Conditions: Type1 Diabetes Mellitus
Keywords: TrialNet
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 273
Healthy volunteers: no

Brief summary:
The study is a 2-arm, double blinded, multicenter, 2:1 randomized, placebo controlled clinical trial. Subjects will receive hydroxychloroquine or placebo and close monitoring for progression of T1D.

Detailed description:
This study is testing a medication, called hydroxychloroquine (HCQ) to assess safety and effectiveness to prevent individuals at risk of type 1 diabetes (T1D) from progressing to type 1 diabetes.

HCQ is approved by the U.S. Food and Drug Administration as a treatment for malaria, lupus, and rheumatoid arthritis. HCQ has been used extensively for treatment of autoimmune disease in adults, children, and during pregnancy. This medication has not previously been studied as a treatment to prevent T1D.

The goal of this study is to learn if HCQ can help prevent or delay progression from normal glucose tolerance (Stage 1) to abnormal glucose tolerance (Stage 2) or type 1 diabetes (Stage 3).

The study involves 5 visits in the first 6 months, then 1 visit every 6 months for the remainder of the study.

Arms:
arm 1: Hydroxychloroquine compound for oral use arm 2: Placebo tablet matching active drug

Interventions: intervention 1: Hydroxychloroquine intervention 2: Placebo
Intervention descriptions: intervention 1: Hydroxychloroquine for oral administration, dosed by weight intervention 2: Placebo tablet identical to active drug

