APPENDIX for

A pragmatic randomized controlled trial reports the efficacy of hydroxychloroquine on coronavirus disease 2019 viral kinetics

Magnus Nakrem Lyngbakken MD PhD1,2, Jan-Erik Berdal MD PhD2,3, Arne Eskesen MD3, Dag Kvale MD

PhD2,4, Inge Christoffer Olsen PhD5, Corina Silvia Rueegg PhD5,6, Anbjørg Rangberg MSc7, Christine

Monceyron Jonassen PhD7, Torbjørn Omland MD PhD MPH1,2, Helge Røsjø MD PhD2,8, Olav Dalgard

MD PhD2,3

1Division of Medicine, Akershus University Hospital, Lørenskog, Norway

2Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway

3Department of Infectious Diseases, Division of Medicine, Akershus University Hospital, Lørenskog,

Norway

4Department of Infectious Diseases, Oslo University Hospital, Oslo, Norway

5Department of Research Support for Clinical Trials, Oslo University Hospital, Oslo, Norway

6Oslo Centre for Biostatistics and Epidemiology, Oslo University Hospital, Oslo, Norway

7Center for Laboratory Medicine, Østfold Hospital Trust, Grålum, Norway

8Division of Research and Innovation, Akershus University Hospital, Lørenskog, Norway

1

Contents

(1) Supplementary Material

(2) Study protocol v1.1

(3) Statistical analysis plan v2.1

2

Supplementary Methods

Procedures for sampling and SARS-CoV-2 viral loads quantitation

Oropharyngeal swab samples were taken from patients at inclusion, at 48 hours and at 96 hours, by a selected group of study physicians. Nasopharyngeal sampling was planned in the initial study protocol, but due to trial conduction in the middle of a global pandemic and an immediate shortage of nasopharyngeal swabs for anything but clinical Covid-19 diagnostics, the study steering committee decided on oropharyngeal sampling using chlamydia swabs. The swabs were rotated for ten seconds on the posterior oropharyngeal mucosal membrane (over both tonsils, soft palate and posterior oropharynx), placed in 1 mL universal in-house virus transport medium, and kept frozen at -80°C until analysis. We validated the oropharyngeal versus nasopharyngeal sampling in seven SARS-CoV-2 positive patients and three SARS-CoV-2 negative healthy controls. There was total agreement in

SARS-CoV-2 qualitative results from paired samples taken either from nasopharynx as for routine diagnosis or from oropharynx with sampling devices used for this study. The mean pairwise difference in Ct values in the SARS-CoV-2 positive samples was of 2.5, with stronger PCR positive results observed in the samples collected with the devices used for this study (Supplementary

Table). The universal in-house virus transport medium was prepared as follows: Two grams of bovine serum albumin (IgG free, protease free) was added to 50mL of phosphate-buffered saline, sterile filtered, and added to sterile distilled water (to 400mL). 200mL of sterile phenol red solution was then added for a total volume of 600mL. pH was adjusted with 1 molar sodium hydroxide for a final pH of 7.48 to 7.50.

3

Supplementary Table. Validation of oropharyngeal versus nasopharyngeal virus sampling for SARS-CoV-2. Ct values

ID A B

1 44.67 36.88

2 Negative Negative

3 30.77 28.61

SARS-CoV-2 positive patients (1-7) 4 35.28 35.73

5 Negative Negative

6 36.93 35.78

7 25.05 23.13

1 Negative Negative

SARS-CoV-2 negative healthy controls (1-3) 2 Negative Negative

3 Negative Negative A, routine nasopharyngeal sampling. B, oropharyngeal sampling with chlamydia swab in universal in-house virus transport medium.

4

NORWEGIAN CORONAVIRUS DISEASE 2019 (NO COVID-19) STUDY: AN OPEN LABELED RANDOMIZED CONTROLLED PRAGMATIC TRIAL TO EVALUATE THE ANTIVIRAL EFFECT OF CHLOROQUINE IN ADULT PATIENTS WITH SARS-COV-2 INFECTION

Protocol Identification Number: Ahus-NO-COVID-19 EudraCT Number: 2020-001010-38

SPONSOR: Akershus University Hospital Head of Department / Associate Professor Jan Erik Berdal Department of Infectious Diseases Akershus University Hospital Sykehusveien 25 1478 Lørenskog, Norway Tel: 67 96 00 00

E-mail: [email protected]

PROTOCOL VERSION NO. 1.1 – 18.03.20

CONTACT DETAILS

Sponsor: Head of Department / Associate Professor Jan Erik Berdal, MD PhD Department of Infectious Diseases, Division of Medicine Akershus University Hospital Sykehusveien 25 1478 Lørenskog, Norway Tel: 67 96 00 00

E-mail: [email protected] Principal Investigator: Professor Olav Dalgard, MD PhD Department of Infectious Diseases, Division of Medicine Akershus University Hospital Sykehusveien 25 1478 Lørenskog, Norway Tel: 92616800

E-mail: [email protected] Coordinating Investigator: Postdoctoral Fellow Magnus Lyngbakken, MD PhD Division of Medicine Akershus University Hospital 1478 Lørenskog, Norway Tel: 67 96 00 00 E-mail: [email protected]

Investigator: Head of Division Helge Røsjø, MD PhD Division of Research and Innovation Akershus University Hospital 1478 Lørenskog, Norway Tel: 67 96 00 00 E-mail: [email protected]

Investigator: Professor Torbjørn Omland, MD PhD MPH Division of Medicine Akershus University Hospital 1478 Lørenskog, Norway Tel: 67 96 00 00 E-mail: [email protected]

Protocol NO-COVID-19 Version no. 1.1, 2020 – 03 – 18 Page 2 of 31

Investigator: Professor Christine Jonassen, PhD Center for Laboratory Medicine, Østfold Hospital Trust/ Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU)

Postboks 300 1714 Grålum Tel: +47 90734557 E-mail: [email protected]

Investigator: Professor Dag Kvale, MD PhD Institute of Clinical Medicine University of Oslo, Norway Tel: +47 22850546 E-mail: [email protected] Investigator: Senior Consultant Arne Eskesen, MD Department of Infectious Diseases, Division of Medicine Akershus University Hospital 1478 Lørenskog, Norway Tel: 67 96 00 00 E-mail: [email protected] Monitor: Clinical Trial Unit Oslo University Hospital HF 0424 Oslo, Norway Tel : +47 23066034

Statistician: Inge Christoffer Olsen, PhD Clinical Trial Unit Oslo University Hospital HF 0424 Oslo, Norway Tel : +47 23066034 E-mail: [email protected]

Protocol NO-COVID-19 Version no. 1.1, 2020 – 03 – 18 Page 3 of 31

SIGNATURE PAGE

Title Norwegian Coronavirus Disease 2019 (NO COVID-19) Study: An open labeled randomized controlled pragmatic trial to evaluate the antiviral effect of chloroquine in adult patients with SARS-CoV-2 infection Protocol ID: NO COVID-19 EudraCT no:

I hereby declare that I will conduct the study in compliance with the Protocol, ICH GCP and the applicable regulatory requirements:

Name Title Role Signature Date

Head of Department Jan Erik Berdal Sponsor’s representative Associate Professor

Olav Dalgard Professor Principal investigator

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SIGNATURE PAGE SITE INVESTIGATORS

Title Norwegian Coronavirus Disease 2019 (NO COVID-19) Study: An open labeled randomized controlled pragmatic trial to evaluate the antiviral effect of chloroquine in adult patients with SARS-CoV-2 infection

Protocol ID: NO-COVID-19

EudraCT no:

I hereby declare that I will conduct the study in compliance with the Protocol, ICH GCP and the applicable regulatory requirements:

Name Title Signature Date

Protocol NO-COVID-19 Version no. 1.1, 2020 – 03 – 18 Page 5 of 31

PROTOCOL SYNOPSIS

Norwegian Coronavirus Disease 2019 (NO COVID-19) Study: An open labeled randomized controlled pragmatic trial to evaluate the antiviral effect of chloroquine in adult patients with SARS-CoV-2 infection

Sponsor Akershus University Hospital HF

Phase and study type Phase 4, treatment trial

Investigational Medical Product Hydroxychloroquine (IMP) (including active comparator and placebo) : No placebo control

Centers: Single center during initial phase and with possible extension to other Norwegian sites pending result from the initial phase. Extension will be according to separate submissions to ethics committee and other regulatory bodies from new centers.

Study Period: Estimated date of first patient enrolled: 23.03.20

Anticipated recruitment period: 6 months

Estimated date of last patient completed: 01.04.21

Estimated study termination: 23.03.25

Treatment Duration: Seven days

Follow-up: Until discharge from the hospital, at 30 and 90 days.

Objectives Main objective:

To assess whether treatment with hydroxychloroquine in patients with COVID-19 will increase the decline rate of SARS-CoV-2 in the nasopharynx

Secondary objectives:

To assess the clinical benefit of chloroquine treatment on

 National Early Warning Score (NEWS)

 Risk of clinical deterioration and mortality

 Biomarker profile

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Endpoints: Primary endpoint:

 Rate of decline in SARS-CoV-2 viral load in nasopharyngeal samples as assessed by RT-PCR in samples collected at baseline, 48 and 96 hours after randomization

Secondary endpoints:

 Change in National Early Warning Score (NEWS) from randomization to 96 hours

 Admission to ICU

 In-hospital mortality

 Duration of hospital admission

 Mortality at 30 and 90 days

 Clinical status as assessed by percentage of subjects reporting each severity rating on a 6-point ordinal scale on day 14 after study inclusion:

1. Death

2. Hospitalized, on invasive mechanical ventilation or extracorporeal membrane oxygenation

3. Hospitalized, on non-invasive ventilation or high flow oxygen devices

4. Hospitalized, requiring supplemental oxygen

5. Hospitalized, not requiring supplemental oxygen

6. Not hospitalized

 Change in protein biomarker profiles from randomization to 96 hours, including C-reactive protein, markers of renal and hepatic injury, and established cardiac biomarkers like cardiac troponin and B-type natriuretic peptide concentrations

Study Design: Two-arm, open label, single center, pragmatic study with possible extension to other Norwegian sites, pending regulatory approvals.

Main Inclusion Criteria: 1. Hospitalised 2. Adults 18 year or older 3. Moderately severe disease (NEWS score ≤ 6) 4. SARS-CoV-2 positive nasopharyngeal swab 5. Signed informed consent must be obtained and documented according to ICH GCP, and national/local regulations.

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Main Exclusion Criteria 1. Requiring ICU admission at screening 2. History of psoriasis 3. Tinnitus, reduced hearing 4. Visual impairment 5. Known adverse reaction to hydroxychloroquine sulphate 6. Pregnancy 7. Prolonged QT interval (>450 ms) Sample Size: Pilot inclusion of 51 patients, with subsequent inclusion and analyses after 101, 151 and 202 completed patients.

Efficacy Assessments: Viral kinetics 96 hours after randomization.

Safety Assessments: Assessment of Adverse Events (AE), Serious Adverse Events (SAE) and Suspected Unexpected Serious Adverse Reactions (SUSAR).

