NPEU-run BOOST-II UK, I2S2, PiPS and SIFT trials). There is evidence that the correlation in pain outcomes between twins is 0.5 (6). Hence the effect of clustering



(technically the design effect) was calculated to be 1.06. This inflated the total sample size required (90% power, 5% two-sided significance level, 10% loss to follow-up and accounting for multiple births) from 148 to 156 (78 per group). 3.9 Intervention allocation The allocation ratio of intervention (morphine) to control (placebo) arms was 1:1. Randomisation was managed via a secure web-based randomisation facility hosted by the National Perinatal Epidemiology Unit Clinical Trials Unit (NPEU CTU) at the University of Oxford. Participants had a roughly equal chance of receiving morphine sulphate or placebo (inactive solution). The randomisation program used a minimisation algorithm to ensure approximate balance between the groups with respect to:

gestational age at the time of randomisation (34+0 to 35+6 weeks, 36+0 to 37+6 weeks,

38+0 to 39+6 weeks, 40+0 to 42+6 weeks)

gestational age at birth (< 28+0 weeks, ≥ 28+0 weeks)

number of days on a ventilator (0, 1 to < 7, 7 or more)

presence of gastric tube at time of randomisation (yes, no)

number of days since morphine has been given (no morphine given, < 3, 3 to < 7, 7

or more)

intra-uterine growth restriction (yes, no)

previous surgery (yes, no).

The users of the system had no insight into the next allocation. Participants were randomised shortly before the 48-hour period of clinical stability monitoring commenced. If exclusion criteria manifested in the first 24 hours of clinical stability monitoring, the IMP or placebo was not administered at this time. If subsequent ROP screening was required and the infant met all eligibility criteria, the IMP or placebo was administered at the required time point for this subsequent ROP screen. Clinical stability measures for this infant were recorded in the 24 hours before and after the clinical intervention related to the subsequent ROP screen.

A Senior Trials Programmer at the NPEU CTU wrote the randomisation program and holds the allocation code. 3.10 Data collection schedule Clinical information was collected using the following electronic case report forms (eCRFs) in the clinical database OpenClinica:

Trial Entry

Clinical Stability

Safety Data

Respiratory Support

PIPP-R

Test Occasion: Heel Lance

Neurophysiology



Withdrawal

Foreseeable SAE

SAE

Adverse Events.

3.11 Interim analyses and stopping rules A Data Monitoring Committee (DMC), independent of the trial organisers and of the TSC, reviewed the trial’s progress and provided advice on the conduct of the trial to the TSC and (via the TSC) to the PMG. The terms of reference for the DMC were agreed at their first meeting, and a DMC Charter was completed and signed by all members. An interim statistical analysis of the primary and secondary outcomes was planned when 50% of trial participants (78 infants) had completed data collection. In the light of the interim data analysis, and other evidence from relevant studies (including updated overviews of relevant randomised controlled trials), the role of the DMC was to inform the TSC, if in their view there was proof beyond reasonable doubt that the data indicate that any part of the protocol under investigation was either clearly indicated or contra-indicated. Guidelines for early cessation due to safety concerns were agreed with the DMC and documented (see 3.11.1 Trial stopping rules). Unless modification or cessation of the trial was recommended by the DMC, the TSC, investigators, collaborators and administrative staff (except those who supply the confidential information) remained ignorant of the results of the safety analyses. See the DMC Charter for more information.

3.11.1 Trial stopping rules

Drug safety was assessed by measuring the number of occurrences of apnoea that required intervention using NeoPuff or ‘bag and mask’, or hypotension that required treatment with inotropes in the 24-hour period after drug administration. The DMC planned to review trial safety outcomes after every 25 patients had been randomised and safety data collected (i.e. n=25, 50, 75, 100 and 125). In addition, the Chief Investigator (or suitably trained delegate) was notified of every such occurrence. A formal sequential safety procedure was applied and presented to the DMC for occurrences of apnoea that required intervention using NeoPuff or ‘bag and mask’. We employed a stopping boundary using group sequential methods with a boundary agreed by the DMC and specified in the DMC Charter. Specifically, a one-sided Gamma spending function (7), with gamma equal to 4.5. This is a type of α-spending function (8), which is a type of stopping boundary where the type I error rate is spread across a number of analyses. They can take many different forms and shapes, distributing the error in different ways. The boundary is a function of the amount of information collected (in this case, the number of infants with safety outcome data available). A type I error rate of 0.2 was used, which was estimated to result in power of 0.79. This boundary was based on the assumption of a control group event rate of 7%, and a difference between the group event rates of 12%.