Protocol document:
Prot_000.pdf: 
TrialNet Protocol: TN22 Hydroxychloroquine Prevention                                                            Protocol Version: 7.0 11Jun20
HYDROXYCHLOROQUINE FOR PREVENTION OF 
ABNORMAL GLUCOSE TOLERANCE AND DIABETES IN 
INDIVIDUALS AT-RISK FOR TYPE 1 DIABETES MELLITUS 
(Protocol TN-22)
VERSION 7.0 
June 11, 2020 
IND # 133,873
EudraCT # 2018-000659-42
Supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), 
and the National Institutes of Health and the JDRF (formerly Juvenile Diabetes Research 
Foundation International) 
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PREFACE
The TrialNet Type 1 Diabetes Protocol TN-22, Hydroxychloroquine for Prevention of Abnormal Glucose 
Tolerance and Diabetes in Individuals At-Risk for Type 1 Diabetes Mellitus, describes the background, 
design, and organization of the study.  The protocol will be maintained by the TrialNet Coordinating 
Center (TNCC) over the course of the study through new releases of the entire protocol, or issuance of 
updates either in the form of revisions of complete chapters or pages thereof, or in the form of 
supplemental protocol memoranda.
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TABLE OF CONTENTS
1.
INTRODUCTION ..........................................................................................................................................6
1.1.
Study Overview .........................................................................................................................6
1.2.
Statement of Purpose .................................................................................................................7
2.
BACKGROUND AND SIGNIFICANCE......................................................................................................8
2.1.
Type 1 Diabetes (T1D) ..............................................................................................................8
2.1.1.
Definition and Metabolic Characteristics of Type 1 Diabetes Mellitus .............................8
2.1.2.
Natural History and Stages of the T1D Disease Process....................................................9
2.2.
Rationale for use of hydroxychloroquine at Stage 1 T1D .......................................................11
2.2.1.
Overview ..........................................................................................................................11
2.2.2 Efficacy.........................................................................................................................................................12
2.2.2.1 FDA and/or EMA Approved Indications ...........................................................................12
2.2.1.2.
Other Clinical Situations...............................................................................................14
2.2.3 Safety and Tolerability of Hydroxychloroquine...........................................................................................14
3.
STUDY DESIGN..........................................................................................................................................15
3.1.
Overview..................................................................................................................................15
3.2.
Objectives ................................................................................................................................15
3.2.1.
Primary Objective.............................................................................................................15
3.2.2.
Secondary Objectives .......................................................................................................15
3.3.
Summary of Inclusion/Exclusion Criteria ...............................................................................15
3.3.1.
Inclusion Criteria ..............................................................................................................15
3.3.2.
Exclusion Criteria.............................................................................................................16
3.4.
Enrollment................................................................................................................................17
3.5.
Description of Study Cohorts...................................................................................................17
3.6.
Description of Treatment Groups ............................................................................................17
3.7.
Treatment Assignment.............................................................................................................18
3.7.1.
Procedures for Unmasking ...............................................................................................18
3.8.
Study Assessments...................................................................................................................18
3.9.
Study Timeline.........................................................................................................................18
3.9.1.
Study Duration..................................................................................................................18
3.9.2.
Follow-up Studies.............................................................................................................19
4.
PATIENT MANAGEMENT........................................................................................................................19
4.1.
Screening Visit and Eligibility Assessment.............................................................................19
4.2.
Hydroxychloroquine Trial for At-risk Participants Screening Visit........................................20
4.3.
Randomization and Baseline visits ..........................................................................................20
4.4.
Close Monitoring .....................................................................................................................20
4.5.
Administration of Hydroxychloroquine...................................................................................21
4.5.1.
Dosing and Dose Withholding .........................................................................................21
4.5.2.
Interruption of Enrollment/Trial Cessation ......................................................................21
5.
STUDY VISIT ASSESSMENTS .................................................................................................................22
5.1.
General Assessments ...............................................................................................................22
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5.2.
Laboratory Assessments ..........................................................................................................22
5.3.
Mechanistic Outcome Assessments.........................................................................................22
5.4.
Metabolic Outcome Assessments ............................................................................................23
5.5.
Visit Windows .........................................................................................................................23
6.
ADVERSE EVENT REPORTING AND SAFETY MONITORING..........................................................23
6.1.
Adverse Event Definition ........................................................................................................23
6.1.1.
Adverse Event...................................................................................................................23
6.1.2.
Adverse Reaction..............................................................................................................24
6.1.3.
Serious Adverse Event/Reaction ......................................................................................24
6.1.4.
Unexpected Adverse Event/Reaction ...............................................................................25
6.1.5.
Grading Event Severity and Causality..............................................................................25
6.2.
Adverse Event Reporting and Monitoring...............................................................................25
7.
PARTICIPANT SAFETY ............................................................................................................................26
7.1.
Risks, Benefits, and Inclusion of Children ..............................................................................26
7.2.
Expected Side Effects and Adverse Events .............................................................................27
7.2.1.
Immunizations ..................................................................................................................28
7.2.2.
Pregnancy .........................................................................................................................29
7.2.3.
Risk of Overdose ..............................................................................................................29
7.2.4.
Prohibited Medications.....................................................................................................29
7.3.
Protecting Against or Minimizing Potential Treatment Risks.................................................30
8.
STATISTICAL CONSIDERATIONS AND ANALYSIS PLAN................................................................30
8.1.
Primary Outcome.....................................................................................................................31
8.2.
Primary Analysis......................................................................................................................32
8.3.
Secondary Outcomes and Analyses.........................................................................................33
8.4.
Study Power and Accrual Target .............................................................................................35
8.5.
Interim Monitoring Plan ..........................................................................................................35
8.6.
Withdrawal Criteria – Individual Participants .........................................................................36
9.
ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD CLINICAL PRACTICE .............36
9.1.
Statement of Compliance.........................................................................................................36
9.2.
Participating Centers................................................................................................................37
9.3.
Informed Consent.....................................................................................................................37
9.4.
Study Participant Confidentiality.............................................................................................38
9.5.
Risks and Benefits....................................................................................................................38
9.6.
Ethics........................................................................................................................................39
10.
STUDY ADMINISTRATION......................................................................................................................39
10.1.
Organizational Structure ......................................................................................................39
10.2.
Groups and Committees.......................................................................................................39
10.2.1.
Hydroxychloroquine Prevention Study Chair Committee............................................39
10.2.2.
TrialNet Chairman’s Office and TNCC........................................................................39
10.2.3.
Clinical Sites .................................................................................................................39
10.2.4.
Eligibility and Events Committee.................................................................................40
10.2.5.
Clinical Site Monitoring ...............................................................................................40
10.3.
Medical Monitor and Data Safety and Monitoring Board (DSMB) ....................................40
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10.4.
Sample and Data Storage .....................................................................................................40
10.5.
Preservation of the Integrity of the Study ............................................................................41
10.6.
Participant Reimbursement and Compensation ...................................................................41
APPENDIX 1: Hydroxychloroquine in At-Risk Individuals:   Study Flow Chart................................................42
APPENDIX 2: Schedule of Assessments..............................................................................................................43
11.
REFERENCES .............................................................................................................................................44
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1. INTRODUCTION
1.1.
Study Overview
Title
Hydroxychloroquine for prevention of abnormal glucose 
tolerance and diabetes in individuals at-risk for type 1 diabetes 
mellitus (T1D)
IND Sponsor
Type 1 Diabetes Trial Network (TrialNet)
Study Supported by
National Institute of Diabetes, Digestive and Kidney Diseases
Conducted By 
Type 1 Diabetes Trial Network (TrialNet)
Protocol Chairs
Dr. Polly Bingley and Dr. Jane Buckner
Accrual Objective
201
Study Design
The study is a 2-arm, double blinded, multicenter, 2:1 
randomized, placebo-controlled clinical trial.  All participants will 
receive close monitoring for progression of T1D.
Treatment Description
Participants will receive hydroxychloroquine or placebo and 
close monitoring for progression to Stage 2 (abnormal glucose 
tolerance) or Stage 3 (clinically overt) T1D. 
Objective
To assess the efficacy, safety and mode of action of 
hydroxychloroquine to prevent progression from Stage 1 to 
Stage 2 or Stage 3 of T1D. 
Primary Outcome
The primary objective is to determine whether intervention with 
hydroxychloroquine will prevent or delay the progression from 
Stage 1 (normal glucose tolerance) to Stage 2 (abnormal 
glucose tolerance) or Stage 3 (clinically overt) T1D. 
Secondary Outcome
Secondary outcomes include: the effect of hydroxychloroquine 
on the risk of T1D; analyses of metabolic and immune changes 
over time; safety and tolerability; evaluation of other possible 
factors on effect (association and effect modification); and 
mechanistic outcomes.
Major Inclusion Criteria
(1) Individuals at Stage 1 T1D (two or more antibodies and 
normal glucose tolerance) (2) ≥ age 3 and previously enrolled 
in TrialNet Pathway to Prevention Study.
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1.2. Statement of Purpose
This protocol describes the background, design, and organization of a study of hydroxychloroquine 
therapy for prevention of progression of Stage 1 (normal glucose tolerance) to Stage 2 (abnormal 
glucose tolerance) or Stage 3 (clinically overt) T1D.  The protocol was written by: the chairs of the 
TrialNet Hydroxychloroquine Prevention Protocol Committee, Dr. Polly Bingley at the University of 
Bristol and Dr. Jane Buckner at the Benaroya Research Institute, the TrialNet Chairman’s Office at the 
Benaroya Research Institute, and the TrialNet Coordinating Center (TNCC).  Significant changes that 
occur to this protocol during the course of the trial require the formal approval of the TrialNet Steering 
Committee.  The study protocol, along with the required informed consent forms, will be approved by a 
Central IRB or each participating institution’s Institutional Review Board (IRB) or Ethics 
Committee/Research Ethics Board (EC/REB).
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2. BACKGROUND AND SIGNIFICANCE
2.1. Type 1 Diabetes (T1D)
2.1.1. Definition and Metabolic Characteristics of Type 1 Diabetes Mellitus
Type 1 diabetes mellitus (T1D) is an immune-mediated disease in which insulin-producing beta cells 
are completely or almost completely destroyed, resulting in life-long dependence on exogenous insulin.  
It is a chronic and potentially disabling disease that represents a major public health and clinical 
concern.  The number of patients being diagnosed with T1D is increasing each year and is approaching 
an epidemic level in many countries1,2.
For individuals living with T1D, continuous exogenous insulin therapy is needed to prevent ketoacidosis 
and other catabolic effects of insulin deficiency, and to promote anabolism and to maintain life.  Insulin 
therapy must be combined with frequent blood glucose monitoring to prevent hypo- and hyperglycemia.  
In addition to the unrelenting financial and psychological burdens on patients and their families from 
day-to-day management of this disease, individuals with T1D are subject to both short-term and long-
term complications.  Short-term complications include severe hyperglycemia resulting in diabetic 
ketoacidosis (DKA), a life-threatening condition.  More than 25% of individuals present with DKA at the 
time of clinical diagnosis; for those under age 5, about 35% have DKA3-5.  Recurrent DKA after the new 
onset period remains a significant clinical problem. A study with over 15 years of follow-up recently 
showed that the presence of DKA at onset of T1D confers worse prognosis, with worse glycemic control 
on follow-up6. Hypoglycemia is a consequence of exogenous insulin therapy which can result in 
impaired cognition and, if severe, coma and death.  Possible long-term complications for those living 