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TABLE OF CONTENTS

CONTACT DETAILS ...... 2 SIGNATURE PAGE ...... 4 SIGNATURE PAGE SITE INVESTIGATORS ...... 5 PROTOCOL SYNOPSIS ...... 6 TABLE OF CONTENTS ...... 9 LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ...... 12 1 INTRODUCTION ...... 12 1.1 Background – Disease ...... 12 1.2 Background - Therapeutic Information ...... 13 1.3 Pre-Clinical & Clinical Experience with Investigational Medicinal Product (IMP) ...... 13 1.4 Rationale for the Study and Purpose ...... 13 2 STUDY OBJECTIVES AND RELATED ENDPOINTS ...... 13 Primary Endpoint ...... 14 Secondary Endpoints ...... 14 3 OVERALL STUDY DESIGN ...... 15 4 STUDY POPULATION ...... 16 4.1 Selection of Study Population ...... 16 4.2 Number of Patients ...... 16 4.3 Inclusion Criteria ...... 16 4.4 Exclusion Criteria ...... 16 5 TREATMENT...... 16 5.1 Drug Identity, Supply and Storage ...... 17 5.2 Dosage and Drug Administration ...... 17 5.3 Duration of Therapy ...... 17 5.4 Concomitant Medication ...... 17 5.5 Subject Compliance ...... 17 5.6 Drug Accountability ...... 17 5.7 Drug Labelling ...... 17 5.8 Subject Numbering ...... 17 6 STUDY PROCEDURES ...... 18 6.1 Flow Chart ...... 18 6.2 Criteria for Patient Discontinuation ...... 18 6.3 Procedures for Discontinuation ...... 19 6.3.1 Patient Discontinuation ...... 19 6.3.2 Trial Discontinuation ...... 19 6.4 Laboratory Tests ...... 19

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6.5 Clinical variables ...... 19 7 ASSESSMENTS...... 21 7.1 Assessment of Efficacy / Response ...... 21 7.2 Safety and Tolerability Assessments ...... 21 8 SAFETY MONITORING AND REPORTING ...... 21 8.1 Definitions ...... 21 8.1.1 Adverse Event (AE) and Adverse Events of Special Intrest (AESI) ...... 21 8.1.2 Serious Adverse Event (SAE) ...... 22 8.1.3 Suspected Unexpected Serious Adverse Reactions (SUSAR) ...... 22 8.2 Expected Adverse Events ...... 22 8.3 Time Period for Reporting AESI and SAE ...... 22 8.4 Recording of Serious AEand AESI ...... 23 8.5 Reporting Procedure ...... 23 8.5.1 SAEs and AESIs ...... 23 8.5.2 SUSARs ...... 24 8.6 Risk–benefit ...... 24 8.7 Data Monitoring Committee (DMC) ...... 24 9 DATA MANAGEMENT AND MONITORING ...... 24 9.1 Electronic Case Report Forms (eCRF) ...... 24 9.2 Source Data...... 25 9.3 Study Monitoring ...... 25 9.4 Confidentiality ...... 26 9.5 Database management ...... 26 10 STATISTICAL METHODS AND DATA ANALYSIS ...... 26 10.1 Determination of Sample Size ...... 26 10.2 Randomization ...... 27 10.2.1 Allocation- sequence generation ...... 27 10.2.2 Allocation- procedure to randomize a patient ...... 27 10.2.3 Blinding and emergency unblinding...... 27 10.3 Population for Analysis ...... 27 10.4 Planned analyses ...... 28 10.5 Statistical Analysis ...... 28 10.5.1 Primary analysis ...... 28 10.5.2 Secondary analyses ...... 28 10.5.3 Safety analyses ...... 29 10.5.4 Descriptive statistics ...... 29 10.5.5 Missing data ...... 29 11 STUDY MANAGEMENT ...... 29

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11.1 Investigator Delegation Procedure ...... 29 11.2 Protocol Adherence ...... 29 11.3 Study Amendments ...... 29 11.4 Audit and Inspections ...... 30 12 ETHICAL AND REGULATORY REQUIREMENTS ...... 30 12.1 Ethics Committee Approval ...... 30 12.2 Other Regulatory Approvals ...... 30 12.3 Informed Consent Procedure ...... 30 12.4 Subject Identification ...... 30 13 TRIAL SPONSORSHIP AND FINANCING ...... 31 14 TRIAL INSURANCE ...... 31 15 PUBLICATION POLICY ...... 31 16 REFERENCES ...... 31 17 LIST OF APPENDICES ...... 31

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LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS

Abbreviation or special term Explanation

AE Adverse Event

CRF Case Report Form (electronic/paper)

CSA Clinical Study Agreement

CTC Common Toxicity Criteria (for cancer trials only)

CTCAE Common Terminology Criteria for Adverse Event (for cancer trials only)

DAE Discontinuation due to Adverse Event

EC Ethics Committee, synonymous to Institutional Review Board (IRB) and Independent Ethics Committee (IEC)

GCP Good Clinical Practice

IB Investigator’s Brochure

ICF Informed Consent Form

ICH International Council for Harmonization

IMP Investigational Medicinal Product (includes active comparator and placebo)

ITT Intention to treat

PP Per-protocol

SAE Serious Adverse Event

SAP Statistical Analysis Plan

SOP Standard Operating Procedure

1 INTRODUCTION

1.1 Background – Disease On 30 December 2019, bronchoalveolar lavage samples were collected from a patient with pneumonia of unknown ethology in Wuhan, China. Real-time polymerase chain reaction (RT-PCR) assays on these samples were positive for pan-Betacoronavirus and genome sequencing indicated that the virus had the closest relationship with the bat SARS-like coronavirus strain BatCov RaTG1. The new virus was named severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) and the respiratory disease it causes COVID-19 (1).

COVID-19 is transmitted via droplets and fomites during close unprotected contact between an infector and infectee and has per March 2020 been detected in approximately 126 000 people worldwide with most cases appearing in the Hubei province of China, South Korea, Iran and Italy. On March 11 2020, the WHO declared COVID-19 a global pandemic (2). In Norway, 629 cases have been detected, and the first patients have been admitted to hospitals and ICUs. Approximately 80% of laboratory confirmed patients in China have had mild to moderate disease, 13.8% have had severe disease and 6.1% critical. The case fatality rate in China has been 3.8 % (5.8% in the city of Wuhan and 0.7% in the remaining China). Mortality increases with age, is higher in men than women and higher in those with important

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comorbidity (cardiac disease, diabetes mellitus, chronic respiratory disease, cancer and hypertension).

1.2 Background - Therapeutic Information To date, there is no specific therapy for COVID-19. However, supportive treatment including oxygen supplementation and mechanical intervention is believed to strongly effect the course of the disease. The effect of several antivirals are currently being investigated in clinical including remdesivir, lopinavir/ritonavir (HIV protease inhibitor), rintatolimod (immune modulator), danoprevir+ritonavir (HIV protease inhibitor), Galidesivir (broad spectrum antiviral drug) and remdesivir (a nucleoside inhibitor of coronavirus polymerase) (3). In addition, chloroquine has been suggested as a treatment against COVID-19.

1.3 Pre-Clinical & Clinical Experience with Investigational Medicinal Product (IMP) Chloroquine is an important anti-malaria drug that has been used since 1947. Furthermore, since the late seventies it has been used as treatment against rheumatoid arthritis and other rheumatic disorders. In short term use, chloroquine has a good safety profile. Its most important side effect being reversible accommodation disturbances and swelling of the cornea. In long term use (months) it may cause chronically reduced vision.

The effect of chloroquine on SARS-CoV-2 is supported by in vitro data showing a strong antiviral effect in non-toxic doses in cell culture (4) and by efficacy in treatment of COVID-19 associated pneumonia in clinical studies (5). The antiviral mechanism of chloroquine is associated with increased endosomal pH needed for fusion-mediated viral entry into host cells, and with alteration of glycosylation of SARS-CoV-2 cellular receptors. Accordingly, the Chinese Expert Consensus on Chloroquine Phosphate for the Treatment of Novel Coronavirus Pneumonia recently recommended short term treatment with chloroquine in patients with mild, moderate and severe cases of novel coronavirus pneumonia and without contraindications to chloroquine use (6).

1.4 Rationale for the Study and Purpose We hypothesize that early treatment with chloroquine in patients with established COVID-19 is safe and will significantly increase the virological clearance rate of SARS-CoV-2. Furthermore, we hypothesize that early treatment with chloroquine is associated with more rapid resolve of clinical symptoms and a decreased admission rate to intensive care units and in-hospital mortality. Considering the immediate and worldwide health emergency associated with the SARS- CoV-2 outbreak and the current lack of evidence based medical interventions for this patient group, studies investigating possible treatment modalities in COVID-19 are direly needed.

2 STUDY OBJECTIVES AND RELATED ENDPOINTS

Specific patient groups, especially elderly patients with comorbidities, are at higher risk of contracting viral respiratory disease and hospitalization than the general population. Relevant clinical end points depend on the patient populations studied, local standards of care, and physician judgment. Studies that focus solely on ICU admission or mortality as primary outcomes are likely to require prolonged time frames for accrual of sufficient numbers of patients and also need to consider many confounding variables that affect clinical outcomes in a possibly heterogeneous patient group. Measures of rate of decline of virus replication as primary end points to evaluate and compare drug efficacy of antivirals is logical and necessary, especially in heterogeneous populations such as hospitalized and immunocompromised patients. Rapid reductions in active viral replication may be essential to prevent tissue damage and to further clinical recovery, as well as reduce risk of viral complications and mortality (7).

The recent years National Early Warning Score (NEWS) has been implemented in Norwegian hospitals as a tool to assess the degree of illness in a patient and to guide level of surveillance and intensity of treatment. This method is based on the degree of abnormality of the vital signs respiratory rate, oxygen saturation, body temperature, systolic blood pressure and heart rate, in addition to the need for supplemental oxygen and mental alertness (8). Scores range from 0-20, with a higher score representing further removal from normal physiology and a higher risk of morbidity and mortality. We expect that approximately 75% of patients will be discharged by day 14 (9) and will therefore analyse clinical status at this day.

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Accordingly, the primary aim of this study is to assess whether treatment with chloroquine in patients with COVID-19 will increase the decline rate of SARS-CoV-2 in the nasopharynx from baseline to 48 and 96 hours. As described above, the use of primary virological end points is strongly supported by the current literature on novel antiviral therapy.

The secondary aims are to assess whether treatment with chloroquine in patients with COVID-19 (1) will improve clinical status as assessed by the NEWS score, improve clinical outcomes, including (2) admission rate to intensive care units and (3) in-hospital mortality, and (4) improve protein biomarker profile (inflammation, cardiac, renal, hepatic).