At the first DMC safety review of 25 and the subsequent requested review of 31 babies, a graph showing evidence of the relative safety of the treatments was provided as a guide, in addition to trial data summarised by trial arm, clinical summaries for participants who had experienced safety events, and the completed case report forms for all infants. The DMC Charter states that if the pre-specified stopping boundary is crossed, then the DMC should consider terminating the trial, taking into account other considerations (e.g. implications on future patients/clinical practice and follow-up, how convincing the evidence is, etc.). It is important to note that the stopping boundary was intended as an aid to the decision to stop the trial, and not as a definitive stopping rule. There was no stopping rule or plan to stop early due to benefit. 3.12 Trial reporting The trial will be reported according to the principles of the CONSORT statement. The final analysis will be conducted for all outcomes collectively, at the end of the trial.

4 PROTOCOL NON-COMPLIANCES A protocol non-compliance is defined as a failure to adhere to the protocol such as the wrong intervention being administered, incorrect data being collected and documented, errors in applying inclusion/exclusion criteria or missed follow-up visits due to error. All protocol non-compliances will be summarised by trial arm in the final report.

4.1 Major

The following are pre-defined major protocol non-compliances with a direct bearing on the primary outcome:

Data considered fraudulent

4.2 Minor

The following are defined as minor protocol non-compliances: Participants randomised in error: These include participants:

born ≥ 32 weeks' gestation and with birth weight ≥ 1501 g

< 34 or > 42 weeks’ gestation at the time of the study

who did not require a clinical heel lance and ROP screening on the same test occasion

with IVH > grade II

with short bowel syndrome

receiving nil by mouth due to documented gut pathology

who received opiates in the last 72 hours

who received other analgesics or sedatives in the last 24 hours

with a previously documented episode of morphine sensitivity

with a congenital malformation or genetic condition known to affect neurological development



born to mothers who regularly use opiates during pregnancy or while breastfeeding or expressing breast milk.

Participants who do not receive allocated treatment: These include participants:

in the morphine arm who did not receive morphine (i.e. received placebo or no study treatment)

in the placebo arm who did not receive the placebo (i.e. received morphine or no study treatment).

Deviations related to treatment:

Participants who receive more or less than 100 μg/kg morphine sulphate or placebo

Incorrect timing of study treatment in relation to the timing of the clinical intervention (heel lance followed by ROP screening).

5 ADHERENCE TO THE INTERVENTION Adherence to the intervention will be reported in the process outcomes table. The median, interquartile range and range of the volume of IMP given, and the number and percentage of infants receiving more than the correct dose for their weight (100 μg/kg), plus a 5% tolerance to account for measurement error, will be reported. Timing of the study treatment in relation to the clinical intervention will also be reported in this table. The time between study treatment administration and the clinical intervention will be summarised by trial arm using median and interquartile range, and ranges.

6 ANALYSIS POPULATIONS 6.1 Post-randomisation exclusions The following will be excluded from the per-protocol analysis population post-randomisation:

Participants who did not receive the study treatment

Participants who did not receive the clinical intervention (heel lance followed by ROP screening)

Participants randomised in error

Participants for whom full consent was not obtained

Participants for whom consent to use their data was withdrawn by the parent(s) (parents can specify whether data collected up to the point of withdrawal can be used. If the response is ‘No’, then they will be considered post-randomisation exclusions. If the response is ‘Yes’, then they will be reported as ‘missing’ for any data not collected after withdrawal)

Participants for whom an entire record of fraudulent data was detected.



The numbers of post-randomisation exclusions will be reported by trial arm, and reasons summarised, in the CONSORT flow diagram. 6.2 Population definitions

6.2.1 Per-protocol population The per-protocol population will be all infants randomised who received both the study treatment and the clinical intervention (heel lance followed by ROP screening), excluding post-randomisation exclusions. Comparative analyses and other descriptive analyses will be presented for this population only. Baseline characteristics at time of trial entry and time of study will be reported for all infants randomised for whom data are available, excluding post-randomisation exclusions.