with T1D include visual impairment and blindness, renal failure, vascular disease and limb amputation, 
peripheral neuropathy, and stroke.  
The 
Diabetes 
Control 
and 
Complications Trial (DCCT) showed 
that long-term complications could be 
reduced with near normal control of 
glucose levels, but at the cost of an 
increased 
frequency 
of 
severe 
hypoglycemia7. While there have 
been significant improvements in 
insulin analogs and insulin delivery 
systems, 
such 
as 
continuous 
subcutaneous insulin infusions with 
wearable pumps, continuous glucose 
monitoring, and closed loop systems, 
normal glucose control, particularly in 
children, is rarely achieved8 (Figure 
1).  Moreover, while the frequency of long-term complications is decreasing, individuals with T1D 
continue to have reduced life expectancy9,10.  
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As described below, individuals identified for enrollment into this clinical trial are destined to develop 
clinically overt diabetes.  Clearly, delay or prevention of disease progression would represent a 
significant advance.  
2.1.2. Natural History and Stages of the T1D Disease Process
Much is known about the natural history of the T1D disease process11.  Individuals with T1D have 
genetic susceptibility for disease; indeed, relatives of individuals with T1D are at much greater risk of 
developing disease.  In the general population, approximately 0.3% of individuals will develop T1D. In 
contrast, those with a relative with T1D have a 5% risk of disease – a 15-fold increase12.  Further risk 
stratification among family members depends upon genetic, immune, and metabolic data13.  
Beta cell destruction is initiated in genetically susceptible individuals years before clinical onset of 
disease14. The autoimmune process that causes beta cell destruction is clinically silent and can only 
be identified by the detection of autoantibodies in the blood, such as islet cell antibodies (ICA), and 
autoantibodies to glutamic acid decarboxylase (GAD65Ab), islet antigen 2 (IA-2Ab/ICA512), insulin 
(mIAA), zinc transporter 8 (ZnT8Ab), and others15-18. Continued immune-mediated beta cell destruction 
involving both B and T cells occurs until physiologic insulin demand cannot be met by the remaining 
beta cells.  Impaired insulin secretion is accompanied by progression from normal to abnormal glucose 
tolerance (both of which are asymptomatic states), until more severe hyperglycemia occurs associated 
with classic symptoms of polyuria and polydipsia and thus clinically overt disease19.  
Data gathered from multiple studies during the past 35 years are remarkably concordant with regard to 
the progression from development of autoantibodies to clinically overt disease.  These data, whether 
from studies of longitudinal cohorts followed from birth20 or cross-sectionally identified populations at 
risk,21-24 indicate that essentially all genetically susceptible individuals with multiple autoantibodies 
(whether or not a relative) will eventually progress to clinical disease.  Thus, T1D is considered to have 
started with the presence of 2 or more autoantibodies.  The presence of two or more autoantibodies 
with normal glucose tolerance is defined as Stage 1 T1D.  Stage 2 T1D is the presence of two or more 
antibodies with abnormal glucose tolerance, and Stage 3 T1D is clinically overt disease25.  
As illustrated in Figure 2, data also suggest that the rate of progression through each stage is age 
dependent, with younger individuals progressing more rapidly21.  
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Overall, about 40% of individuals at Stage 1 T1D will progress to Stage 2 within two years (Figure 3), 
and almost 70% of those at Stage 2 will develop clinically overt T1D (Stage 3) within four years (Figure 
4).  More children than adults will be expected to progress from Stage 1 to 2 and from Stage 2 to Stage 
3 during these time periods.  
2.1.3 Disease-Modifying Therapy in T1D
Since the first use of insulin in 1922, treatment of T1D has been targeted at controlling the symptoms 
and consequences of hyperglycemia. This is analogous to symptom control for individuals with 
rheumatoid and juvenile arthritis, where the aim of therapy was previously to control pain and/or provide 
devices to adapt to the presence of disability.  Yet, control of symptoms of arthritis - and almost all other 
autoimmune disease - is no longer the primary aim of treatment; instead, the standard of care is 
disease-modifying therapy with the intent to fundamentally alter the course of the condition.  This 
approach has dramatically changed the lives of those with these autoimmune diseases.  The aim of 
disease-modifying therapy in T1D is the same: to move away from symptom management (e.g., 
glycemic control) as the mainstay of treatment and find approaches to alter the disease course.  
At least four therapies have been shown to have some effect on preservation of insulin secretion at 
Stage 3 (clinical onset) of disease26-29. Two of these (Abatacept or CTLA4-Ig) and Teplizumab (Anti-
CD3) are now under trial at Stage 1 and Stage 2 of T1D, testing the concept that treating earlier in 
disease is likely to have greater benefits.  Early treatment has been shown to be useful in other 
autoimmune diseases, such as juvenile arthritis.  Moreover, successful application of disease-
modifying therapy at Stage 1 or Stage 2 of T1D would have clear clinical benefit by the delay or 
prevention of clinically overt disease (Stage 3).  
In summary, the rationale for this study is that individuals at Stage 1 T1D, defined as those with 
immunologic markers and normal glucose tolerance, will inevitably develop Stage 3 or clinically overt 
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T1D.  Prior to development of clinical T1D, they will progress from Stage 1 T1D (normal glucose 
tolerance) to Stage 2 T1D (abnormal glucose tolerance). Intervention early in the course of disease is 
likely to be more effective than intervention in those with abnormal glucose tolerance or clinical T1D.  
As described below, this is particularly likely to be true with the therapy being tested in this trial – 
hydroxychloroquine.  
2.2.
Rationale for use of hydroxychloroquine at Stage 1 T1D
2.2.1. Overview
Since T1D results from immune-mediated beta cell destruction, disease-modifying therapy will require 
approaches that alter the autoimmune process.  Both B and T cells are involved in disease 
pathogenesis, as evident by the efficacy of anti-T cell (Teplizumab), anti-B cell (Rituximab), and co-
stimulation blockade (Abatacept) therapies in preserving beta cell function at Stage 3 T1D (clinical 
onset).   Other studies have demonstrated alterations in T and B cell phenotype and function when 
measured in peripheral blood from those with T1D as compared with unaffected controls. 
While the mechanism of action of hydroxychloroquine is poorly understood, it appears to act on 
autoimmune responses which are important in early pathogenesis of T1D.  It has a long history of use 
in children, is well tolerated, is relatively low cost, and is thought to target B cell responses. In particular, 
hydroxychloroquine has been shown to decrease cellular activation, including hindering of antigen 
presentation to CD4+ T cells and reducing the expression of the CD4+ T cell activation marker 
CD15430,31. Hydroxychloroquine is also thought to have a number of other immune actions that may 
lead to decreased autoantibody production, including (i) alkalinization of intracellular vacuoles leading 
to inhibition of proteolysis, chemotaxis, phagocytosis, and antigen processing32, (ii) reduction of 
macrophage-mediated cytokine production33 and (iii) inhibition of function of toll-like receptors34. 
Hydroxychloroquine has also been found to inhibit calcium-mediated signaling in T and B cell lines, 
interfering with downstream effects including TCR-dependent CD69 expression35. 
In a preclinical study, oral hydroxychloroquine was able to partially protect against streptozotocin-
induced diabetes in a rat model,36 with greater preservation of islet structure, β-cell mass and 
proliferation over the untreated group. Hydroxychloroquine lowered the pancreatic levels of IL-1β, IL-
6, TNF-α, and TGF-β1, as well as the serum level of MCP-1, compared to the untreated group, 
suggesting an anti-inflammatory effect. Lower pancreatic levels of the apoptotic marker caspase-3 were 
also found in hydroxychloroquine treated animals. 
Importantly, the drug not only has been shown to ameliorate other autoimmune diseases, but 
hydroxychloroquine also abrogates the progression of autoimmunity in at-risk populations37-41.  This 
drug is therefore particularly suitable to test early in the T1D process, in this study, targeting those with 
multiple islet autoantibodies but normal glucose tolerance (Stage 1 of T1D).  It also has the potential to 
be efficacious at an even earlier stage of the disease process, prior to the development of multiple 
autoantibodies. 
Initially developed as a therapy for malaria, hydroxychloroquine is approved by the Food and Drug 
Administration (FDA) and the European Medicines Agency (EMA) for that indication, as well as for 
rheumatoid arthritis (RA) and discoid and systemic lupus erythematous (SLE).  It is widely used in 
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clinical practice for these indications, including in pediatric populations. Efficacy in autoimmune disease 
has been well established in RA42-44, SLE45-47, antiphospholipid syndrome48 and Sjögren’s 
syndrome49,50.  Particularly relevant for this study is that hydroxychloroquine is effective when used 
early in the course of autoimmune disease, having been shown to slow or halt progression in 
palindromic rheumatism37-40 and delay onset of SLE in antibody-positive individuals41.
Other beneficial actions of hydroxychloroquine have been identified in epidemiologic and randomized 
studies of other autoimmune diseases, including improvement in cardiovascular risk factors, such as 
reduction of LDL cholesterol51, thrombosis52, glycaemia52,53, insulin resistance52,54,55, and reduction of 
risk of incident diabetes mellitus in SLE54 and RA55,56. A randomized trial of hydroxychloroquine in obese 
adults demonstrated improved clamp-assessed insulin sensitivity, as well as increased first phase 
insulin secretion, possibly mediated through anti-inflammatory effects57. Because 1/3rd of our pediatric 
population is currently overweight, and elevated BMI may increase risk of progression to T1D in 
children58 and adults59, these actions may provide an additional benefit to this therapy. 
In summary, the rationale for testing hydroxychloroquine as disease-modifying therapy in Stage 1 T1D 
is that it is a well-established, cost-effective treatment for various autoimmune diseases used safely in 
both adult and pediatric populations, which may work through multiple mechanisms as an agent to 
prevent progression of autoimmunity. In addition, hydroxychloroquine has a positive metabolic profile 
that has the potential to be of particular benefit in the context of the T1D pathogenesis.
2.2.2 Efficacy
2.2.2.1 FDA and/or EMA Approved Indications 
Hydroxychloroquine is approved by FDA and EMA for use in malaria, rheumatoid arthritis (including 
juvenile idiopathic arthritis), and systemic and chronic discoid lupus erythematosus in adults and 
children. It is also approved for use in dermatological conditions caused or aggravated by sunlight in 
adults, with unlicensed use for this indication in children.
Table 1 provides a summary of the types of studies, patient populations, and number of patients treated 
with hydroxychloroquine (or matching placebo) in completed clinical studies in these conditions.
Table 1 
Indications
Exposure N 
Age groups
Notes
Malaria
Widespread use 
Adults & children
No clinical trials published.  Usual daily dose:  5 - 6.5 mg/kg 
Rheumatoid arthritis (including 
juvenile idiopathic arthritis)
Widespread use 
Adults & children
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HERA study 
Esdaile J et al, American 
Journal of Medicine 199544
N=120
Adults
Dose: 7 mg/kg (max 400 mg) vs 
placebo Duration: 36 weeks
Outcome: Significant benefit on synovitis, pain, and 
physical disability of recent-onset RA
3 year follow up 
Tsakonas E et al., J Rheumatol 
200060
N=104
Early treatment with 
hydroxychloroquine was 
associated with less pain 
and physical disability after 3 
years compared with 
placebo.
Clark P et al. Ann Intern Med 
199343
N=126
Adults
Dose: 400 mg/day vs 
placebo Duration 24 weeks
Outcome: Moderate clinical effect in early 
Rheumatoid Arthritis
Rantalaiho V et al. Arthritis Res 
Therr 2010 61
N=199
Adults
Use of hydroxychloroquine in combination 
therapy vs single DMARD +/- prednisolone.
Duration 2 years
Outcome: Combination 
therapy results in less long- 
term radiological damage
Das SK et al. Curr Med Res Opin 
200762
N= 122
Adults
Dose: 400 mg/day vs placebo (+Nimesulide) 
Duration 8 weeks
Outcome: Improved 
American College of 
Radiology (ACR) criteria. Well 
tolerated.
Lupus Erythematosus (chronic 
discoid and systemic)
Approved since 1955 
Used in adults & children
ACR and EULAR guidelines for lupus nephritis accept 
hydroxychloroquine as adjunctive therapy.
Costedoat-Chalumeau N et al. Ann 
Rheum Dis 201363
N=573
Adults
Low hydroxychloroquine levels associated 
with higher SLE activity, but this was not 
responsive to changes in dose.
Clowse ME et al. Arthritis Rheum 
200664
N=257
Pregnant 
women
Higher risk of flare if 
hydroxychloroquine stopped. 
Steroid sparing
Absence of fetal toxicity
N diff
i
t
Levy RA et al. Lupus 200165
N=20
Pregnant 
women
Lower SLE activity scores in treatment 
group. Steroid sparing.
Dermatological conditions 
caused or aggravated by 
sunlight
Adults
Singal AK et al.Clin 
Gastroenterol Hepatol 201266
N=48 Adults 
with porphyria 
cutanea tarda
Dose: 100 mg twice a week versus phlebotomy for 
12 months.
Hydroxychloroquine as effective and safe as 
placebo.
Petersen CS et al. J Am Acad 
Dermatol 199267
N=93 Adults 
with porphyria 
cutanea tarda
Dose: 250 mg TDS for 3 days. Efficacious 
and safe.
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2.2.1.2.  Other Clinical Situations
Hyperglycemia is a result of the intersection of insulin secretion (impacted by immune-mediated beta 
cell destruction) and insulin sensitivity.  While preserving insulin secretion through immune modulation 
is the primary rationale for this study, improvement in insulin sensitivity would also impact progression 
to Stage 2 and 3 of disease. It is assumed that the association of increasing BMI and accelerated 
progression to T1D in both pediatric and adult relatives at risk is due to obesity-induced insulin 
resistance58,59.  Thus, it is relevant to highlight the salutary effects of hydroxychloroquine on this aspect 
of disease pathophysiology.  Clinical trials of metabolic and other effects have been completed in non-
diabetic adult participants with obesity and other risk factors for insulin resistance and these 
demonstrated improved insulin sensitivity and beta cell function57,68.  In a further trial in 135 adult 
participants with type 2 diabetes, treatment with up to 600 mg of daily hydroxychloroquine for 18 months 
was associated with improved glycemic control53.   Also of interest, since those with T1D are at risk for 
cardiovascular complications, is a Phase I trial conducted by Achuthan et al. (2015) comparing the 
efficacy of hydroxychloroquine (400 mg daily) with aspirin or combined aspirin/clopidogrel as 
antiplatelet therapy in 20 adult participants which showed that hydroxychloroquine treatment was 
associated with a reduction in platelet aggregation by arachidonic acid69.
2.2.3 Safety and Tolerability of Hydroxychloroquine
Hydroxychloroquine has been in clinical use for >60 years. It is generic and its pharmacological 
characteristics and safety profile have been established in pediatrics, adults, and pregnancy. It is 
considered a far safer drug than its close relative chloroquine. It is well tolerated, with the most common 
side effects being transient nausea experienced by 1-10% of patients. Few individuals stop therapy 
due to side effects, and ‘once daily’ dosing facilitates adherence. The recommended pediatric dose is 
5–6.5 mg/kg once daily (max. dose 400 mg). 
The most important adverse effect of hydroxychloroquine is retinal toxicity (see Section 7.2).  This is 
correlated with dose of drug and duration of therapy. There is negligible risk for duration of therapy <6 
months,70 and it is unlikely to occur with less than 5 years of therapy if dose is <5 mg/kg/day up to 400 
mg/day71.  Despite longstanding use of hydroxychloroquine for malaria therapy in very young children, 
there is a dearth of information about retinopathy, suggesting its rarity72.  
In this study, hydroxychloroquine dose will be targeted at a maximum of 5.0 mg/kg/day actual body 
weight up to 400 mg/day and individuals will undergo annual ophthalmological exams.  
Section 7.2 describes other reported adverse effects. 
3. STUDY DESIGN
3.1.
Overview
This is a double-masked, multicenter, randomized, placebo-controlled trial to determine whether 
treatment of participants with Stage 1 T1D (two or more antibodies with normal glucose tolerance) with 
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hydroxychloroquine results in delay or prevention of progression to Stage 2 T1D (abnormal glucose 
tolerance) or Stage 3 T1D (clinical onset). 
3.2.
Objectives
3.2.1. Primary Objective
The primary objective of the study is to determine whether treatment of participants at Stage 1 T1D 
with hydroxychloroquine results in delay or prevention of progression to Stage 2 T1D or Stage 3 T1D. 
3.2.2. Secondary Objectives