Specific objectives

 Assess the impact of early treatment with chloroquine in patients with established COVID-19 on SARS-CoV-2 presence in nasopharyngeal specimens at 48 and 96 hours after inclusion in the trial  Assess the impact of early treatment with chloroquine in patients with established COVID-19 on changes in NEWS score  Assess the impact of early treatment with chloroquine in patients with established COVID-19 on admission rate to ICU  Assess the impact of early treatment with chloroquine in patients with established COVID-19 on in-hospital mortality and mortality after 30 and 90 days  Assess the impact of early treatment with chloroquine in patients with established COVID-19 on clinical outcomes after 14 days  Assess the impact of early treatment with chloroquine in patients with established COVID-19 on markers of inflammation, and cardiac, renal and hepatic injury at 96 hours after inclusion in the trial

Primary Endpoint  Rate of decline in SARS-CoV-2 viral load in nasopharyngeal samples as assessed by RT-PCR in samples collected at baseline, 48 and 96 hours after randomization

Secondary Endpoints  Change in NEWS score at 96 hours after randomization

 Admission to ICU

 In-hospital mortality

 Duration of hospital admission

 Mortality at 30 and 90 days

 Clinical status will be assessed by percentage of subjects reporting each severity rating on a 6-point ordinal scale 14 days after randomization: 1. Death 2. Hospitalized, on invasive mechanical ventilation or extracorporeal membrane oxygenation 3. Hospitalized, on non-invasive ventilation or high flow oxygen devices 4. Hospitalized, requiring supplemental oxygen 5. Hospitalized, not requiring supplemental oxygen 6. Not hospitalized

 Change in protein biomarker profiles at 96 hours after randomization, including C-reactive protein, markers of renal and hepatic injury, and established cardiac biomarkers like cardiac troponin and B-type natriuretic peptide concentrations

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3 OVERALL STUDY DESIGN

The study is a two-arm, open label, pragmatic randomized controlled trial (RCT) designed to assess the virological and clinical effect of chloroquine therapy in patients with established COVID-19. Pragmatic clinical trials (PCT) are characterized by 3 attributes: (1) focus on informing decision-makers (e.g. patients, politicians, administrators) on optimal clinical medicine practice, as opposed to elucidating a biological or social process; (2) intent to enrol a population representative to the decision in practice and for whom the decision is relevant; and (3) either an intent to streamline procedures and data collection in the trial or to measure a broad range of outcomes. By utilizing resources already paid for by the hospitals (physicians and nurses in daily clinical practice), pragmatic clinical trials can include a larger number of patients at a short time duration and at a lower cost than studies utilizing traditional RCT designs with an external study organization (e.g. study nurses, study physicians). Due to the immediate need for study commencement and the time frame of the current proposal, a pragmatic approach will enable swift initiation of randomization and treatment. We will especially use data from the data warehouse at Akershus University Hospital for eligible patient identification (i.e. electronic surveillance) and for automatic data extraction to the study specific database. The study will not be able to procure an acceptable placebo treatment and the study will accordingly not be placebo-controlled.

In the initial phase of the study, patients will be included from a single center (Akershus University Hospital). The study is designed as a sequential adaptive trial where analyses are planned after the inclusion of 51 patients, with subsequent analyses after 101, 151 and 202 completed patients. This approach will enable frequent assessment of all outcome measures. After inclusion of the initial 51 patients, analyses will be performed for the predefined outcome measures. Pending these result, the study will possibly be extended to other Norwegian centres, pending separate submission to ethics committee and other regulatory bodies from new centers. See Section 10 Statistical methods and data analysis for further details.

All patients at Akershus University Hospital with suspicion of acute respiratory tract infections are examined with a nasopharyngeal swab, with subsequent microbiological examination, including SARS-CoV-2 specific polymerase chain reaction (PCR). Laboratory reports from the Department of Microbiology are surveilled real-time for SARS-CoV-2 positive samples in the local data warehouse, which will allow for immediate screening and randomization in all eligible subjects. Figure 1 gives a schematic overview of the study.

Figure 1. Trial design

Study Period of initial Estimated date of first patient enrolled: 23.03.20 study phase: Anticipated recruitment period: 6 months Estimated date of last patient completed: 01.04.21 Estimated study termination: 23.03.25 Treatment Duration: Patients allocated to the treatment arm will be treated with 400 mg hydroxychloroquine sulphate (equalling 310 mg base) twice daily for seven days Follow-up: Continuous of follow-up at admission, as well as assessment of mortality at day 30 and 90

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4 STUDY POPULATION

4.1 Selection of Study Population Participants will be recruited from the entirety of the inpatients at the participating hospitals. Electronic real-time surveillance of laboratory reports from the Department of Microbiology will be examined regularly, with maximum interval 24 hours, for SARS-CoV-2 positive subjects.

4.2 Number of Patients We aim to include patients by a sequential adaptive approach, where analyses are planned after the inclusion of 51 patients, with subsequent analyses after 101, 151 and 202 completed patients. All patients included in each sequence will be used for the final analyses of the entire study. See Section 10 Statistical methods and data analysis for further details.

4.3 Inclusion Criteria All of the following conditions must apply to the prospective patient at screening prior to inclusion:

1. Hospitalised

2. Adults 18 year or older

3. Moderately severe disease (NEWS score ≤ 6)

4. SARS-CoV-2 positive nasopharyngeal swab

5. Signed informed consent must be obtained and documented according to ICH GCP, and national/local regulations.

4.4 Exclusion Criteria Patients will be excluded from participation in the study if they meet any of the following criteria:

1. Requiring ICU admission at screening

2. History of psoriasis

3. Tinnitus, reduced hearing

4. Visual impairment

5. Known adverse reaction to hydroxychloroquine sulphate

6. Pregnancy

7. Prolonged QT interval (>450 ms)

5 TREATMENT

Recent reports document inhibition of SARS-CoV-2 by chloroquine in vitro, as well as in experimental clinical trials conducted in China. No apparent adverse effects of chloroquine treatment has been observed in these trials on human subjects. Hydroxychloroquine has a more favourable side effect profile compared to chloroquine and is more potent than chloroquine to inhibit SARS-CoV-2 in vitro (10). Accordingly, in the current proposal, and in accordance with the Chinese

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Expert Consensus on Chloroquine Phosphate for the Treatment of Novel Coronavirus Pneumonia (6), we will utilize a short term treatment with 400 mg hydroxychloroquine sulphate (equalling 310 mg base) twice daily for seven days. Standard of care will apply to both treatment arms at the discretion of the treating physician. Due to the lack of randomized evidence based therapy in COVID-19 and possibly a large proportion of patients who will present with serious to life-threatening disease, treatment by compassion will be allowed at any time at the discretion of the treating physician.

5.1 Drug Identity, Supply and Storage For this study, hydroxychloroquine sulphate is defined as the Investigational Medicinal Product (IMP). The IMP has marketing authorization as a medicinal product, is produced by Sanofi Aventis under the trade name “Plaquenil”, and is distributed through licensed pharmacists.

The drug will be provided by the trial sponsor (Akershus University Hospital) to patients included in the treatment arm free of charge.

5.2 Dosage and Drug Administration At enrolment, all patients randomized to treatment will be allocated a per oral dosage of 400 mg hydroxychloroquine sulphate (equalling 310 mg base) twice daily for seven days. This is in agreement with the SPC of the IMP. The drug will be dispensed at the regular drug administration rounds of the clinical wards.

5.3 Duration of Therapy Duration of therapy is per protocol seven days, equalling 14 doses of 400 mg hydroxychloroquine sulphate (equalling 310 mg base). The duration of treatment is based on a Chinese Expert Consensus that 5-10 days of chloroquine should be administered to patients with coronavirus pneumonia (6).

5.4 Concomitant Medication All concomitant medication used by the patient will be recorded at admission and retrieved from the hospital electronic prescribing system. Care should be taken when patients are on antiepileptic or antidiabetic medication, as well as on therapy associated with risk of QT time prolongation.

5.5 Subject Compliance Compliance to the protocol will be monitored by the hospital electronic prescribing system, where administration status of every dose prescribed is registered.

5.6 Drug Accountability The IMP has a marketing authorization in Norway, is routinely ordered by the pharmacy and will be dispensed from the pharmacy's own stock.

5.7 Drug Labelling The IMP is labeled according to the standard production by the commercial manufacturer (Sanofi Aventis).

5.8 Subject Numbering Each subject is identified in the study by a unique subject number that is assigned when subject signs the Informed Consent Form. Once assigned the subject number cannot be reused for any other subject.

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6 STUDY PROCEDURES

As described in Section 3, the current study will utilize a pragmatic approach. In this regard, we will utilize resources already paid for by the hospitals, and all study procedures will be performed at the discretion of the treating physician, including laboratory testing, medical imaging and other procedures deemed clinically necessary.

6.1 Flow Chart Table. Trial flow chart Time after randomization 24 48 72 96 120 144 168 192 216 14 30 90 Screening Randomization h h h h h h h h h d d d Nasopharyngeal X 1X 2X 2X 2X swab Inclusion/exclusion X evaluation Informed consent X Physical 3X 4X 4X 4X 4X 4X 4X 4X 4X 4X examination Blood samples 4, 5X 2X 4X 4X 4X 4X 4X 4X 4X 4X 4X AESI and SAEs6-7 X X X X X X ECG X 8X 8X 8X 8X 8X 8X 8X 8X 8X Record of concomitant X X X X X X X X X X medication Mortality X X X X X X X X X X X Clinical status 9X 1If > 12 hours since screening nasopharyngeal swab 2Study specific biobanking 3Clinical examination at the discretion of the admitting physician (full clinical examination including heart, lungs, abdomen, extremities), as well as medical history, substance use, allergies, current medication, etc. 4At the discretion of the treating physician 5Women of child bearing age must take a pregnancy test 6Every day whilst under treatment 7AESIs for this protocol is defined as gastrointestinal discomfort, visual disturbances, diarrhoea, headache, nausea and dizziness 8Daily ECG as long as a drug known to prolong QT interval is taken in combination with chloroquine 9Death, hospitalized (on invasive mechanical ventilation or extracorporeal membrane oxygenation), hospitalized (on non-invasive ventilation or high flow oxygen devices), hospitalized (requiring supplemental oxygen), hospitalized (not requiring supplemental oxygen), not hospitalized

6.2 Criteria for Patient Discontinuation Patients may be discontinued from the study any time. Specific reasons for discontinuing a patient for this study are:

 Voluntary discontinuation by the patient who is at any time free to discontinue his/her participation in the study, without prejudice to further treatment.

 Incorrect enrolment i.e. the patient does not meet the required inclusion/exclusion criteria for the study

 In the occurrence of severe adverse events, possibly or probably associated to the IMP, the IMP should be stopped unless a clinical risk/benefit assessment at the treating physician's discretion warrants continuation.

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6.3 Procedures for Discontinuation

6.3.1 Patient Discontinuation The reason for discontinuation will be recorded, if the patient chooses to disclose it. Management of patients who withdraw or are withdrawn from the study will have to be individualized and should in all cases be discussed with the Principal Investigator. All patients randomized will be included in the study population. The following may trigger individual patient discontinuation from the trial:  When chloroquine is used in combination with drug therapy known to prolong QT interval, an increase in QTc above 470ms for males or 480ms for females, or the QTc interval increases 60 ms or more from pre-treatment values (11).

6.3.2 Trial Discontinuation The whole trial may be discontinued at the discretion of the sponsor in the event of any of the following:

 Occurrence of AEs unknown to date in respect of their nature, severity and duration that may negatively affect the benefit/risk of the trial.  Medical or ethical reasons affecting the continued performance of the trial  Difficulties in the recruitment of patients

The sponsor and principal investigator(s) will inform all investigators, the relevant Competent Authorities and Ethics Committees of the termination of the trial along with the reasons for such action. If the study is terminated early on grounds of safety, the Competent Authorities and Ethics Committees will be informed within 15 days.

6.4 Laboratory Tests As described in Section 3, the current study will utilize a pragmatic approach. In this regard, we will utilize resources already paid for by the hospitals, among others laboratory tests, except for quantitative virological investigations and a pregnancy test of women of childbearing age. All laboratory sampling will be done at the discretion of the treating physician. Glucose concentrations are routinely measured in all admitted patient with diabetes mellitus and/or on antidiabetic therapy. On admission, a standard panel of laboratory test will be performed on all patients (covering haematology, electrolytes, liver- and renal function, standard serology, inflammatory markers), and these will be performed locally in accordance with hospital/laboratory standard procedures. Additionally, serum/plasma will be collected for biobanking on study inclusion. The samples will be labelled with study specific ID only (i.e. the samples will be handled de-identified).