6.2.2 Safety analysis population

Safety analyses will include all infants randomised who received the study treatment.

7 DESCRIPTIVE ANALYSES 7.1 Representativeness of trial population and participant throughput The flow of participants through each stage of the trial will be summarised using a CONSORT

diagram. This will describe the numbers of participants:

Randomised

o Randomised in error

Allocated to each intervention

o Received allocated intervention

o Did not receive allocated intervention

Received other intervention

Received no study treatment

Withdrawn from trial

o Data already collected can be used

o Clinical data can be collected from baby’s notes for remaining study period

o Vital signs monitor data can be recorded for remaining study period

o Reason for withdrawal

Clinical decision

Parental request

Other reasons

Included in the analysis

Excluded post-randomisation (with reasons)



7.2 Baseline comparability of randomised groups The per-protocol population groups will be described by allocation group with respect to their demographic and clinical characteristics at trial entry and at the time of study. The time of study is defined as the time of the start of the clinical intervention (heel lance followed by ROP screening). The following will be described (* denotes minimisation criteria): Characteristics at trial entry

Gestational age at birth*

Gestational age at randomisation*

Birth weight

Birth weight z-score (adjusted for gestational age and gender at birth)

Weight at randomisation

Apgar score at 10 minutes of age

Mode of delivery

Sex

Days ventilated*

Days since morphine was given*

Presence of gastric tube*

Intrauterine growth restriction*

Baby has had surgery*

Intraventricular haemorrhage (IVH) grade 1 or 2

Baby is one of a multiple pregnancy

Conceived using IVF treatment. Characteristics at time of study (clinical intervention)

Gestational age at time of study

Postnatal age at time of study

Weight at time of study

Level of care received o ITU o HDU o LDU o Maternity ward

Respiratory support modality o Self-ventilating o Low flow therapy o High flow therapy

Time since last feed

Comfort measures at heel lance o Kangaroo care o Swaddling, containment, facilitated tucking o Rocking/holding o Non-nutritive sucking



o Oral sucrose

Duration of ROP screening exam

Diagnosis of ROP.

The number and percentage will be presented for binary and categorical variables. The mean and standard deviation, or the median and the interquartile range, and the range if appropriate, will be presented for continuous variables. There will be no tests of statistical significance performed nor confidence intervals calculated for differences between randomised groups on any baseline variable. 7.3 Other descriptive analysis Other data collected during the trial and the 48-hour observation period will be summarised by allocation group for descriptive purposes, as follows:

PIPP-R score 30 seconds after control heel lance

Magnitude of nociceptive-specific brain activity evoked by control heel lance

Amplitude of reflex withdrawal following control heel lance

Mean blood pressures (systolic, diastolic and mean) during, and in the 6 and 24-hour periods before and after the clinical intervention.

The number and percentage will be presented for binary and categorical variables. The mean and standard deviation, or the median and the interquartile range, and the range if appropriate, will be presented for continuous variables. There will be no tests of statistical significance performed nor confidence intervals calculated for differences between randomised groups on any of these endpoints. To assess reliability of the PIPP-R, it will be re-scored by the same individual who originally scored it, and by a different individual, for the heel lance and ROP screening occasions for all infants. The inter-rater and intra-rater reliability will be reported for the derived PIPP-R scores overall using intra-class correlation from a random effects model with 95% confidence intervals. The inter-rater reliability will be based upon the first entry by the first rater, and the entry by the second rater. 7.4 Losses to follow-up The number (with percentages) of losses to follow-up will be reported for the two trial arms, and the reasons will be recorded. Any deaths (and their causes) will be reported separately.

8 COMPARATIVE ANALYSES Infants will be analysed on a per-protocol basis, i.e. only infants receiving the allocated treatment and the clinical intervention will be analysed, according to their allocation. The placebo group will be used as the reference group in all analyses. Effect estimates will be presented with confidence intervals and p-values. Comparative analyses will not adjust for minimisation criteria due to the small final sample size. Due to the small final sample size, models using maximum likelihood estimation are not appropriate for these data.