To determine whether treatment with hydroxychloroquine is superior to placebo with respect 
to the risk of T1D

To determine whether treatment with hydroxychloroquine is superior to placebo with respect 
to other measures as obtained from timed collections during longitudinal tests:
a.
metabolic assessment of insulin secretion and insulin resistance calculated from 
the oral glucose tolerance test (OGTT) data 
b.
immunologic measures of disease activity or progression 

To compare the safety and tolerability of hydroxychloroquine to placebo

To assess the effects of treatment on mechanistic outcomes
3.3.
Summary of Inclusion/Exclusion Criteria
Participants must meet all entry criteria for the protocol as outlined below.
3.3.1. Inclusion Criteria 
Study participants must be or have:
1.
A participant in TrialNet Pathway to Prevention Study (TN01). 
2.
Age ≥ 3 years at the time of randomization in this trial.           
3.
Willing to provide informed consent or, if the participant is <18 years of age, have a parent or 
legal guardian provide informed consent.
4. Normal glucose tolerance by OGTT within 7 weeks (no more than 52 days) prior to randomization.  If 
previous abnormal glucose tolerance, has had two consecutive OGTTs with normal glucose 
tolerance:  
a. Fasting plasma glucose < 110 mg/dL (6.1 mmol/L), and
b.
2-hour plasma glucose <140 mg/dL (7.8 mmol/L), and
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c.
30, 60, and 90 minute value on OGTT<200 mg/dL (11.1 mmol/L).
5.
Two or more diabetes-related autoantibodies present on two separate samples, one of which 
was drawn within the past six months (210 days). Confirmation does not have to involve the 
same 2 autoantibodies.
6.
Weight ≥12 kg at the time of screening. 
7.
If a female participant with reproductive potential, willing to avoid pregnancy and undergo 
pregnancy testing prior to randomization and at each study visit.
8.
Anticipated ability to swallow study medication.
3.3.2. Exclusion Criteria
Potential participants must not meet any of the following exclusion criteria:
1.
 Abnormal Glucose Tolerance or Diabetes 
a. Fasting plasma glucose ≥110 mg/dL (6.1 mmol/L), or
b. 2 hour plasma glucose ≥140 mg/dL (7.8 mmol/L), or
c. 30, 60, or 90 minute plasma glucose during OGTT ≥200 mg/dL (11.1 mmol/L)
2.
History of treatment with insulin or other diabetes therapies. 
3.
Ongoing use of medications known to influence glucose tolerance, e.g. glucocorticoids, growth 
hormone, anticonvulsants, thiazide or potassium-depleting diuretics, beta adrenergic blockers, 
niacin and antipsychotics. Participants on such medications should be changed to a suitable 
alternative, if available, by their primary care provider, and will become eligible one month after 
medication is discontinued. 
4.
Ongoing or anticipated future use of any medications known to impact T1D progression or have 
untoward interactions with hydroxychloroquine, as described in section 7.2.4.
5.
Known hypersensitivity to 4-aminoquinoline compounds.
6.
G6PD deficiency.
7.
History of retinopathy.
8.
Active infection at time of randomization.
9.
Serologic evidence of current or past HIV, Hepatitis B (positive for Hepatitis B core antibody or 
surface antigen), or Hepatitis C infection. 
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10. Deemed unlikely or unable to comply with the protocol or have any complicating medical issues,  
including prolonged QT interval, a disease previously or likely in the future to require 
immunosuppression, or abnormal clinical laboratory results that interfere with study conduct or 
cause increased risk. 
11.  Be pregnant or breastfeeding.
3.4.
Enrollment
Potential study participants will be identified through the ongoing TrialNet Pathway to Prevention Study. 
Autoantibody-positive individuals in that study are then further evaluated with the performance of 
OGTTs, additional blood tests, detailed medical history, and physical exam. The results of these 
evaluations will be used to determine eligibility for this protocol.
3.5.
Description of Study Cohorts 
While enrollment for the TrialNet Abatacept Prevention Trial (TN18) is open, enrollment in this study 
for individuals age 6 or older will be limited to those with mIAA confirmed within 6 months of enrollment 
as one of their two or more autoantibodies.   However, those who are age 3-5 years or who reside in 
countries not participating in TN18 and have normal glucose tolerance and two or more autoantibodies 
(regardless of mIAA status) are eligible.  Otherwise, after completion of enrollment for TN18, all 
individuals ≥ 3 years of age with normal glucose tolerance and two or more autoantibodies regardless 
of mIAA status are eligible to screen for this study.  
3.6.
Description of Treatment Groups
The intervention will only be administered to those who provide informed consent to participate.  
Participants will be randomized to receive either oral hydroxychloroquine or placebo along with close 
monitoring for abnormal glucose tolerance or diabetes. Blood and serum samples for the primary and 
secondary outcome determinations will be sent to the Core Laboratories for analysis. Clinical laboratory 
studies may be done at the local sites.
Participants will be randomly assigned in a 2:1 ratio to treatment arms within strata defined by two 
stratification factors: prior treatment for T1D prevention (prior enrollment in a prevention study and 
treatment with placebo vs. prior enrollment in a prevention study and treatment with active therapy vs. 
no prior enrollment in a prevention study) and age group (<8 years old vs. 8 years old or older).
3.7.Treatment Assignment
After participants (or parents/legal guardians as applicable) sign the consent form, complete the 
screening visit(s), and meet all of the inclusion criteria and none of the exclusion criteria, they will be 
randomized to receive either hydroxychloroquine or placebo; each treatment arm will include close 
monitoring for study endpoints.
3.7.1. Procedures for Unmasking
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Emergency unmasking occurs as follows: Investigators can break the treatment code by contacting the 
TrialNet Central Pharmacy via the 24-hour emergency number. No approval or intervention by the study 
sponsor or TrialNet administration is required in emergency situations. The TrialNet Chair, NIDDK 
TrialNet program officer and TrialNet Medical Monitor will be notified of the unmasking within 24 hours 
of its occurrence. Non-emergent unmasking will occur upon notification of the TNCC and approval by 
TrialNet Chair or NIDDK TrialNet program officer. If unmasking is approved, the study sponsor and 
appropriate TrialNet committees (e.g. Safety Monitoring) will be notified of the event as soon as 
reasonably possible; however, they will not be unmasked. 
3.8.Study Assessments
During the course of the study, participants will undergo frequent assessments of their glucose 
tolerance status, insulin production, immunologic status, and overall health and well-being (see 
Schedule of Assessments).  
Samples will be drawn for storage in the National Institute for Diabetes and Digestive and Kidney 
Disease (NIDDK) Repository and at TrialNet Laboratory Sites for future analysis related to T1D. 
3.9.Study Timeline
3.9.1. Study Duration
The study has been designed to provide 83% power to detect a 50% risk reduction in the progression 
from Stage 1 to Stage 2 or 3 T1D using a two-sided test at the 0.05 level after six years of study duration 
(approximately 4 years of accrual and a minimum of 2 years of follow-up on all subjects).  A total of 
approximately 201 participants will be allocated in a 2:1 ratio to the two groups.    
The assumptions underlying the sample size  are: 1) 40% two-year event rate in the placebo group for 
the progression from Stage 1 to Stage 2 or 3 T1D, 2) detectable hazard ratio of 0.50 (i.e., reducing the 
hazard rate by 50%, which here translates to 40% vs. 22.5% in the two-year event rates) in the 
hydroxychloroquine group, 3) a 10% or less dropout rate for a minimum of 2 years of follow-up on all 
participants in both groups, and 4) assumption that the distribution of time to T1D stage progression is 
approximately exponentially distributed. Under the above assumptions, the total target number of 
events that would trigger the final analysis is 80; i.e., the final analysis will be conducted once 80 events 
(abnormal glucose tolerance or diabetes) have been observed. If the accrual rate is 50 patients per 
year, with the above assumptions it is estimated that the enrollment period will last approximately 4 
years, with a total study duration of approximately 6 years to provide a sufficient number of events to 
detect the powered detectable difference.
3.9.2. Follow-up Studies
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Participants who have progressed to Stage 2 T1D (confirmed abnormal glucose tolerance) will have 
reached the primary study endpoint. These participants who have developed abnormal glucose 
tolerance but have not progressed to Stage 3 T1D (clinical onset) will have the option to remain in this 
study without further therapy, undergoing close monitoring and safety assessments until the 
development of Stage 3 T1D (clinically overt diabetes) or until they enroll in another clinical trial, 
whichever comes first.   
Once participants reach Stage 3 (diagnosis of clinical diabetes), they will no longer be followed in the 
context of this protocol; however, they will be offered follow-up in the Long Term Investigative Follow-
Up in TrialNet (LIFT) Study (TN16). Alternatively, individuals who progress to Stage 3 may be eligible 
for interventional studies sponsored by TrialNet or other organizations under separate INDs. In the 
event that a participant wishes to participate in another investigational study whose entry criteria 
exclude participants who have previously received immunomodulating therapy and is otherwise eligible 
for the new study, the participant may request and be told of their treatment group assignment for the 
hydroxychloroquine prevention study.  Every attempt will be made to minimize the potential bias that 
this may introduce.  The TNCC will make treatment assignment information available to the site 
investigator of the new study after the participant is determined to be willing to participate and not 
otherwise excluded from the new study. Other study group members will not be informed of the 
treatment assignment information.  Mitigation of bias issues must be balanced against safety and 
interests of participants. 
4. PATIENT MANAGEMENT
4.1.
Screening Visit and Eligibility Assessment
This study will draw participants from the TrialNet Pathway to Prevention Study.  
The initial testing for islet autoantibodies, HLA type, and OGTT will generally be done as part of 
Pathway to Prevention Study.  Potential participants in the hydroxychloroquine prevention trial are 
those with two or more autoantibodies present on two separate samples. Initially, for those age 6 or 
older, enrollment will require the presence of mIAA antibodies unless the individual resides in a country 
not participating in the Abatacept Prevention Trial (TN18).  After completion of enrollment for the 
Abatacept Prevention Trial (TN18), any two confirmed autoantibodies will meet criteria for this study.  
The autoantibodies that may be confirmed are mIAA, GAD65Ab, IA-2/ICA512Ab, ZnT8Ab, and/or ICA. 
The confirmation of two positive autoantibodies must occur within the six months prior to study drug 
administration, but the confirmation does not have to involve the same two autoantibodies. 
Those individuals with two diabetes-related autoantibodies will then be eligible for additional screening 
tests and possible enrollment into the hydroxychloroquine prevention trial. A participant must have a 
normal OGTT (done either as part of the Pathway to Prevention Study or during screening for the 
hydroxychloroquine prevention trial) within 7 weeks (52 days) prior to randomization.  If a participant 
has a previous abnormal glucose tolerance, he/she must have two consecutive OGTTs with normal 
glucose tolerance in order to be eligible. 
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Appendix 1 summarizes the flow of participants from the Pathway to Prevention Study into the 
hydroxychloroquine prevention trial.
4.2. Hydroxychloroquine Trial for At-risk Participants Screening Visit
Prior to the initial visit, the hydroxychloroquine prevention trial will be described to the potential 
participant.  The participant/parent/guardian will be asked to sign an informed consent document, and 
minors will be asked to sign an age appropriate assent document, describing the purpose, risks, and 
benefits of screening for the trial.  A participant’s signature indicates that he/she understands the 
potential risks and benefits of study participation.  During these visits, clinical tests will be done to 
determine eligibility. 
Any antibody positive relative either not eligible or not willing to be randomized into the 
hydroxychloroquine prevention trial is eligible for continued follow-up as part of the TrialNet Pathway to 
Prevention Study.  
4.3.
Randomization and Baseline visits
Participants will be randomized to either the treatment arm or the placebo arm.  Randomization must 
occur within 7 weeks of a normal OGTT in order to ensure that participants have normal glucose 
tolerance, and study drug administration must occur within 4 weeks after randomization.   
 