A specific research biobank will be established in accordance with national regulations. Nasopharyngeal swabs will be collected in a uniform fashion at randomization if > 12 hours have passed since the screening sample, as well as at 48 hours and 96 hours after randomization. Nasopharyngeal swab at screening from the clinical laboratories will additionally be transferred to the research biobank upon study inclusion. Stored material will in addition consist of serum/plasma, which will be processed and stored in an ultra-freezer at Akershus University Hospital. The main purpose of the biobank will be to search for biomarkers of prognosis and disease severity. Since the field is in constant development, new potential biomarkers can be discovered as the study progresses.

6.5 Clinical variables Collection of clinical variables will start at admission and for the entirety of the hospital stay. Data will be collected from the hospital electronic record system, including electronic patient records, laboratory and medical imaging systems, and prescribing systems. The data warehouse at Akershus University Hospital will be utilized for automatic data extraction to the study specific database. All clinical variables will be registered in the study eCRF system, including clinical endpoints and quantitative virological results from serial nasopharyngeal specimens.

Data retrieved from hospital electronic record system

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Date of birth Sex Admission date Discharge date Date of symptom onset Admission signs and symptoms Fever Chills Cough Sputum production Sore throat Chest pain Shortness of breath Headache Confusion Myalgia or arthralgia Abdominal pain Diarrhoea Vomiting/Nausea Skin rash Epidemiological factors History of travel to an area with documented cases of COVID-19 infection? Contact with a confirmed/probable case of a symptomatic COVID-19 patient? Imaging on admission (chest X-ray, CT, etc) Infiltrates Pleural effusion Ground glass opacities Co-morbidities Cardiovascular disease Chronic obstructive pulmonary disease Chronic kidney disease Autoimmune disease Diabetes mellitus Cognitive impairment/dementia Cancer Substance use Tobacco and alcohol consumption Clinical biochemistry (upon availability, on admission and Haemoglobin as long as admitted) Platelet count Leukocyte count with differential count C-reactive protein (CRP) Glucose Na+ K+ Creatinine with estimated glomerular filtration rate Urea International Normalized Ratio (INR) Bilirubin Alanine aminotransferase (ALT) Aspartate aminotransferase (AST) Alkaline phosphatase (ALP) Gamma-glutamyl transferase (GT) Microbiology Blood culture results Urine culture results Anthropometrics Height (cm) Weight (kg) ECG recordings (upon availability, on admission and as Heart rate, rhythm, intervals, ST-T assessment, pathology long as admitted) Echocardiography (upon availability, on admission and as Left and right ventricular structure and function,

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long as admitted) pericardial effusion, pulmonary pressure, other pathology Daily NEWS score on three separate time point with > 5 Respiratory rate hours apart (at regular nurse rounds) Oxygen saturation Supplemental oxygen Temperature Systolic blood pressure Heart rate Level of consciousness (alert, verbal, pain, unresponsive) Medical therapy/medication All medical therapy/drug prescribed during hospitalization Clinical outcomes Admission to ICU Mortality (in-hospital, after 30 and 90 days) Hospitalization status 14 days hours after randomization 1. Death 2. Hospitalized, on invasive mechanical ventilation or extracorporeal membrane oxygenation 3. Hospitalized, on non-invasive ventilation or high flow oxygen devices 4. Hospitalized, requiring supplemental oxygen 5. Hospitalized, not requiring supplemental oxygen 6. Not hospitalized Safety monitoring and reporting Adverse events Serious adverse events Suspected unexpected serious adverse reactions 7 ASSESSMENTS

7.1 Assessment of Efficacy / Response Viral kinetics at 96 hours after randomization is the primary efficacy measure. We will assess the kinetics of SARS-CoV- 2 by serial nasopharyngeal samples with quantification of viral load, measured by RT-PCR (log10 copies/mL) (12).

7.2 Safety and Tolerability Assessments Safety will be monitored by the assessments described below as well as the collection of AEs, SAEs and SUSARs. For details on collection and reporting, refer to Section 8.

8 SAFETY MONITORING AND REPORTING

The investigator is responsible for the detection and documentation of events meeting the criteria and definition of an adverse event (AE) or serious adverse event (SAE). The methods for collection of safety data are described below.

8.1 Definitions

8.1.1 Adverse Event (AE) and Adverse Events of Special Interest (AESI) An adverse event (AE) is any untoward medical occurrence in a patient in relation to administration of a pharmaceutical product and which does not necessarily have a causal relationship with this treatment.

An AE can therefore be any unfavourable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal (investigational) product, whether or not related to the medicinal (investigational) product.

The term AE is used to include both serious and non-serious AEs.

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If an abnormal laboratory value/vital sign are associated with clinical signs and symptoms, the sign/symptom should be reported as an AE.

AESIs for this protocol is defines as gastrointestinal discomfort, visual disturbances, diarrhoea, headache, nausea and dizziness

8.1.2 Serious Adverse Event (SAE) Any untoward medical occurrence that at any dose:

 Results in death

 Is immediately life-threatening

 Requires in-patient hospitalization or prolongation of existing hospitalization

 Results in persistent or significant disability or incapacity

 Is a congenital abnormality or birth defect

 Is an important medical event that may jeopardize the subject or may require medical intervention to prevent one of the outcomes listed above,

Medical and scientific judgment is to be exercised in deciding on the seriousness of a case. Important medical events may not be immediately life-threatening or result in death or hospitalization, but may jeopardize the subject or may require intervention to prevent one of the listed outcomes in the definitions above. In such situations, or in doubtful cases, the case should be considered as serious.

8.1.3 Suspected Unexpected Serious Adverse Reactions (SUSAR) Suspected Unexpected Serious Adverse Reaction: SAE (see section 8.1.2) that is unexpected as defined in section 8.2 and possibly related to the IMP. Any event other than those mentioned in the SPC of the IMP may be classified as SUSAR.

8.2 Expected Adverse Events The most common SAE in short-term therapy with the IMP is gastrointestinal discomfort. In addition, the following symptoms will be registered in the CRF: visual disturbances, diarrhoea, headache, nausea and dizziness. These AE and gastrointestinal discomfort is considered adverse events of special interest (AESI). Will be reported according to standard adverse event criteria. On day 1, 3 and 6 during the treatment patients will be asked specifically about AESI. Further adverse events listed in the SPC for the IMP will also be registered as AE/SAE if they occur (see Appendix Summary of Product Characteristics “PLAQUENIL”).

8.3 Time Period for Reporting AESI and SAE For each patient the standard time period for collecting and recording SAEs will begin at the day of IMP treatment start and will continue for the entirety of the duration of treatment. During the course of the study all SAEs will be proactively followed up for each patient; events should be followed up to resolution, unless the event is considered by the investigator to be unlikely to resolve due to the underlying disease. Every effort should be made to obtain a resolution for all events.

Except AESI, AEs will not be reported, unless they meet the definition of an SAE.

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8.4 Recording of Serious AE and AESI If the patient has experienced serious adverse event(s), the investigator will record the following information in the CRF:

 The nature of the event(s) will be described by the investigator in precise standard medical terminology and assigned an ICD-10 diagnosis (i.e. not necessarily the exact words used by the patient).  The duration of the event will be described in terms of event onset date and event ended data.  The intensity of the adverse event will be graded as follows (18): . Mild: asymptomatic or mild symptoms; clinical or diagnostic observations only; intervention not indicated. . Moderate: minimal, local or non-invasive intervention indicated; limiting age-appropriate instrumental Activities of Daily Living (ADL) . Severe or medically significant but not immediately life-threatening: hospitalization or prolongation of hospitalization indicated; disabling; limiting self-care ADL.  The causal relationship of the event to treatment cessation will be assessed as one of the following:

Unrelated: There is not a temporal relationship to treatment cessation, or there is a reasonable causal relationship between non-investigational product, concurrent disease or circumstance and the AE.

Unlikely: There is a temporal relationship to treatment cessation, but there is not a reasonable causal relationship between treatment cessation and the AE.

Possible: There is reasonable causal relationship between treatment cessation and the AE.

Probable: There is a reasonable causal relationship treatment cessation and the AE.

Definite: There is a reasonable causal relationship between treatment cessation and the AE.

 Action taken.

 The outcome of the adverse event – whether the event is resolved or still ongoing.

It is important to distinguish between serious and severe AEs. Severity is a measure of intensity whereas seriousness is defined by the criteria in Section 8.1. An AE of severe intensity need not necessarily be considered serious. For example, nausea that persists for several hours may be considered severe nausea, but is not an SAE. On the other hand, a stroke that results in only a limited degree of disability may be considered a mild stroke, but would be an SAE.

8.5 Reporting Procedure

8.5.1 SAEs and AESIs All adverse events of special interest and serious adverse events that should be reported as defined in section 8.1.1 and 8.1.2 will be recorded in the patient's CRF.

SAEs must be reported within 24 hours after the site has gained knowledge of the SAE. Every SAE must be documented by the investigator on the AE forms in Viedoc and signed electronically. The initial report shall promptly be followed by

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detailed, written reports if necessary. The initial and follow-up reports shall identify the trial subjects by unique code numbers assigned to the latter.

The coordinating investigator keeps detailed records of all SAEs reported by the investigators and performs an evaluation with respect to causality and expectedness. Based on, among other, SAE reports the sponsor will evaluate whether the risk/benefit ratio associated with study is changed.

8.5.2 SUSARs SUSARs will be reported to the Norwegian Medicines Agency and REK Sør-Øst according to national regulation. The sponsor will ensure that all relevant information about SUSARs that are fatal or life-threatening is recorded and reported as soon as possible and in no case later than seven (7) days after knowledge by the sponsor of such a case, and that relevant follow-up information is subsequently communicated within an additional eight (8) days.

SUSARs will be reported to The Norwegian Medicines Agency ([email protected]) using the CIOMS form no later than 7 days after the incident.

8.6 Risk–benefit As described previously, the inherent risk associated with IMP is low, and expected adverse events are mild and self-limiting especially in short-term use. The risk of mortality from COVID-19 is yet undetermined, but preliminary reports document significant mortality risk, especially in elderly with pre-existing chronic disease. WHO has declared COVID-19 a global pandemic, and novel measures aimed at decreasing morbidity and mortality are imminently needed. Accordingly, the possible benefit of the IMP on individual, national and international scale greatly outweighs individual risk.

8.7 Data Monitoring Committee (DMC) A data monitoring committee (DMC) will be established to monitor the safety and efficacy of the study treatment and will consist of one statistician and two physicians with experience from virology/infectious disease and clinical studies. All members will be independent from the sponsor and will not be investigators or collaborators in the current study. The DMC will review all SAEs. The study investigators may call on the DMC to review specific SAEs. The DMC will conduct its tasks according to the EMA guideline (http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003635.pdf). A DMC charter based on the NORCRIN (http://www.norcrin.no/dokumentoversikt/) template will be signed and included in the Trial Master File.

9 DATA MANAGEMENT AND MONITORING

9.1 Electronic Case Report Forms (eCRF) The Clinical Data Management System (CDMS) used for the eCRF in this study is Viedoc. The eCRF system will be FDA Code of Federal Regulations 21 Part 11 compliant.