8.1 Detailed definition of outcomes Derivations of the outcomes are described in the data derivation document (associated document). 8.2 Primary analysis

8.2.1 Analysis of the primary and co-primary outcomes

The co-primary outcomes (PIPP-R score during the 30 second period after ROP screening, and nociceptive-specific brain activity) are both continuous and will be compared in the morphine and placebo groups. Means and standard deviations will be presented for each group, and a t-test will be used to estimate the mean difference with 95% confidence interval. If the outcome data are skewed, medians with interquartile ranges will be presented and the median difference with 95% confidence interval will be estimated using the Hodges-Lehmann estimator. In this case, a Wilcoxon rank-sum test will be used to compare the distributions between the morphine and placebo groups, with a p-value presented. 8.2.2 Analysis of secondary outcomes The secondary outcomes PIPP-R score following heel lance and reflex withdrawal (average RMS activity in the 1000 ms post the heel lance) are both continuous and will be compared in the morphine and placebo groups. Means and standard deviations will be presented for each group, and a t-test will be used to estimate the mean difference with 95% confidence interval. If the outcome data are skewed, medians with interquartile ranges will be presented and the median difference with 95% confidence interval will be estimated using the Hodges-Lehmann estimator. In this case, a Wilcoxon rank-sum test will be used to compare the distributions between the morphine and placebo groups, with a p-value presented. Clinical stability analysis The number of episodes of bradycardia, tachycardia, desaturation and apnoea are measured as counts for each infant. For episodes of bradycardia, tachycardia and desaturation, the difference between the 6 and 24-hour periods before and after will be standardised to adjust for the number of pre-intervention episodes for each infant. Medians, interquartile ranges and ranges of these standardised differences, and of the absolute change in number of episodes will be presented by trial arm. The median difference of the standardised differences with 95% confidence interval will be estimated using the Hodges-Lehmann estimator. A Wilcoxon rank-sum test will be used to compare the distribution of these standardised differences between the morphine and placebo groups, with a p-value presented. The standardised differences in number of episodes in the periods before and after the clinical intervention will be calculated as the difference in number of episodes as a proportion of the total number of episodes, for each infant. To avoid issues caused by zero counts, a negligible constant term will be added to each observed value prior to standardisation. These standardised differences will be calculated symmetrically – so, the



difference in the number of episodes in the 6-hour period before and the 6-hour period after the clinical intervention as a proportion of the total number of episodes in the 12-hour period, and the difference in the number of episodes in the 24-hour period before and the 24-hour period after the clinical intervention as a proportion of the total number of episodes in the 48-hour period will be used. For episodes of apnoea, a composite of infants who either developed new-onset apnoeas or had an increased number of apnoeas in the 6 and 24-hour periods after the clinical intervention will be used as a binary outcome. These comparisons will also be symmetrical, comparing the 6-hour period after the clinical intervention with the 6-hour period before, and the 24-hour period after with the 24-hour period before. The number and proportion of infants who had this composite outcome will be presented, and risk ratios comparing the allocation groups for the 6 and 24-hour period after the clinical intervention will be estimated with 95% confidence intervals. Infants requiring an increase in respiratory support is a binary measure. The number and percentage of infants requiring this in the 6 and 24-hour periods before and after the clinical intervention will be reported for each group. Risk ratios for the 6 and 24-hour period after the clinical intervention will be estimated with 95% confidence intervals. Due to the small final sample size, models using maximum likelihood estimation are not appropriate for these data.

8.2.3 Safety analysis

Drug safety will be assessed by measuring the number of occurrences of apnoea that require intervention using NeoPuff or ‘bag and mask’ and the number of episodes of hypotension that require treatment with inotropes in the 24-hour period after drug administration. The number of occurrences will be compared in the morphine and placebo groups. Because these events are expected to be infrequent, we will treat these outcomes as binary (infant either did or did not have at least one occurrence of the events). For each of these outcomes, risk differences will be estimated and presented with 95% confidence intervals. 8.3 Secondary analyses No pre-specified secondary analyses. 8.4 Pre-specified subgroup analyses No pre-specified subgroup analyses.

8.5 Sensitivity analyses No pre-specified sensitivity analyses. 8.6 Significance levels For all primary and secondary outcomes, 95% confidence intervals will be reported, as well as p-values. However, due to the small sample size and the trial being underpowered, p-values will be interpreted with caution.



8.7 Missing data The analysis will be conducted using the available data, and the number of missing observations will be reported for each outcome.

8.8 Statistical software employed The statistical software Stata/SE will be used for analyses. For some additional analyses, MATLAB will be used.