4.4.
Close Monitoring
During the study period, all participants will receive close monitoring for development of abnormal 
glucose tolerance and diabetes.  OGTT tests will be performed at approximately six-month intervals 
thereafter.  At each visit, participants will be asked questions about the presence or absence of 
symptoms associated with diabetes.  If participants respond affirmatively or have other laboratory or 
clinical evidence of disease progression, further evaluation will be performed.  Individuals in both of the 
study arms will have laboratory and mechanistic studies performed as detailed in the Schedule of 
Assessments.
4.5.
Administration of Hydroxychloroquine
The hydroxychloroquine used in these human trials is a 4-aminoquinoline compound 
(hydroxychloroquine sulfate) supplied as a 200mg tablet for oral administration. As described below, 
these tablets will be prepared in 100mg increments and over encapsulated for this trial.
Hydroxychloroquine must be stored below 25°C/77°F.
4.5.1. Dosing and Dose Withholding
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The target dose is 5 mg/kg/day (actual body weight), up to a maximum of 400mg/day, of 
hydroxychloroquine or placebo.  The dose was chosen based on demonstrated safety and efficacy in 
other human autoimmune diseases.  Dose by body weight will be calculated in 100mg increments and 
will be equivalent to ≤ 5 mg/kg/day averaged over a one-week period. 
Dose Withholding
1.
Study drug will be withheld in those with 3 or more days of fever >101°F (38.3°C) until 
resolved.  
2.
If needed, in individuals who have gastrointestinal or other symptoms limiting their tolerance 
of study medication, the dose may be temporarily reduced at the discretion of the 
investigator in consultation with the medical monitor.
3.
Any abnormal ophthalmologic examination finding possibly related to study medication, 
confirmed by repeat measure and central reading within 4 months, will result in stopping the 
study medication (permanently).
4.
Grade 2 anemia or thrombocytopenia, i.e. hemoglobin <10 gm/dl and platelets 
<75,000/mm3.    In consultation with the medical monitor, study medication may be restarted 
after resolution of the Grade 2 anemia or thrombocytopenia.The complete blood count 
(CBC) will be repeated no more than one month after restarting study medication. If the 
abnormality recurs, then medication will be discontinued permanently.
5.
Study medication should be withheld if a participant develops an adverse event of severe 
intensity (grade 3 or higher) or a serious adverse event at least possibly related to study 
medication
Individuals who are unable to continue on study medication due to adverse effects will remain on study 
and continue with study visits. 
4.5.2.   Interruption of Enrollment/Trial Cessation
Section 4.5.1 describes the circumstances for potential withholding or reduction of drug treatment in 
individual patients. This section lists events that will necessitate interruption of enrollment in the trial as 
a whole, pending DSMB review. As part of their ongoing safety review, the DSMB will make 
independent judgments regarding other adverse events requiring trial interruption.
1.
Any death that occurs in the study which is assessed as possibly, probably, or definitely 
related to study treatment regimen by the study site investigator and the medical monitor.
2.
Two or more of the first 10 treated participants or ≥20% of all treated participants experience 
a clinically significant, drug-related adverse event, resulting in the permanent 
discontinuation of study treatment.
3.
If more than one of the first 10 treated participants or ≥10% of all treated participants develop 
clinically significant retinal changes, the study will be placed on hold pending review by 
DSMB.
5. STUDY VISIT ASSESSMENTS
The schedule of evaluations and laboratory studies is presented in Appendix 2, Schedule of 
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Assessments.  A summary of assessments for the Protocol is given below.
5.1.
General Assessments
General assessments for this Protocol will include:

Informed consent

Inclusion/exclusion criteria

Medical history 

Complete or directed physical examination including height/weight, waist circumference and 
Tanner staging

Concomitant medications

Adverse events

Treatment compliance

Ophthalmology examination
5.2.
Laboratory Assessments
The following clinical laboratory assessments will be performed during the study as described in the 
Schedule of Assessments:

Chemistry (sodium, potassium, chloride, CO2, glucose, BUN, creatinine)

Liver function tests (ALT, AST, LDH, alkaline phosphatase, total protein, albumin, total and 
direct bilirubin)

Hematology (CBC with differential and platelets)

Urine pregnancy test as appropriate

Antibodies to HIV, hepatitis B (anti-HBcAb, HBsAg), hepatitis C (HCV)

G6PD screening
5.3.
Mechanistic Outcome Assessments
TrialNet will perform pharmacokinetic, immune, and genetic assays to further understand mechanisms 
that may be underlying the T1D disease process and response to therapy.  For this purpose, samples 
for PBMC, DNA, RNA, plasma, and serum may be obtained.  Islet autoantibodies will be measured at 
approximately six-month intervals in the study. HLA or other testing for diabetes-related genes may be 
done under the auspices of either the TrialNet Pathway to Prevention Study or this protocol.
Responses to influenza or other clinically indicated immunizations may be determined through analysis 
of pre- and post-dose samples. Samples may also be collected for thyroid autoantibodies, virology and 
other immunization titers. Details of the mechanistic outcome assessments can be found in the 
mechanistic analysis plan. 
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5.4.
Metabolic Outcome Assessments
Metabolic assessments will consist of:
1.
OGTT                                                                                                                                                              

The OGTT will be performed approximately every 6 months, or more frequently if clinically 
indicated based on a random glucose level of ≥ 200 mg/dL (11.1 mmol/L).

The C-peptide and insulin data from the OGTT will be used to determine insulin secretion 
with assessment of early and late insulin responses.

The insulin, glucose and C-peptide data from the OGTT may be used to determine insulin 
sensitivity.       