The designated investigator staff will enter the data required by the protocol into the eCase report forms (eCRF). The Investigator is responsible for assuring that data entered into the eCRF is complete, accurate, and that entry is performed in a timely manner. The signature of the investigator will attest the accuracy of the data on each eCRF. If any assessments are omitted, the reason for such omissions will be noted on the eCRFs. Corrections, with the reason for the corrections will also be recorded.

After database lock, the investigator will receive a digital copy of the subject data for archiving at the investigational site.

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9.2 Source Data Source data are all information in original records and certified copies of original records of clinical findings, observations, or other activities in a clinical trial necessary for the reconstruction and evaluation of the trial. Source data are contained in source documents (original records or certified copies).

The medical records for each patient should contain information which is important for the patient’s safety and continued care, and to fulfil the requirement that critical study data should be verifiable.

To achieve this, the medical records of each patient should clearly describe at least:

 That the patient is participating in the study, e.g. by including the enrolment number and the study code or other study identification;

 Date when Informed Consent was obtained from the patient and statement that patient received a copy of the signed and dated Informed Consent;

 Results of all assessments confirming a patient’s eligibility for the study;

 Diseases (past and current; both the disease studied and others, as relevant);

 Results of assessments performed during the study;

 Treatments given, changes in treatments during the study and the time points for the changes;

 Serious Adverse Events (if any) including causality assessments;

 Date of, and reason for, withdrawal from study;

Ethnicity will be recorded directly into the Case Report Form (meaning that CRF is source data and not the hospital records).

A source data list will be agreed upon for each site specifying the source at a module or a variable level.

9.3 Study Monitoring The investigator will be visited on a regular basis by the Clinical Study Monitor, who will check the following:

 Informed consent process

 Reporting of adverse events and all other safety data

 Adherence to protocol

 Maintenance of required regulatory documents

 Facilities and equipment (example: laboratory, pharmacy, ECG machine, etc…) if applicable

 Data completion on the CRFs including source data verification (SDV).

The monitor will review the relevant CRFs for accuracy and completeness and will ask the site staff to adjust any discrepancies as required.

Sponsor’s representatives (e.g. monitors, auditors) and/or competent authorities will be allowed access to source data for source data verification in which case a review of those parts of the hospital records relevant to the study will be required.

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9.4 Confidentiality The investigator shall arrange for the secure retention of the patient identification and the code list. Patient files shall be kept for the maximum period of time permitted by each hospital. The study documentation (CRFs, Site File etc) shall be retained and stored during the study and for 15 years after study closure. All information concerning the study will be stored in a safe place inaccessible to unauthorized personnel.

9.5 Database management Data management will be performed by the data management unit at the Clinical Trials Unit, Oslo University Hospital. The Data management procedures will be performed in accordance with the department’s SOPs and ICH guidelines. The data management process will be described in the study specific Data Handling Plan and the study specific Data Handling Report after database closure.

Data entered into the eCRF will be validated as defined in the Data Validation Plan. Validation includes, but is not limited to, validity checks (e.g. range checks), consistency checks and customised checks (logical checks between variables to ensure that study data are accurately reported) for eCRF data and external data (e.g. laboratory data). A majority of edit checks will be triggered during data entry and will therefore facilitate efficient ‘point of entry’ data cleaning.

Data management personnel will perform both manual eCRF review and review of additional electronic edit checks to ensure that the data are complete, consistent and reasonable. The electronic edit checks will run continually throughout the course of the study and the issues will be reviewed manually online to determine what action needs to be taken.

Manual queries may be added to the system by clinical data management or study monitor. Clinical data managers and study monitors are able to remotely and proactively monitor the patient eCRFs to improve data quality.

All updates to queried data will be made by authorised study centre personnel only and all modifications to the database will be recorded in an audit trail. Once the queries have been resolved, eCRFs will be signed by electronic signature. Any changes to signed eCRFs will be approved and resigned by the Investigator.

Adverse events and medical history will be coded from the verbatim description (Investigator term) using the Medical Dictionary for Regulatory Activities (MedDRA).

Once the full set of eCRFs have been completed and locked, the Sponsor will authorise database lock and all electronic data will be sent to the designated statistician for analysis. Subsequent changes to the database will then be made only by written agreement.

The data will be stored in a dedicated and secured area at Oslo University Hospital. Data will be stored in a de-identified manner, where each study participant is recognisable by his/her unique trial subject number. The data will be stored until 15 years following the last patient’s final study visit.

10 STATISTICAL METHODS AND DATA ANALYSIS

10.1 Determination of Sample Size The rate of decline in SARS-CoV-2 viral load from baseline to 96 hours will be used as the basis for determination of sample size. Little is known about the variance of the viral load decline rate under standard of care and the possible efficacy of the intervention. Thus, we are planning this trial as an adaptive group sequential trial assuming a rate reduction from baseline of 6 log10 copies/mL/24 hours for the control group and 8 for the active group with a standard deviation of 4 within the first 96 hours after inclusion. Under these assumptions and with a Pocock type uniform alpha spending function (see Figure 2) with 4 looks the maximum sample size will be 202 using a 2.5% one-sided significance level to reach 90% power to detect a difference between the treatments. The interim analyses will be at 51, 101, 151 and 202 completed trial subjects. The assumptions underlying the sample size calculation will be re-evaluated at each interim analysis possibly warranting changes in the sample size without affecting the Type 1 error rate.

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Figure 2.

10.2 Randomization

10.2.1 Allocation- sequence generation Eligible patients will be allocated in a 1:1 ratio, using a computer randomization procedure. The allocation sequence will be prepared by an independent statistician.

10.2.2 Allocation- procedure to randomize a patient The computer-generated randomized allocation sequence will be imported into the eCRF system and made available to site personnel responsible for the participant enrolment. Randomization allocation will automatically be visible when enrolling a new eligible patient. This is an open-label study and no steps to conceal allocation are necessary.

The study statistician will be blinded to the randomization allocation for the writing of the statistical analysis plan (SAP). The authorisations bound to role of the study statistician in the eCRF when reading or downloading data will ensure that the statistician won’t see the treatment allocation until database lock.

10.2.3 Blinding and emergency unblinding This is an open-label study. However, the staff at the central laboratory at OUS as well as the statistician responsible for analysis of the data will be blinded to the treatment allocation for the writing of the SAP.

10.3 Population for Analysis The following populations will be considered for the analyses:

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- Intention to treat (ITT) population: All randomized participants will be included in the main ITT analyses, regardless of protocol adherence.

- Per-protocol population (PP): Includes all patients in the ITT population having completed the study treatment without major protocol violations. Criteria for inclusion in the PP population will be specified in the SAP and the final criteria will be defined prior to database lock

- Safety population: Includes all subjects with any safety information after baseline. Patients randomised to hydroxychloroquine without receiving any amount of the treatment will be excluded from the safety population.

- Total population: All enrolled participants independent of study arm will be used for additional analyses in the total population.

The primary population is the ITT population.

10.4 Planned analyses

This is a group sequential adaptive trial where analyses are planned after 51, 101, 151 and 202 completed patients.

Each statistical analysis is planned when

 The planned number of patients have been included

 All included patients have either finalized their last assessment discharge or has/is withdrawn/lost to follow-up according to protocol procedures

 All data have been entered, verified and validated according to the data management plan

Prior to each statistical analysis, the data in the data base will be exported and the exported data will be locked for further altering of data. A SAP will provide details on the planned statistical analyses. The SAP will be finalized, signed and dated prior to first interim analysis. The statistical interim analysis will be performed by an unblinded Data Monitoring Committee (DMC) statistician based on program code from the trial statistician. The trial statistician will remain blinded to treatment when finalising the SAP prior to first interim analysis and throughout the trial until final database lock. The unblinded statistician performing the analysis will only provide the study group with information on whether the trial should be stopped or continued. Details of the DMC and their procedures will be given in a separate DMC charter.

10.5 Statistical Analysis

10.5.1 Primary analysis  The primary outcome is rate of decline in SARS-CoV-2 viral load in nasopharyngeal samples as assessed by RT-PCR in samples collected at baseline, 48 and 96 hours after randomization.

The primary outcome will be analysed using a generalized linear mixed model, with subject-specific random intercept and slope, adjusted for stratification factors in the randomization.

10.5.2 Secondary analyses Between group comparisons will be performed for the primary variable on the per-protocol population in addition to secondary efficacy endpoints on both efficacy populations (ITT and PP populations).

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The between-group comparisons for secondary variables will be tested as for the primary variable where applicable and additional analyses will be performed based on the following methods (but not limited to):

 Continuous secondary variables will be subject to repeated measures mixed models or appropriate non- parametric alternatives

 Binary response variables will be analysed using logistic regression (possibly adjusting for within-subject dependencies by generalized estimating equations or mixed models) or chi-square/Mantel-Haenszel tests

 Time-to-event variables will be analysed using the Kaplan-Meier method and comparisons between the two groups will be performed using the log rank test, Cox regression analyses or appropriate parametric models such as the gamma or Weibull model.

Unless otherwise specified, all statistical hypotheses will be tested as the primary variable, i.e. with an assessment of superiority of the estimated difference between the groups. All efficacy analyses will be presented with the results from the hypothesis testing (by p-value) in addition to estimates and 95% confidence limits of the treatment effect.

10.5.3 Safety analyses The safety analyses population will include all patients. Safety analyses will be descriptive and presented as summary tables by treatment group.

10.5.4 Descriptive statistics Descriptive statistics will be presented with number and percentages for categorical variables, and means, standard deviation, and range for continuous variables. In case of clearly skewed continuous variables, they will be presented with median, interquartile range (25th and 75th percentiles) and range. Demographics and baseline characteristics will be presented with descriptive statistics without any hypothesis testing.

10.5.5 Missing data If missing data is regarded as having a significant effect on the conclusions of the trial, sensitivity analyses with different methods for handling missing data will be included. Such methods may include complete case analyses, last observation carried forward, worst case/best case imputation and multiple imputation techniques.

11 STUDY MANAGEMENT

11.1 Investigator Delegation Procedure The principal investigator is responsible for making and updating a “delegation of tasks” document listing all the involved co-workers and their role in the project. He will ensure that appropriate training relevant to the study is given to all of these staff, and that any new information of relevance to the performance of this study is forwarded to the staff involved.

11.2 Protocol Adherence Investigators ascertain that they will apply due diligence to avoid protocol deviations. All significant protocol deviations will be recorded and reported in the Clinical Study Report (CSR). 11.3 Study Amendments If it is necessary for the study protocol to be amended, the amendment and/or a new version of the study protocol (Amended Protocol) must be notified to and approved by the Competent Authority and the Ethics Committee according to EU and national regulations.

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11.4 Audit and Inspections Authorized representatives of a Competent Authority and Ethics Committee will visit the centre to perform inspections, including source data verification. Likewise the representatives from sponsor may visit the center to perform an audit. The purpose of an audit or inspection is to systematically and independently examine all study-related activities and documents to determine whether these activities were conducted, and data were recorded, analysed, and accurately reported according to the protocol, Good Clinical Practice (ICH GCP), and any applicable regulatory requirements. The principal investigator will ensure that the inspectors and auditors will be provided with access to source data/documents.

12 ETHICAL AND REGULATORY REQUIREMENTS

The study will be conducted in accordance with ethical principles that have their origin in the Declaration of Helsinki and are consistent with ICH/Good Clinical Practice and applicable regulatory requirements. Registration of patient data will be carried out in accordance with national personal data laws.

12.1 Ethics Committee Approval The study protocol, including the patient information and informed consent form to be used, must be approved by the regional ethics committee before enrolment of any patients into the study.