9 SAFETY DATA ANALYSIS Foreseeable serious adverse events (SAEs), unforeseeable SAEs, suspected unexpected serious adverse reactions (SUSARs), and adverse events (AEs) will be listed by trial allocation. Details of incidences of apnoea that require intervention using NeoPuff or ‘bag and mask’, and incidences of hypotension that require treatment with inotropes in the 24-hour period following drug administration will also be listed by trial allocation.

10 ADDITIONAL ANALYSIS These additional analyses were not pre-specified in the protocol. The following additional objectives have been added in light of early suspension of trial recruitment.

10.1 Additional objectives These objectives aim (i) to quantify whether within the study population, the magnitude of the noxious-evoked brain activity, reflex withdrawal activity and PIPP-R scores can discriminate noxious and non-noxious input, and (ii) to further understand how the heart rate, oxygen saturation and respiratory rate are influenced by the clinical intervention (heel lance and ROP screening) and the administration of morphine.

We will investigate whether:

(i) the nociceptive-specific brain activity and reflex withdrawal activity are different

with the heel lance compared with the background activity and control heel

lance, independent of the intervention allocation.

(ii) the PIPP-R scores are different following the heel lance and the ROP screening

compared with control heel lance, independent of the intervention allocation.

(iii) the average time courses for the heart rate, oxygen saturation and respiratory

rate following the clinical intervention differ between the morphine group and

the placebo group.



10.2 Analysis of additional objectives Validation of pain measures

Nociceptive-specific brain activity and reflex withdrawal activity will be summarised at

background, control heel lance and heel lance using means with standard error of the mean

and ranges, or medians with standard error of the median (using bootstrap estimation) and

ranges if the data are skewed. These summary statistics will also be presented graphically.

PIPP-R scores will be summarised at control heel lance, heel lance and ROP screening using

means with standard error of the mean and ranges, or medians with standard error of the

median (using bootstrap estimation) and ranges if the data are skewed. These summary

statistics will also be presented graphically.

Extended exploratory analysis of physiological stability

The average changes in heart rate, oxygen saturation and respiratory rate will be presented graphically for the 24 hours pre and post the clinical intervention, separately for the morphine and placebo groups. The time courses (which describe the variation in these measures with time) will be described and compared between the two groups using non-parametric cluster analysis (9). Time periods with differences between the morphine and placebo groups will be identified by first calculating a t-statistic at each time point. Significant differences in the time courses will be defined to have occurred when the t-statistic is above a threshold set at 97.5th percentile of the t-distribution. Time periods (clusters) of significant activity will be identified based on temporal adjacency of above threshold points. A cluster-based test statistic will be calculated from 1000 random permutations of the data.



11 REFERENCES 11.1 Trial documents ST 105 v3.0 Statistical Analysis Plan SOP Poppi Trial Protocol version 6.0 Poppi Dummy tables Poppi Data derivation

11.2 Other references 1. Lemyre B, Hogan DL, Gaboury I, Sherlock R, Blanchard C, Moher D. How effective is tetracaine 4% gel, before a venipuncture, in reducing procedural pain in infants: a randomized double-blind placebo controlled trial. BMC Pediatr. 2007;7:7. 2. Stevens BJ, Gibbins S, Yamada J, Dionne K, Lee G, Johnston C, et al. The premature infant pain profile-revised (PIPP-R): initial validation and feasibility. Clin J Pain. 2014;30(3):238-43. 3. Gal P, Kissling GE, Young WO, Dunaway KK, Marsh VA, Jones SM, et al. Efficacy of sucrose to reduce pain in premature infants during eye examinations for retinopathy of prematurity. Ann Pharmacother. 2005;39(6):1029-33. 4. Marsh VA, Young WO, Dunaway KK, Kissling GE, Carlos RQ, Jones SM, et al. Efficacy of topical anesthetics to reduce pain in premature infants during eye examinations for retinopathy of prematurity. Ann Pharmacother. 2005;39(5):829-33. 5. Mehta M, Mansfield T, VanderVeen DK. Effect of topical anesthesia and age on pain scores during retinopathy of prematurity screening. J Perinatol. 2010;30(11):731-5. 6. Norbury TA, MacGregor AJ, Urwin J, Spector TD, McMahon SB. Heritability of responses to painful stimuli in women: a classical twin study. Brain. 2007;130(Pt 11):3041-9. 7. Hwang IK, Shih WJ, Decani JS. Group sequential designs using a family of type I error probability spending functions. Statistics in Medicine, vol. 9, no. 12, pp. 1439-45, 1990. 8. Lan KKG, DeMets DL. Discrete sequential boundaries for clinical trials. Biometrika 70(3):659-63, 1983. 9. Maris E, Oostenveld R. Nonparametric statistical testing of EEG- and MEG-data. J. Neurosci. Methods 164:177-90, 2007.