Other hormones and metabolites such as glucagon and proinsulin may also be measured.   
2.
HbA1c  

Measure of glycemic control.
5.5.
Visit Windows
Randomization must occur within 7 weeks (no more than 52 days) of the OGTT, and initial treatment 
administration must begin no more than 4 weeks after randomization.  Visit target dates will be 
determined as of the baseline visit and will not be changed.  Subsequent study visits should be +/- 2 
weeks of the target date.  
6.  ADVERSE EVENT REPORTING AND SAFETY MONITORING
6.1.Adverse Event Definition
6.1.1. Adverse Event
In this clinical trial, an adverse event (AE) is any occurrence or worsening of an undesirable or 
unintended sign, symptom or disease, whether or not related to the treatment and study procedures.  
Throughout the study, the investigator must maintain source documentation of all adverse events.  
Events not related to diabetes onset which are Grade 2 or greater per the NCI CTCAE (see Section 
6.1.5. Grading Event Severity below) must be reported to TNCC.  The investigator should treat 
participants with adverse events appropriately and observe them at suitable intervals until the events 
resolve or stabilize.
Adverse events may be discovered through:

observation of the participant;

questioning the participant;

unsolicited complaint by the participant.
Questioning of the participant should be conducted in an objective manner.
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6.1.2. Adverse Reaction
An adverse reaction means any adverse event caused by a drug. Since diabetes onset is a study 
endpoint, this is not considered an adverse reaction unless associated with death. Adverse reactions 
are a subset of all suspected adverse events for which there is reason to conclude that the drug caused 
the event.  Suspected adverse reaction means any adverse event for which there is a reasonable 
possibility that the drug caused the adverse event.  For the purposes of safety reporting, “reasonable 
possibility” means there is evidence to suggest a causal relationship between the drug and the adverse 
event. A suspected adverse reaction implies a lesser degree of certainty about causality than an 
adverse reaction, which means any adverse event definitely caused by a drug.  Examples of evidence 
that suggest a causal relationship (reasonable possibility) between the drug and the adverse event 
include:

A single occurrence of an event that is uncommon and known to be strongly associated with 
drug exposure.

One or more occurrences of an event that is not commonly associated with drug exposure, but 
is otherwise uncommon in the populations exposed to the drug.