12.2 Other Regulatory Approvals The protocol will be submitted and approved by the applicable competent authorities before commencement of the study. Amendments to the protocol will be submitted to the competent authorities according to local regulations.

The protocol will be registered in www.clinicaltrials.gov before inclusion of the first patient.

Application to the Norwegian Medicines Agency will be approved before inclusion of the first patient.

Collection, storage and analyses of all data and sensitive information will be conducted according to current General Data Protection Regulation (GDPR) and in accordance with approval from the local Data Protection Official.

12.3 Informed Consent Procedure The investigator is responsible for giving the patients full and adequate verbal and written information about the nature, purpose, possible risk and benefit of the study. They will be informed as to the strict confidentiality of their patient data, but that their medical records may be reviewed for trial purposes by authorized individuals other than their treating physician.

It will be emphasized that the participation is voluntary and that the patient is allowed to refuse further participation in the protocol whenever she/he wants. This will not prejudice the patient’s subsequent care. Documented informed consent must be obtained for all patients included in the study before they are registered in the study. This will be done in accordance with the national and local regulatory requirements. The investigator is responsible for obtaining signed informed consent.

A copy of the patient information and consent will be given to the patients. The signed and dated patient consent forms will be filed in the Investigator Site File binder.

In cases where the patient does not speak Norwegian, a professional translator will be used.

12.4 Subject Identification The investigator is responsible for keeping a list of all patients (who have stopped treatment or undergone any study specific procedure) including patient’s date of birth and personal number, full names and last known addresses.

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The patients will be identified in the CRFs by patient number.

13 TRIAL SPONSORSHIP AND FINANCING

The study is sponsored by Akershus University Hospital.

14 TRIAL INSURANCE

The Principal investigator has insurance coverage for this study through membership of the Drug Liability Association (see http://www.laf.no for more details).

15 PUBLICATION POLICY

Upon study completion and finalization of the study report the results of this study will either be submitted for publication and/or posted in a publicly assessable database of clinical study results.

The results of this study will also be submitted to the Competent Authority and the Ethics Committee according to EU and national regulations.

All personnel who have contributed significantly with the planning and performance of the study (Vancouver convention 1988) may be included in the list of authors.

16 REFERENCES

1. Ren LL, et al. Identification of a novel coronavirus causing severe pneumonia in human: A descriptive study. Chin Med J (Engl) 2020;Publish Ahead of Print. 2. WHO. Coronavirus disease (covid-19) outbreak. https://www.who.int/emergencies/diseases/novel-coronavirus-2019 (Accessed March 12 2020). 3. WHO. R&d blueprint. Informal consultation on prioritization of candidate therapeutic agents for use in novel coronavirus 2019 infection. 2020. 4. Wang M, et al. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro. Cell Res 2020;30:269-71. 5. Gao J, et al. Breakthrough: Chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies. Bioscience trends 2020:10.5582/bst.2020.01047. 6. Multicenter Collaboration Group of Department of Science Technology of Guangdong Province Health Commission of Guangdong Province for Chloroquine in the Treatment of Novel Coronavirus Pneumonia. [expert consensus on chloroquine phosphate for the treatment of novel coronavirus pneumonia]. Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese journal of tuberculosis and respiratory diseases 2020;43:E019. 7. Ison MG, et al. End points for testing influenza antiviral treatments for patients at high risk of severe and life-threatening disease. The Journal of infectious diseases 2010;201:1654-62. 8. McGinley A, et al. A national early warning score for acutely ill patients. BMJ 2012;345:e5310. 9. Zhou F, et al. Clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: A retrospective cohort study. Lancet 2020. 10. Yao X, et al. In vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2). Clin Infect Dis 2020. 11. Trinkley KE, et al. Qt interval prolongation and the risk of torsades de pointes: Essentials for clinicians. Curr Med Res Opin 2013;29:1719-26. 12. Zou L, et al. Sars-cov-2 viral load in upper respiratory specimens of infected patients. N Engl J Med 2020.

17 LIST OF APPENDICES

Summary of Product Characteristics “PLAQUENIL”

Protocol NO-COVID-19 Version no. 1.1, 2020 – 03 – 18 Page 31 of 31 STATISTICAL ANALYSIS PLAN for Ahus-NO-COVID-19

Akershus University Hospital Sponsor name Jan Erik Berdal

Akershus University Hospital Head of Department / Associate Professor Jan Erik Berdal Department of Infectious Diseases Akershus University Hospital Sponsor address Sykehusveien 25 1478 Lørenskog, Norway Tel: 67 96 00 00

E-mail: [email protected]

EudraCT number 2020-001010-38

Trial ID Ahus-NO-COVID-19

Norwegian Coronavirus Disease 2019 (NO COVID-19) Study: An open labelled randomized controlled pragmatic trial to evaluate the antiviral effect of chloroquine in adult patients with SARS-CoV-2 infection

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Statistical Analysis Plan for the final analysis

Version 2.1 - Final

Date: 28 May 2020

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SIGNATURE PAGE

SPONSOR REPRESENTATIVE:

Head of Department / Associate Professor Jan Erik Berdal, MD PhD

Department of Infectious Diseases, Division of Medicine Akershus University Hospital

Signature Date (dd/mmm/yyyy)

PRINCIPAL/COORDINATING INVESTIGATOR:

Professor Olav Dalgard, MD PhD

Department of Infectious Diseases, Division of Medicine Akershus University Hospital

Signature Date (dd/mmm/yyyy)

TRIAL STATISTICIANS:

Inge Christoffer Olsen, PhD

Clinical Trial Unit Oslo University Hospital HF

Signature Date (dd/mmm/yyyy)

Corina S. Rueegg, PhD

Oslo Centre for Biostatistics & Epidemiology Oslo University Hospital HF

Signature Date (dd/mmm/yyyy)

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ABBREVIATIONS

AE Adverse Event

ATC Anatomical/ Therapeutic/ Chemical

BMI Body Mass Index

CI Confidence Interval

COVID-19 Coronavirus Disease

DMC Data Monitoring Committee eCRF electronic Case Report Form

EDC Electronic Data Capture

ICU Intensive Care Unit

ID Identification/ Identifier

IMP Investigational Medicinal Product

ITT Intention to treat

MedDRA Medical Dictionary for Regulatory Activities

NEWS National Early Warning Score

PCR Polymerase Chain Reaction

PCT Pragmatic Clinical Trial

PP Per-protocol

RCT Randomised Controlled Trial

SAE Serious Adverse Event

SAP Statistical Analysis Plan

SD Standard Deviation

SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2

SOC System Organ Class

SPC Summary of Product Characteristics

SUSAR Suspected Unexpected Serious Adverse Reactions

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WHO World Health Organization

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TABLE OF CONTENTS

1 STUDY OBJECTIVES ...... 6 1.1 Primary Objective ...... 6 1.2 Secondary Objectives ...... 6 2 OVERALL STUDY DESIGN ...... 6 3 GENERAL STATISTICAL CONSIDERATIONS ...... 7 4 HYPOTHESES AND DECISION RULES ...... 8 4.1 Statistical Hypotheses ...... 8 4.2 Statistical Decision Rule ...... 9 5 DEFINITIONS AND DERIVED VARIABLES...... 9 5.1 Change from baseline ...... 9 5.2 Other calculations ...... 10 5.3 Safety definitions ...... 10 5.4 Previous and Concomitant medications ...... 10 6 EFFICACY AND SAFETY ENDPOINTS / VARIABLES ...... 11 6.1 Primary Endpoint ...... 11 6.2 Secondary Endpoints ...... 11 6.3 Exploratory Endpoints ...... 11 6.4 Safety Parameters ...... 12 7 DETERMINATION OF SAMPLE SIZE ...... 12 8 DATA SETS TO BE ANALYSED ...... 13 8.1 Treatment Misallocation ...... 13 8.2 Protocol violations ...... 13 9 BLIND REVIEW ...... 14 10 STATISTICAL METHODOLOGY ...... 14 10.1 Adjustment for covariates ...... 14 10.2 Centre effect ...... 14 10.3 Multiplicity adjustments ...... 14 10.4 Demographic and other baseline characteristics ...... 14 10.5 Primary efficacy analysis ...... 14 10.6 Secondary efficacy analyses ...... 15 10.7 Patient Characteristics ...... 15 10.8 Exploratory Analysis ...... 16 11 SAFETY ANALYSIS ...... 16 11.1 Adverse events ...... 16 11.2 Other safety assessments ...... 17 12 INTERIM ANALYSIS ...... 17 13 DATA MONITORING ...... 17 14 TABLE OF CONTENTS OF TABLES, LISTINGS AND FIGURES ...... 17 14.1 List of planned Tables ...... 17 14.2 List of planned Listings ...... 18 14.3 List of planned Figures ...... 18 15 REFERENCES ...... 18 16 APPENDICES ...... 18

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AMENDMENTS TO THE SAP Not applicable

1 STUDY OBJECTIVES

1.1 Primary Objective The primary aim of this study is to assess whether treatment with chloroquine in patients with COVID-19 will increase the decline rate of SARS-CoV-2 in the oropharynx from baseline to 48 and 96 hours.

1.2 Secondary Objectives The secondary aims are to assess whether treatment with chloroquine in patients with COVID-19 (1) will improve clinical status as assessed by the NEWS score, improve clinical outcomes, including (2) admission rate to intensive care units and (3) in-hospital mortality, and (4) improve protein biomarker profile (inflammation, cardiac, renal, hepatic).

2 OVERALL STUDY DESIGN The study is a two-arm, open label, pragmatic randomized controlled trial (RCT) designed to assess the virological and clinical effect of chloroquine therapy in addition to standard of care versus standard of care in patients with established COVID-19. Pragmatic clinical trials (PCT) are characterized by 3 attributes: (1) focus on informing decision-makers (e.g. patients, politicians, administrators) on optimal clinical medicine practice, as opposed to elucidating a biological or social process; (2) intent to enrol a population representative to the decision in practice and for whom the decision is relevant; and (3) either an intent to streamline procedures and data collection in the trial or to measure a broad range of outcomes. By utilizing resources already paid for by the hospitals (physicians and nurses in daily clinical practice), pragmatic clinical trials can include a larger number of patients at a short time duration and at a lower cost than studies utilizing traditional RCT designs with an external study organization (e.g. study nurses, study physicians). Due to the immediate need for study commencement and the time frame of the current proposal, a pragmatic approach will enable swift initiation of randomization and treatment. We will especially use data from the data warehouse at Akershus University Hospital for eligible patient identification (i.e. electronic surveillance) and for automatic data extraction to the study specific database. The study will not be able to procure an acceptable placebo treatment and the study will accordingly not be placebo- controlled.

In the initial phase of the study, patients will be included from a single center (Akershus University Hospital). The study is designed as a sequential adaptive trial where interim efficacy analyses are planned after the inclusion of 51 patients, with subsequent analyses after 101, 151 and 202 completed patients. This approach will enable frequent assessment of the primary outcome measure. After inclusion of the initial 51 patients, analyses will be performed for the predefined primary endpoint. Pending this result, the study will possibly be extended to other Norwegian centres, pending separate submission to ethics committee and other regulatory bodies from new centers.