12 APPROVAL

Senior Trial Statistician

Name: Dr Louise Linsell

Signature

Date

Chief Investigator

Name: Dr Eleri Adams

Signature

Date

Principal Investigator

Name: Dr Rebeccah Slater

Signature

Date

Chair of Trial Steering Committee

Name: Professor Neena Modi

Signature

Date

13 DOCUMENT HISTORY

Version Date Edited by

Comments/Justification Timing in relation to interim analysis/unblinding

0.1 08/04/2016 JB First draft by Jennifer Bell Prior to first safety analysis

0.2 06/05/2016 JB Changes made to primary and secondary analysis. Minor amendments throughout.

Prior to first safety analysis

0.3 21/11/2016 JB Moved to new SAP template. Added detail of stopping boundaries. Many other changes and additions throughout.


0.4 02/12/2016 JB Amended Analysis of Secondary Outcomes, added Other References


0.5 20/10/2017 JB Updated following changes to protocol (wording of outcomes)


0.6 30/10/2017 JB Updated following review by Louise Linsell


0.7 01/12/2017 JB Moved to final SAP template. Minor amendments following review by Louise Linsell.

JB & LL unblinded for first safety analysis.

0.8 16/01/2018 JB Amended in preparation for final analysis following temporary halt to trial, and discussion with LL.

JB & LL unblinded for first two safety analyses

0.9 29/01/2018 JB, LL Amended following discussion with LL, VM, RS, CH, FM.




0.10 14/02/2018 JB, LL Amended following further discussion with PMG. Changed clinical stability analysis of counts to use standardised differences with a transformation. Changed safety analysis to risk difference. Renamed Exploratory Analysis to ‘Additional Analysis’, reduced additional objectives and amended analysis methods for these. Added section 6.3 Other descriptive analysis, including PIPP-R inter and intra-rater reliability. Updated NPEU logo. Other minor changes.


0.11 16/02/2018 JB Reviewed by Rebeccah Slater, Caroline Hartley, Louise Linsell, Andrew King & David Murray. Reworded PIPP-R re-scoring in descriptive analysis, and amended intra-rater reliability measure. Amended method of calculating median differences in primary analysis, and added use of Wilcoxon rank-sum test for these. Removed addition of a constant from the clinical stability analyses, and replaced with imputing a 0 value for babies with no episodes. Amended additional analysis of heart rate, oxygen saturation and respiratory rate. Other minor changes.


0.12 08/03/2018 JB, LL Reviewed by TSC & DMC. Amended introduction to clarify and update the sequence of events. Moved section 10 Deviations from Analysis described in Protocol to become section 2 (all other section numbers onwards subsequently change).


0.13 29/03/2018 JB Reviewed by Andrew King. Additional detail on clinical stability analysis (section 8.2.2). Other minor clarifications and amendments to grammar and formatting.


0.14 05/04/2018 JB Corrected minor typographical errors.


1.0 06/04/2018 JB Reviewed and agreed by TSC & DMC. Corrected minor typographical errors. Final version.


2.0 05/06/2018 JB Corrected typographical errors in section 3.11.1 - description of the

JB, LL, Vaneesha Monk (trial manager) and trial investigators



safety outcomes is now consistent with the section 5 of the Protocol (Objectives and Outcome Measures).

Corrected typographical error in section 4.2 – the criterion for a participant randomised in error is now correct, reflecting the reverse of the inclusion criteria.

Postnatal age at time of study and Respiratory support modality are added to Characteristics at time of study (in section 7.2), which were originally omitted in error.

Presence of gastric tube at time of study was removed from this section, as data are not available to report this.

(Eleri Adams, Rebeccah Slater, Caroline Hartley, Fiona Moultrie) unblinded post-data lock.

statistical analysis plan version 2.0 date: 06/06/2018...poppi version date: 06/06/2018 version...

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