An aggregate analysis of specific events observed in a clinical trial (such as known 
consequences of the underlying disease or condition under investigation or other events that 
commonly occur in the study population independent of drug therapy) that indicates those 
events occur more frequently in the drug treatment group than in a concurrent or historical 
control group.
6.1.3. Serious Adverse Event/Reaction
A serious adverse event (SAE) or reaction is defined as any untoward medical occurrence at any dose 
that suggests a significant hazard, contraindication, side effect, or precaution. This includes but is not 
limited to any of the following events with the exception of an adverse event associated with, the initial 
diagnosis of type 1 diabetes including hospitalization related to that diagnosis. In the event that a DKA 
related hospital admission occurs, all relevant details of the admission should be reported to the TNCC. 
The diagnosis of diabetes will be reported as an SAE only in the event of death. 
1.
Death.  A death that occurs during the study or that comes to the attention of the 
investigator during the protocol-defined follow-up after the completion of therapy must be 
reported whether it is considered to be treatment-related or not.
2.
A life-threatening adverse event.  A life-threatening event is any adverse event that, in the 
view of the investigator, places the participant at immediate risk of death from the reaction 
as it occurred.
3.
Inpatient hospitalization or prolongation of existing hospitalization, with the exception of 
hospitalization relating to initial diagnosis of type 1 diabetes.
4.
Persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions.
5.
Congenital anomaly or birth defect.
6.
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical 
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judgment, they may jeopardize the participant and may require medical or surgical 
intervention to prevent one of the outcomes listed above. 
An adverse event or suspected adverse reaction is considered “life-threatening” if, in the view of either 
the investigator or sponsor, its occurrence places the participant at immediate risk of death. It does not 
include an adverse event or suspected adverse reaction that, had it occurred in a more severe form, 
might have caused death.
Regardless of the relationship of the adverse event to study drug, the event must be reported as a 
serious adverse event if it meets any of the above definitions.
6.1.4. Unexpected Adverse Event/Reaction
An adverse event/reaction is considered unexpected when the nature (specificity) or severity of the 
event is not consistent with the risks described in the package insert, this protocol, or the informed 
consent document. Unexpected refers to an experience that has not been previously observed.  This 
includes events that occur more frequently than expected.
6.1.5. Grading Event Severity and Causality
TrialNet has adopted usage of the National Cancer Institute (NCI) Common Terminology Criteria for 
Adverse Events (CTCAE) and/or study-specific criteria for classification to describe the severity of 
adverse events with the exception of hypoglycemia, hyperglycemia or diabetes onset (which will be 
reported as an AE only in the case of death). TrialNet Investigators will also provide an assessment of 
relationship of AE to study drug as: not, unlikely, possibly, probably, or definitely related. 
6.2.
Adverse Event Reporting and Monitoring
Adverse events will be reported to the TrialNet Coordinating Center.  The investigator will grade their 
severity according to common toxicity criteria or study-specific criteria and will make a determination of 
their relation to therapy with the exception of the initial diagnosis of diabetes which is considered an 
adverse event only in the case of death.  Events will be assessed and reported consistent with the ICH 
Guideline for Good Clinical Practice, 21 CFR 312.32 for expedited safety reporting, and per the 
guidance of the DHHS Office for Human Research Protections (OHRP).  
The adverse event case report form for the protocol must be completed for all AEs ≥ Grade 2.  In 
addition to the reporting requirements for the TrialNet network (as described above), the FDA requires 
reporting of any adverse event, if the event is serious, related, AND unexpected (21CFR312.32 
(c)(i)(A)-(B)). When required, the FDA form 3500A (MedWatch form) should also be completed and 
sent to the TNCC within 24 hours of when the site was notified of the event.  This will be reviewed by 
the TrialNet Medical Monitor, the TrialNet Safety Monitoring Committee, and the DSMB, as appropriate.  
Deaths must be reported immediately.  Event outcome and other follow-up information regarding the 
treatment and resolution of the event will be obtained and reported when available, if not known at the 
time the event is initially reported.  The follow-up information should contain sufficient detail to allow for 
a complete medical assessment of the case and an independent determination of possible causality. 
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Adverse events will be assessed by the TrialNet Medical Monitor.  The DSMB will conduct regular 
safety reviews approximately every three to six months (and otherwise as needed) of adverse events 
by treatment group assignment.  Serious adverse events, as well as adverse events leading to study 
discontinuation, will be reviewed by the DSMB. 
For SAEs that are unexpected and considered possibly or probably drug-related, the Medical Monitor 
will provide information on frequency of similar events, and will review FDA form 3500A reports for 
distribution to FDA (and equivalent regulatory bodies for non-US sites), NIDDK, DSMB, and site 
investigators.  Expedited safety reports will be submitted to the IND by the TNCC on behalf of NIDDK.
7. PARTICIPANT SAFETY
7.1. Risks, Benefits, and Inclusion of Children
Component analysis: Monitoring - There is the prospect of direct benefit to the individual participants 
for their participation in the study.  These potential benefits include the recognized benefits of being in 
a clinical study, including the close monitoring offered to all participants regardless of group 
assignment.  As noted in the introduction, marked hyperglycemia and DKA occur frequently at the time 
of clinical diagnosis.  Close monitoring of autoantibody positive individuals using an OGTT has been 
demonstrated to have direct clinical benefit3-5.  The presence of DKA at diagnosis is reduced from more 
than 25% to 3-4% when in close monitoring.  For those under 5, the frequency of DKA at diagnosis is 
reduced by two thirds.  Moreover, in the general T1D population, the mean HbA1c at diagnosis is 11%.  
This contrasts with a mean HbA1c of 6-8% when closely monitored, indicating less exposure to 
hyperglycemia.  OGTTs are generally considered a minimal risk procedure, as they just involve an IV 
placement and the ingestion of oral glucose. Other procedures, venipuncture, laboratory tests, 
histories, physical examinations, and ophthalmologic examinations are likewise generally considered 
to be minimal risk.   The monitoring aspects of this study are therefore consistent with the United States 
Department of Health and Human Services, Protection of Human Subjects, Subpart D, section 46.404 
(research not involving greater than minimal risk) and with the Food and Drug Administration, Protection 
of Human Subjects, Subpart D, section 50. 51 (Clinical investigations not involving greater than minimal 
risk).
Component analysis: Intervention Arm - hydroxychloroquine – The use of hydroxychloroquine 
offers the prospect of direct benefit. As described above in this protocol, hydroxychloroquine is a Food 
and Drug Administration (FDA)-approved agent for malaria as well as rheumatoid arthritis (RA) and 
discoid and systemic lupus erythematous (SLE).  It is widely used in clinical practice for these 
indications, including in pediatric populations. Efficacy in autoimmune disease has been well 
established in RA42-44, SLE45-47, antiphospholipid syndrome48, and Sjögren’s syndrome49,50.  Particularly 
relevant for this study is that hydroxychloroquine is effectively used early in the course of autoimmune 
disease, having been shown to slow or halt progression in palindromic rheumatism37-40 and in delaying 
onset of SLE in antibody positive individuals41. Other beneficial actions of hydroxychloroquine have 
been identified with use of the drug in other autoimmune diseases, including improvement in 
cardiovascular risk factors, such as reduction of LDL cholesterol51, thrombosis52, glycemia52,53, insulin 
resistance52,54,55, and reduction of risk of incident diabetes mellitus in SLE54 and RA55,56. Positive effects 
of hydroxychloroquine on beta cell function and insulin sensitivity have also been demonstrated in 
healthy volunteers, possibly mediated through anti-inflammatory effects57.  The risks of 
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hydroxychloroquine are thus justified by the anticipated benefit to the participants and the relation of 
the anticipated benefit to the risk is at least as favorable to the participants as that presented by 
available alternative approaches.
This research proposal in children is therefore consistent with the United States Department of Health 
and Human Services, Protection of Human Subjects, Subpart D, section 46.405 (research involving 
greater than minimal risk but presenting the prospect of direct benefit to the individual participants) and 
with the Food and Drug Administration, Protection of Human Subjects, Subpart D, section 50. 52 
(Clinical investigations involving greater than minimal risk but presenting the prospect of direct benefit 
to individual participants) for those in the hydroxychloroquine treatment arm.
Component analysis: Placebo Arm - The placebo intervention has no known risks and is therefore 
consistent with the United States Department of Health and Human Services, Protection of Human 
Subjects, Subpart D, section 46.404 (research not involving greater than minimal risk) and with the 
Food and Drug Administration, Protection of Human Subjects, Subpart D, section 50. 51 (Clinical 
investigations not involving greater than minimal risk).
Summary: Assent of children, along with consent of both parents/legal guardians, as applicable 
according to country and IRB or ethics board requirements, will be obtained prior to any study 
procedures.  This research proposal in children is consistent with the United States Department of 
Health and Human Services, Protection of Human Subjects, Subpart D, section 46.405 (research 
involving greater than minimal risk but presenting the prospect of direct benefit to the individual 
participants) and with the Food and Drug Administration, Protection of Human Subjects, Subpart D 
section 50. 52 (Clinical investigations involving greater than minimal risk but presenting the prospect of 
direct benefit to individual participants) for those in the active treatment group and is consistent with 
the United States Department of Health and Human Services, Protection of Human Subjects, Subpart 
D, section 46.404 (research not involving greater than minimal risk) and with the Food and Drug 
Administration, Protection of Human Subjects, Subpart D, section 50. 51 (Clinical investigations not 
involving greater than minimal risk) for those in the placebo group. 
7.2.
Expected Side Effects and Adverse Events 
A full description of the adverse events linked to hydroxychloroquine is available within the prescribing 
information found in the medication packet insert.  The descriptions below highlight the most important 
and/or common drug-related events and potential adverse events70.
Gastrointestinal:  
Gastrointestinal side effects are common, affecting between 1-10% of users.  These include nausea, 
diarrhea, abdominal discomfort and vomiting.  These symptoms are dose-related and stop with 
cessation of drug.  Less than 10% of those with these symptoms choose to discontinue therapy.  
Ophthalmologic: 
The most important adverse effect of hydroxychloroquine is retinal toxicity.  In a retrospective case-
control study of 2,361 individuals who had taken hydroxychloroquine for at least 5 years, retinopathy 
was seen in 7.5% of users when assessed with spectral domain optical coherence tomography and 
10 degree standard automated perimetry visual fields; as duration of use increases, an unknown, but 
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higher percentage that varies with the daily dose was seen.   The risk of retinopathy was duration- 
and dose-dependent, and was less than 2% within 10 years in those taking 4-5 mg/kg/day71.  There is 
negligible risk when duration of therapy <6 months70 and it is unlikely to occur if dose is <5 mg/kg/day 
actual body weight (generally equivalent to 6.5 mg/kg/day ideal body weight) up to 400 mg/day.  
Moreover, retinopathy detected by these sensitive techniques is not equivalent to clinically apparent 
or significant visual effects.  Despite longstanding use of hydroxychloroquine for malaria therapy in 
very young children, there is a dearth of information about retinopathy, suggesting its rarity72.  Current 
practice recommendation by the American Academy of Ophthalmology is annual examination after 5 
years of drug exposure in those on daily doses 5.0 mg/kg with no major risk factors73.  
Dermatologic AEs (pruritus, allergic rashes, and increased skin pigmentation), neuromuscular AEs 
(muscle aches), and insomnia each occur 1% of the time and resolve within 1-2 weeks with continued 
therapy.
Cardiovascular: Hydroxychloroquine has the potential to cause QT prolongation and should not be 
administered with other drugs that have a potential effect on the QT interval (see Drugs Interactions; 
section  7.2.4.2). In addition, rare cases of cardiomyopathy have been reported in those over age 50 
who received high doses of hydroxychloroquine over a long duration. 
Thrombocytopenia and hemolysis are rare and are probably associated with high doses, other drugs, 
or G6PD deficiency.  
There are case reports of allergic reactions (urticarial, angioedema), abnormal liver function tests, 
and skeletal muscle myopathy, which are reversible over time.
7.2.1. Immunizations
With widespread clinical use, there is no evidence that hydroxychloroquine has an impact on the safety 
or efficacy of vaccines.  As such, all participants will be instructed to maintain their standard clinically 
indicated immunization schedules.  However, the mechanism of action of the drug raises the theoretical 
possibility that live cellular vaccines could be less effective while receiving hydroxychloroquine.  
7.2.2. Pregnancy
Hydroxychloroquine is widely used in pregnancy without safety concerns.  However, due to the impact 
of pregnancy on study outcome measures and the fact that the drug is being tested for a new indication, 
female participants with reproductive potential will be instructed to avoid pregnancy and use birth 
control while on study. They will undergo urine pregnancy testing at the start of every study visit.  All 
pregnancies that are identified during the study must be requested to be followed to conclusion and the 
outcome of each must be reported.  The investigator should be informed immediately of any pregnancy 
occurring in a female participant.  Monitoring of the participant should continue until the conclusion of 
the pregnancy. Participants who are found to be pregnant while on this study shall have treatment 
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withheld, but will still be followed for safety. Treatment may only be resumed when these participants 
are no longer pregnant or breast-feeding.
7.2.3. Risk of Overdose
A number of fatalities have been reported in children following the accidental ingestion of the 4-
aminoquinoline compound chloroquine, sometimes in 
relatively small doses. However, 
hydroxychloroquine is a different drug.  It has less toxicity overall, and a higher therapeutic ratio. 
7.2.4. Prohibited Medications 
Participants will be asked to consult with their study team before taking any new therapy.  
7.2.4.1.
Drugs that may impact T1D progression or effect of Hydroxychloroquine
Participants will be instructed not to use prednisone, other immunosuppressive agents, or chronic 
inhaled or nasal corticosteroids during this trial in order to prevent possible impact on progression to 
diabetes. They will also be instructed not to use proton pump inhibitors or antacids, which may inhibit 
accumulation of hydroxychloroquine in lysosomes by decreasing the pH of these organelles, and thus 
potentially antagonizing immunomodulating effects.  However, as an intention-to-treat study, no 
individual will be withdrawn from analysis if this occurs.
7.2.4.2.
Hydroxychloroquine drug interactions
Hydroxychloroquine has known drug interactions with therapies that are unlikely to be used by the 
population enrolled in this study.  Upon review by the TrialNet medical monitor, participants who have 
a clinical indication to take these medications after enrollment may be withdrawn temporarily from  study 
treatment while on these other medications, or permanently, but with continued monitoring and safety 
assessments for this trial.  These medications include monoamine oxidase inhibitors, anti-
arrhythmogenic drugs such as amiodarone, cyclosporine, digoxin, halofantrine, other anti-malarials, 
anti-epileptic drugs, praziquantel, agalsidase, CYP34A inhibitors such as cimetidine and ketoconazole 
(which may increase hydroxychloroquine levels), penicillamine, bosutinib, droperidol, lanthanum, 
laronidase, cholinesterase inhibitors, and kaolin. Hydroxychloroquine potentially prolongs the QT 
interval, and therefore should not be co-administered with other drugs that potentially prolong the QT 
interval, including SSRIs.
7.3.
Protecting Against or Minimizing Potential Treatment Risks
Participants who have other active serious medical problems will not be enrolled. Frequent monitoring 
of participants with history, physical examination, and laboratory studies will allow for early identification 
of adverse events. Every attempt will be made to minimize the number of venipunctures. Participants 
will be screened for G6PD deficiency and excluded if present. 
To mitigate potential ophthalmologic risk, a fundus photograph or examination will be attempted at 
Baseline, and OCT and visual field examinations will be attempted annually in all participants. 
Participants with a successful OCT examination may continue with the study per protocol. If neither the 
OCT nor visual field examination can be successfully completed (e.g., in very young children), these 
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examinations will be re-attempted on an annual basis while the participant continues on study drug.  
The target dose of hydroxychloroquine will be 5.0 mg/kg/day to a maximum of 400mg.  This is below 
the dose associated with ophthalmologic risk. Moreover, individuals who reach study endpoint 
(abnormal glucose tolerance) will be considered to be a non-responder and withdrawn from therapy, 
thus decreasing duration of exposure.
Individuals will be informed of potential impact on skin, sleep, gastrointestinal symptoms, and muscle 
aches.  If symptoms continue or are intolerable, drug dose may be temporarily reduced or stopped, at 
the discretion of the investigator in consultation with the medical monitor.  If signs or symptoms are 
consistent with infectious mononucleosis, evaluation may include measurements of EBV serology and 
viral load as clinically indicated. 
8. STATISTICAL CONSIDERATIONS AND ANALYSIS PLAN  
This is a randomized, double-blinded, placebo-controlled clinical trial designed to assess the impact of 
hydroxychloroquine to reduce the risk of progression from Stage 1 T1D (multiple autoantibodies, normal 
glucose tolerance) to Stage 2 (abnormal glucose tolerance) or Stage 3 (clinical diagnosis of diabetes). 
Eligible participants will be randomized in a 2:1 allocation ratio to treatment with hydroxychloroquine 
vs. placebo. Randomization will be stratified by two factors to ensure balance in the proportions who 
are randomized to each of the treatment arms: (1) prior enrollment on a T1D prevention trial and (2) 
age (<8 vs. ≥8 yrs. old). Specifically, participants will be randomized to hydroxychloroquine vs. placebo 
with stratification on whether they: (1) had prior enrollment on a T1D prevention trial and received 
placebo vs. had prior enrollment on a T1D prevention trial and received active therapy vs. had no prior 
enrollment on a T1D prevention trial, and (2) are younger than 8 years old vs. 8 years old or older. Age 
is a known significant factor in relation to clinical outcomes of interest even in the T1D prevention 
setting. Further, this cutoff of 8 years old at study entry was selected based on analyses that showed 
a significant effect modification on clinical outcomes based on this74. 
Analyses of study data will be conducted to address the primary and secondary objectives of the trial, 
other stated objectives, and other interrelationships among elements of study data of interest to the 
investigators and of relevance to the objectives of the study. Analyses by age, gender, and 
race/ethnicity, as appropriate, are also planned.
All analyses will be conducted under an intention-to-treat principle, whereby all outcome data in all 
randomized participants will be included in all analyses, as appropriate. Details of the analysis can be 
found within the statistical analysis plan. 
8.1.Primary Outcome 
The primary outcome is the elapsed time from random treatment assignment to the development of 
confirmed abnormal glucose tolerance or clinical diabetes among those enrolled in the primary analysis 
cohort consisting of participants with islet cell autoimmunity and absence of metabolic abnormalities 
(normal OGTT).  The date of the confirmatory abnormal OGTT or diabetes will be considered as the 
date of reaching these study endpoints. It is expected that abnormal glucose tolerance will be detected 
prior to diabetes onset by OGTT; however, the presence of diabetes, without a previous abnormal 
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OGTT, is also considered an endpoint.
While the hydroxychloroquine prevention study is ongoing, any participant who has progressed to 
abnormal glucose tolerance will have met the primary endpoint criteria. These participants who have 
developed abnormal glucose tolerance but have not progressed to Stage 3 T1D will have the option to 
remain in this study without further therapy; they will receive close monitoring and safety assessments 
until the development of Stage 3 T1D (clinical diabetes) or until they enroll in another clinical trial, 
whichever comes first. 
The study endpoint is realized with either confirmed OGTT criteria for abnormal glucose tolerance or 
diabetes or clinical criteria for diabetes.  
OGTT criteria for abnormal glucose tolerance or diabetes: 
The presence of an OGTT consistent with impaired glucose tolerance or diabetes on two 
sequential dates.  A participant with abnormal glucose tolerance or diabetes on an OGTT 
should undergo a repeat OGTT as soon as possible, but no less than one day apart.  Aim to 
repeat within one month.  
The definition of abnormal glucose tolerance is:
a.  Fasting plasma glucose ≥ 110 mg/dL (6.1 mmol/L) and < 126 mg/dL (7.0 mmol/L), or
b.
 2 hour plasma glucose ≥ 140 mg/dL (7.8 mmol/L) and < 200 mg/dl (11.1 mmol/L), or
c.
 30, 60, 90 minute plasma glucose during OGTT ≥ 200 mg/dL (11.1 mmol/L).
Criteria for Diabetes Diagnosis: 
A. Diabetic Ketoacidosis (DKA) or unequivocally symptomatic* AND at least one of the 
following:
1) Random glucose ≥ 200 mg/dl (11.1 mmol/L) OR
2) Fasting Glucose ≥ 126 mg/dl (7.0 mmol/L) OR
3) OGTT = diabetic** OR
4) HbA1C ≥ 6.5%.
*Unequivocal symptoms: Severe/persistent polyuria, polydipsia and/or significant unexplained 
weight loss
**A diabetic OGTT is defined as:
a.
Fasting plasma glucose ≥ 126 mg/dL (7.0 mmol/L) AND/OR
b.
2 hour plasma glucose ≥ 200 mg/dL (11.1 mmol/L)
B. If not unequivocally symptomatic, two separate objective measures are required for 
diagnosis. These tests must occur within 1 year of each other and must be consecutive, 
defined as without an intervening non-diabetic OGTT.