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All patients at Akershus University Hospital with suspicion of acute respiratory tract infections are examined with a nasopharyngeal swab, with subsequent microbiological examination, including SARS-CoV-2 specific polymerase chain reaction (PCR). Laboratory reports from the Department of Microbiology are surveilled real-time for SARS-CoV-2 positive samples in the local data warehouse, which will allow for immediate screening and randomization in all eligible subjects. Figure 1 gives a schematic overview of the study.

Figure 1. Trial design

Study Period of initial Estimated date of first patient enrolled: 23.03.20 study phase: Anticipated recruitment period: 6 months

Estimated date of last patient completed: 01.04.21 Estimated study termination: 23.03.25 Treatment Duration: Patients allocated to the treatment arm will be treated with 400 mg hydroxychloroquine sulphate (equalling 310 mg base) twice daily for seven days. Follow-up: Continuous of follow-up at admission, as well as assessment of clinical status at 14 days and mortality at day 30 and 90.

3 GENERAL STATISTICAL CONSIDERATIONS All analyses described in this plan are considered a priori analyses in that they have been defined in the protocol and/or this SAP. All post hoc analyses will be identified as such in the Clinical Study Report. Even though this is an open trial, the SAP and statistical programs were written prior to any analyses, tabulations, figures or listings by treatment arm. Thus, for all practical purposes, the statisticians writing this SAP can be considered blinded.

All categorical (binary and ordinal) data will be summarised using frequency counts and percentages of patient incidence. Percentages will be calculated using the study population; any exceptions to this will be highlighted in the table footnote. The continuous variables will be summarised using number of patients (N), mean, standard deviation (SD), median, 25/75 percentile and range (minimum/maximum), depending on the skewness of the distribution. In general, minimum and

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Effect estimates will be accompanied by a 95% confidence interval (CI) and p-value.

P values will be displayed to three decimal places after rounding. P values below 0.001 will be displayed as “<0.001”.

Data will be presented, unless otherwise indicated, according to the two treatment groups.

In general, analyses presented in the tables, listings, and figures will be confined to those subjects included in the safety analysis set, full analysis set and per protocol set (see below), or a specific subset thereof. Data listings will include all randomised study subjects unless otherwise noted. Subjects will be identified in the listings by a variable which uniquely combines site number and subject number. Listings will be sorted by this unique id.

Study days will be computed from the date of first IMP administration, with study day 1 representing the first treatment date.

AE duration will be calculated as (Stop Date – Start Date) + 1.

This trial is designed for 4 looks, 3 interim analyses and one final analysis with a cumulative one- sided alpha of 0.025 uniformly spent (Pocock). The one-sided local significance levels are 0.0091 at each stage. The interim analyses will be performed by a designated Data Monitoring Committee (DMC) statistician, who will be unblinded to treatment allocation.

At the writing of this SAP, the first interim analysis has been performed, with a recommendation to continue the trial. Due to low recruitment the sponsor has decided to stop the trial and report the findings. The decision to stop the trial was not based on internal efficacy analyses, and there is therefore no need to adjust the significance level based on this decision. We therefore redesign the trial as a group sequential trial with two looks, the performed interim and the final analysis. With an interim analysis with 0.0091 alpha spent at 0.9 of the information, the final one-sided local significance level will be 0.0244 to claim superiority.

4 HYPOTHESES AND DECISION RULES

4.1 Statistical Hypotheses This protocol is designed to establish the superiority of hydroxychloroquine sulphate (+ standard care) compared to standard care only with regard to decrease in viral load from randomisation to 48 and 96 hours.

The primary null hypothesis is that there is no difference in the slope of the viral load from randomisation to 48 and 96 hours between the two treatment regimes.

The primary alternative hypothesis is that there is a difference in the slope of the viral load from randomisation to 48 and 96 hours between the two treatment regimes.

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The primary null hypothesis will be tested using an appropriate statistical test as described in chapter 10.5.

There will be no formal hypothesis testing for the secondary endpoints.

4.2 Statistical Decision Rule This protocol is designed to address a single primary endpoint at four times. Statistical significance is claimed if the null hypothesis is rejected on the significance level (alpha) of 0.0182 (two-sided) at each stage. That is, if the p-value of the null hypothesis test is less than 0.0182.

Due to the premature stopping of the trial due to low recruitment, the position above position had to be reassessed because there only was one interim look. The final one-sided local significance level will be 0.0244 to claim superiority, see explanation in Section 3.

5 DEFINITIONS AND DERIVED VARIABLES For analysis and tabulation purposes, we define the study time points as followed: Time Point Label Target Hour/Day Definition (Hour/Day window)

TP1. Baseline Hour 0 (Randomisation) Information up to randomisation

TP2. 24 h Hour 24 0-35 hours

TP3. 48 h Hour 48 36-59 hours

TP4. 72 h Hour 72 60-83 hours

TP5. 96 h Hour 96 84-107 hours

TP6. 120 h Hour 120 108-131 hours

TP7. 144 h* Hour 144 132-155 hours

TP8. 168 h Hour 168 156-179 hours

TP9. 192 h Hour 192 180-203 hours

TP10. 216 h Hour 216 204-227 hours

TP11. 14 d Day 14 13-15 days

TP12. 30 d Day 30 29-31 days

TP13. 90 d Day 90 89-91 days

*Study treatment stops at day 7 (144h)

5.1 Change from baseline Change from baseline (Δ) = time-point value - baseline value.

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% change from baseline (%Δ) = [(time-point value – baseline value) / baseline value] *100%

5.2 Other calculations Age (years) = [(date of baseline – date of birth)/365.25].

Time of withdrawal = (date of withdrawal – date of randomization) +1

Follow-up time = (last date in study – date of randomisation) + 1

In-hospital time = (discharge date – admission date) + 1

The viral load values will be log transformed with base 10.

BMI = weight in kilograms / (height in metres) x (height in metres)

BMI will be categorized according to the WHO definitions for underweight, normal, overweight and obese: <18.5 kg/m2 (underweight), 18.5–24.9 kg/m2 (normal range), 25-29.9 (overweight), ≥30 (obesity)

NEWS score = mean of available score assessments per day with >5 hours apart.

Time since debut of symptoms = (Date of randomization - Date of debut of symptoms) + 1

5.3 Safety definitions

5.3.1 Treatment emerging adverse events Treatment emerging adverse events (TEAEs) are defined as AEs with a start date on or after the first treatment day.

5.3.2 Past disease and concomitant disease A disease/condition is considered as past disease/condition if it is not ongoing at randomisation.

A disease/condition is considered as concomitant disease/condition if it is ongoing at randomisation.

5.4 Previous and Concomitant medications The following definitions will be used:

 previous medication (stop date < date of randomisation);

 concomitant medication (stop date ≥ date of randomisation or ongoing at study end)

In case of missing or incomplete dates/times not directly allowing allocation to any of the two categories of medications, a worst-case allocation will be performed according to the available parts of the start and the end dates. The medication will be allocated to the first category allowed by the available data, according to the following order:

 concomitant medication

 previous medication

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6 EFFICACY AND SAFETY ENDPOINTS / VARIABLES

6.1 Primary Endpoint The primary endpoint consists of the SARS-CoV-2 viral load (log10) outcome measured at 48h and 96h post-randomisation. The slope of the viral load from baseline to 48h and 96h after randomisation modelled with a mixed model is the primary endpoint (see also chapter 10.5 on the primary efficacy analysis).

6.2 Secondary Endpoints The secondary endpoints will not be analysed or described in the three interim analyses.

6.2.1 Efficacy - The change (Δ) in SARS-CoV-2 viral load (log10) from baseline to 96 hours

- The change (Δ) in NEWS score from baseline to 96 hours after randomisation. The NEWS score is an ordinal scale with range 0-20. A higher score indicates clinical deterioration. Depending on the clinical status of the patient, NEWS is assessed from once daily to quite frequently (at the discretion of the nurses). If more than one NEWS score per day is recorded and they are >5 hours apart, the average will be calculated and used as the score of the respective day.

- Number of participants referred to the ICU at any time from baseline to discharge or end of study (whatever comes first).

- In-hospital mortality: Number of participants who died at any time from baseline to discharge or end of study follow-up (whatever comes first).

- Duration of hospital stay: Number of days from admission to discharge.

- Mortality: Number of participants who died from baseline to 1) 30 days and 2) 90 days after randomisation.

- Clinical status 14 days after randomisation on an ordinal score ranging from 1 to 6: death (1); hospitalized, on invasive mechanical ventilation or extracorporeal membrane oxygenation (2); hospitalized, on non-invasive ventilation or high flow oxygen devices (3); hospitalized, requiring supplemental oxygen (4); hospitalized, not requiring supplemental oxygen (5); not hospitalized (6).

- The change in corrected QT time from randomisation to 48 hours post-randomisation

6.2.2 Patient Reported Outcomes (PRO) None

6.3 Exploratory Endpoints

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- Change in protein biomarker profiles at 96 hours after randomization, including C-reactive protein, markers of renal and hepatic injury, and established cardiac biomarkers like cardiac troponin and B-type natriuretic peptide concentrations.

6.4 Safety Parameters For each patient the standard time period for collecting and recording SAEs will begin at the day of IMP treatment start and will continue for the entirety of the duration of treatment. In the case of patient discharge before the end of treatment, patients will be followed up by phone for assessment of AEs. Except AESI, AEs will not be reported, unless they meet the definition of an SAE. Further, adverse events listed in the Summary of Product Characteristics (SPC) for the IMP will also be registered as AE/SAE if they occur (see protocol Appendix for the SPC of “PLAQUENIL”).

 Adverse events (AEs): any unfavourable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal (investigational) product, whether or not related to the medicinal (investigational) product. The term AE is used to include both serious and non-serious AEs.

 Adverse events of special interest (AESI): gastrointestinal discomfort, visual disturbances, diarrhoea, headache, nausea and dizziness. AESIs will be reported according to standard adverse event criteria. On day 1, 3 and 6 of treatment patients will be asked specifically about AESIs.

 Serious adverse events (SAEs): any untoward medical occurrence that at any dose 1) Results in death; 2) Is immediately life-threatening; 3) Requires in-patient hospitalization or prolongation of existing hospitalization; 4) Results in persistent or significant disability or incapacity; 5) Is a congenital abnormality or birth defect; or 6) Is an important medical event that may jeopardize the subject or may require medical intervention to prevent one of the outcomes listed above. The most common SAE in short-term therapy with the IMP is gastrointestinal discomfort

 Suspected unexpected serious adverse reactions (SUSARs): an SAE that is unexpected as defined after and possibly related to the IMP. Any event other than those mentioned in the SPC of the IMP will be classified as SUSAR (see protocol Appendix for SPC of “PLAQUENIL”).

7 DETERMINATION OF SAMPLE SIZE The rate of decline in SARS-CoV-2 viral load from baseline to 96 hours will be used as the basis for determination of sample size. Little is known about the variance of the viral load decline rate under standard of care and the possible efficacy of the intervention. Thus, we are planning this trial as an adaptive group sequential trial assuming a rate reduction from baseline of 6 log10 copies/mL/24 hours for the control group and 8 for the active group with a standard deviation of 4 within the first 96 hours after inclusion. Under these assumptions and with a Pocock type uniform alpha spending function with 4 looks the maximum sample size will be 202 using a 2.5% one-sided significance level to reach 90% power to detect a difference between the treatments. The interim analyses will be at 51, 101, 151 and 202 completed trial subjects. The assumptions underlying the sample size calculation will be re-evaluated at each interim analysis possibly warranting changes in the sample size without affecting the Type 1 error rate.

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With the trial stopped at 51 patients, and local significance levels at 0.0091 and 0.0244, the power under the above assumptions will be 42%.

8 DATA SETS TO BE ANALYSED The Full Analysis Set (FAS) will include all randomised subjects who have had at least one baseline and one post-randomisation evaluation of efficacy.

The per-protocol (PP) set is a subset of the FAS including all randomised participants without major protocol violations as defined under section 8.2.

The Safety Analysis Set Includes all subjects with any safety information after baseline. Patients randomised to hydroxychloroquine without receiving any amount of the treatment will be excluded from the safety population.

The primary efficacy analysis will be based on the FAS (intention-to-treat, ITT). Secondary efficacy analyses will be based on both the FAS and PP set. The safety analysis will be based on the safety analysis set.

8.1 Treatment Misallocation If patients were:

 Randomized but not treated: patient will appear on the patient evaluation table as randomized but not treated; this is the extent of how much the patient will be reported.

 Treated but not randomized: then by definition the patient will be excluded from both the efficacy and safety analyses since randomized treatment is missing.

 Randomized but did not follow protocol according to allocation: then they will be reported under their randomized treatment group for all efficacy and safety analyses as part of the FAS and safety analyses but omitted from the PP set.

Note that treated in this section is defined as having completed at least one post-baseline efficacy assessment.

8.2 Protocol violations The following criteria need to be fulfilled for participants to be a member of the PP set:

- Participants of the hydroxychloroquine arm who took at least 80% of the target number of tablets within the first 96 hours. Participants are supposed to take 400mg hydroxychloroquine sulphate twice daily for 7 days resulting in 14 doses. Participants who took ≥11 doses are considered to be compliant, while patients having taken at least 7 of the first 8 doses will be included in the PP set. Drug compliance is monitored by the hospital electronic prescribing system, where administration status of every dose prescribed is registered. - Participants of the standard care arm who have not received any hydroxychloroquine from randomisation until 90 days post randomisation.

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9 BLIND REVIEW Prior to each statistical analysis, the data in the data base will be exported and the exported data will be locked for further altering of data. The study statistician will be blinded to the randomization allocation for the writing of the statistical analysis plan (SAP). The SAP will be finalized, signed and dated prior to first interim analysis. The statistical interim analysis will be performed by an unblinded Data Monitoring Committee (DMC) statistician based on program code from the trial statistician. The trial statistician will remain blinded to treatment throughout the trial until final database lock. The unblinded statistician performing the interim analyses will only provide the study group with information on whether the trial should be stopped or continued.

The final blind review has been performed, and all discovered inconsistencies and errors have been corrected.

10 STATISTICAL METHODOLOGY The primary efficacy analyses will be based on the Full Analysis Set. Secondary efficacy analyses will be based both on the Full Analysis and Per Protocol set. As there is only one identified primary analysis, there will be no adjustments for multiple testing in the secondary analyses.

When random numbers are warranted for inference (such as for bootstrapping and multiple imputation), the seed will be set to the date the statistical analysis plan is signed-off (in the format yyyymmdd).

All efficacy analyses will be presented by the size (point estimate) of the difference between the treatments and the associated 95% confidence interval. 10.1 Adjustment for covariates Continuous change from baseline endpoints will be adjusted for the baseline value. Otherwise there will be no adjustment for baseline or other covariates.

10.2 Centre effect Not applicable, this is a single-centre study.

10.3 Multiplicity adjustments As there is only one identified primary analysis, there will be no adjustments for multiple testing in the secondary analyses.

10.4 Demographic and other baseline characteristics Demographic and baseline characteristics will be summarised descriptively by treatment group using the FAS. Categorical data will be displayed as numbers and %. Continuous data will be displayed as mean, SD, and range or if not normally distributed as median, IQR and range.

For the interim analysis, only age and sex will be included in the table of baseline characteristics. For the final analysis, more demographic and baseline characteristics will be defined to be included in the baseline table, such as symptoms, imaging, co-morbidities, substance use, clinical biochemistry, microbiology, anthropometrics, ECG, NEWS score, current medications

10.5 Primary efficacy analysis

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The primary endpoint (slope of the viral load (log10) from baseline to 48h and 96 hours post- randomisation) will be analysed using a mixed model with fixed and random intercept and slope. The mathematical expression of the model is as follow:

푌푖푗 = 퐵0푖 + 훽0 + 훽0푇 + (퐵1푖 + 훽1 + 훽1푇)푥푖푗 + 휀푖푗 where 푖 is the patient, 푗 is the time (1 = baseline, 2 = 48h, 3 = 96h), 퐵0푖 and 퐵1푖 are the random intercept and slope for patient 푖, 훽0 and 훽0 + 훽0푇 is the fixed intercept for the standard of care and hydroxychloroquine groups, 훽1 is the fixed slope for the standard of care group, 훽1 + 훽1푇 is the fixed slope for patients in the hydroxychloroquine group, 푥푖푗 is the time from baseline to timepoint 푗 for patient 푖, 휀푖푗 is the residual, and 푌푖푗 is the viral load (log10) for patient 푖 at timepoint 푗.

The null hypothesis to be tested is that 훽1푇 = 0, against the alternative hypothesis that 훽1푇 ≠ 0. The p-value will be calculated using the z-statistic based on the maximum likelihood estimates.

The primary analysis will be performed on the Full Analysis Set.

10.5.1 Missing data imputation and sensitivity analyses Missing data will be handled implicitly in the mixed model. Mixed model analyses provide unbiased estimates under the assumption of Missing at Random, meaning that the missing random process is conditionally independent on the unobserved outcome values. Given that these are hospitalised patients, this assumption is plausible.

10.6 Secondary efficacy analyses Secondary efficacy analyses will be defined at a later time point and are not performed or specified at the first interim analysis. Later versions of this analysis plan will detail the secondary efficacy analyses.

10.7 Patient Characteristics

10.7.1 Patient Disposition The disposition of all patients will be listed and summarised by treatment arm. The number and percentage of patients who are randomised, received any study treatment, prematurely discontinued from treatment and lost to follow-up will be summarised.

The number and percentage of patients will be categorized by the reason(s) for

1) Discontinued from treatment: This is when it is decided that a patient will not receive further treatment according to study protocol. Reasons can be: Violation of eligibility criteria, withdrawal of informed consent, adverse event, lack of efficacy or disease worsening, low drug concentrations or anti-drug antibody formation and other.

2) Lost to follow-up: This is when the patient withdraws or is withdrawn from the study. Reasons can be: violation of eligibility criteria, patient withdrawal of informed consent, adverse event, lost to follow-up, death, investigator decision or other.

10.7.2 Protocol Deviations Protocol deviations resulting in exclusion from the Per Protocol set will be determined and summarised by treatment group. See section 8.2 for protocol deviation categories.

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10.7.3 Treatment Compliance Data summarizing the proportions of patients complying with the treatment regimen according to protocol will be presented by treatment arm. Treatment compliance is defined as the number of medication doses actually taken divided by the number of medication doses which should have been taken according to protocol. The treatment compliance is set to 100% in the standard of care arm.

10.7.4 Background and Demographic Characteristics Patient demographics and baseline characteristics will be summarised for the Full Analysis Set.

Patient demographics and baseline characteristics will be summarised by randomised treatment arm using descriptive statistics (N, mean, standard deviation, median, 25/75 percentiles, minimum, and maximum) for continuous variables, and number and percentages of patients for categorical variables.

10.7.5 Concomitant Medications and Other Therapies Concomitant medication information will be coded by the Anatomical Therapeutic Chemical (ATC) classification system. Concomitant medications taken during the study will be summarised by generic name. The number and percentage of patients who took at least one drug within each specific preferred term will be presented. Patients will only be counted once if they are taking more than one medication (within the same code) or take the same generic medication more than once. If it cannot be determined whether a medication is concomitant (based on stop date or, if the stop date is missing, start date), then the medication will be considered to be concomitant. Information on concomitant medication will be extracted from the hospital electronic journal system and merged with the Viedoc and laboratory datasets.

For the first interim analysis, only any of the following concomitant medications will be recorded and reported (any medication known to possibly prolong QT-time): haloperidol, carbamazepine, phenytoin, rifampin, phenobarbital, isoniazid, pyrazinamide, nevirapine, ritonavir, sodium valproate/valproic acid.

10.8 Exploratory Analysis Exploratory analyses will be defined at a later time point and are not performed or specified at the first interim analysis. Later versions of this analysis plan will detail the exploratory analyses.

Exploratory endpoints will not be limited to those mentioned here, and will include variables/endpoints and statistical methods/modelling as necessary to explore the secondary objectives of the study as described in the protocol.

11 Safety Analysis Safety analysis will be based on the safety analysis set. Safety analyses will be descriptive and presented as summary tables by treatment group.

11.1 Adverse events We will calculate the numbers and proportions of participants in the safety analysis set that experienced an AE, SAE, and SUSAR by study arm for the time interval from baseline to 144 hours post-randomisation. We will also present the number and proportions of participants with 1, 2, or ≥3 AEs, SAEs, and SUSARs; the number and proportion of participants with any treatment related AE

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MedDRA will be used to code all AEs recorded in the CRF to system organ class (SOC), higher level group term (HLGT) and preferred term (PT).

All deaths recorded during this trial will be presented in a data listing. All reported SAEs will be presented in a data listing. Any AEs for which treatment was discontinued will be presented in a data listing.

11.2 Other safety assessments No further safety parameters will be assessed or reported.

12 Interim analysis This trial is designed for 4 looks, 3 interim analyses and one final analysis with a cumulative one- sided alpha of 0.025 uniformly spent (Pocock). The one-sided local significance levels are 0.0091 at each stage.

The first 3 interim analysis will include efficacy evaluation of the primary analysis of the primary endpoint as well as safety analysis. Details about the statistical analyses and decision rules for the interim analyses are provided in the DMC charter.

13 Data monitoring Details on data monitoring, Data Monitoring Committee (DMC) and interim analyses are provided in the DMC charter.

The DMC will meet 3 times after 51, 101 and 151 patients have completed the assessment of the primary endpoint (96 hours post randomisation) to review unblended safety and efficacy data. Stopping and decision rules are defined in the DMC charter.

14 Table of Contents of Tables, Listings and Figures The study group will receive a mock report (complete report but with a dummy study group allocation) from the trial statistician for each of the interim and the final analysis. Based on this report, the study group will find consensus on the final Tables, Figures and Listings generated in the analysis.

14.1 List of planned Tables Table 1: Patient disposition

Table 2: Demographic and baseline characteristics by study arm (only age and sex for interim analysis)

Table 3: Study drug exposure

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Table 3: Results of the primary efficacy analysis

Tables 4: Results of the secondary efficacy analyses (not relevant for interim analyses)

Tables 5: Safety tables (AESIs, related AEs, SAEs, AEs leading to withdrawal, AEs of severe intensity, laboratory values)

Table 6: Protocol deviations and treatment adherence (not relevant for the interim analyses)

Table 7: Concomitant medications (only medications known to possibly prolong QT-time as defined under 10.7.5 for the interim analysis)

14.2 List of planned Listings Listing 1: Safety listing on deaths, SAEs and AEs leading to treatment discontinuation

14.3 List of planned Figures Figure 1: Summary of patient enrolment

Figure 2: Estimated treatment effect of the primary analysis, with 95% CIs

Figures 3: Estimated treatment effects of the secondary analyses, with 95% Cis (not relevant for the interim analyses)

15 References

16 Appendices

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