The preferred objective measures for diagnosis are:
 
* 2 diabetic OGTTs, not on the same day (the confirmatory OGTT is the date of  
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                          diagnosis)

However, if an individual is diagnosed without the consecutive OGTTs, the following 
also constitute diagnosis in TrialNet:
* Diabetic OGTT + FPG ≥ 126 mg/dl [7 mmol/L] (not on the same day)
* Diabetic OGTT + HbA1c ≥ 6.5% (these may be on the same day)
* FPG ≥ 126 mg/dl [7 mmol/L] + HbA1c ≥ 6.5% (these may be on the same day)
The TrialNet Eligibility and Events Committee will review and adjudicate any cases with only a single 
objective measure, cases which do not meet the criteria above, and/or cases which have uncertain 
results of tests and/or the test procedures. Qualifying glucose values should be from laboratory tests, 
rather than capillary blood glucose meter readings.
8.2.Primary Analysis 
The primary objective of the study is to assess the effect of hydroxychloroquine therapy versus 
placebo on the risk of progression to Stage 2 or 3 T1D, which is measured from an OGTT or clinically 
diagnosed with confirmation.
Time to progression to Stage 2 or 3 T1D will be calculated across participants within each treatment 
group. Time to T1D stage progression will be estimated based on Kaplan-Meier methodology, where 
participants who were "free of abnormal glucose tolerance and/or T1D" at their last follow-up will be 
censored at that time point. 
This time-to-event outcome will be analyzed using a Cox proportional hazards regression model that 
stratifies by prior T1D prevention treatment (none vs. placebo vs. active therapy) and age at the time 
of randomization (<8 yr vs ≥8 yr), where treatment effect in this model will be tested using the Wald 
statistic. Incorporating these as stratification factors in the Cox regression model will allow separate 
baseline hazard functions to be fitted for each stratum. We will further evaluate differences in event 
rates in those in the prior treatment stratification factor; specifically, we will assess if there are 
differential event rates in those enrolling after participation in another prevention trial and ensure that 
this is accommodated by corresponding stratification in the Cox regression model. 
In addition to stratifying on these factors, we will also evaluate if they are effect modifiers on any 
differences observed in the time to T1D stage progression between these prior treatment groups; i.e., 
we will formally test for interactions of treatment effect with prior treatment for T1D prevention as well 
as age on time to T1D stage progression (i.e. progression from Stage 1 to Stages 2 or 3 T1D).     
Proportional hazards can be tested to ensure that we can assume that these effects will be parallel. If 
the assumption of proportional hazard is not appropriate, the data will be examined to determine the 
cause of non-proportional hazard, such as the presence of a decaying, diverging, or crossing effect of 
hazard ratios over time. Based on the cause of the non-proportional hazard, post-hoc analyses such 
as frailty models, parametric models, or models with interactions and time-dependent covariates may 
be employed.
Additional factors may be defined before unmasking of the study data to the investigators. The analyses 
will distinguish between factors specified prior to unmasking, and those identified post-hoc during 
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analysis. Data from the TrialNet Pathway to Prevention Study may be used to determine participant 
eligibility. Outcomes from other studies may be used in meta-analyses, but not in the evaluation of this 
study’s efficacy.
8.3.Secondary Outcomes and Analyses
A variety of secondary analyses are planned, including the following: 
Time to progression to Stage 3 T1D (clinical diabetes) will also be evaluated. Those participants who 
progress to Stage 2 T1D (abnormal glucose tolerance) prior to developing clinical diabetes will also be 
included in the time to (Stage 3) T1D analyses. Any participants who develop abnormal glucose 
tolerance and enroll in another treatment trial or receive other prevention therapy prior to development 
of Stage 3 T1D will be censored at that time. The treatment arms will be compared in terms of the 
corresponding distributions of time to progression to Stage 3 T1D (clinical diabetes) using the same 
methods described for the primary endpoint. Subgroup analyses analogous to those described for time 
to the composite event definition of progression to Stage 2 or 3 T1D will be conducted on the endpoint 
of time to progression to Stage 3 T1D. 
Longitudinal analyses will assess the effects of hydroxychloroquine versus placebo treatment on 
immunologic and metabolic markers over time up to the onset of progression to Stage 2 or 3 T1D. 
Differences between groups in the mean levels of quantitative factors over time will be assessed using 
a normal errors linear model for repeated measures. Differences between groups in the prevalence of 
qualitative factors over time will be assessed using generalized estimating equations for categorical 
measures. Generalized estimating equations may also be employed for the analysis of quantitative 
factors if the assumption of multivariate normal random errors is violated.
Immunologic and metabolic markers will be modeled to determine the effects of hydroxychloroquine 
versus placebo treatment while adjusting for participant characteristics for each follow-up time point of 
interest. For continuous endpoints that lend themselves to normal error linear models, ANOVA and 
ANCOVA models will be employed. Generalized linear modeling will be employed for dichotomous and 
categorical endpoints by using the most appropriate link functions. Longitudinal analyses may be 
employed in order to characterize the relationship among the repeated measures during the treatment 
period and possibly beyond. Due to the exploratory nature of the longitudinal modeling, treatment effect 
hypothesis testing will not be conducted.
The association of demographic, genetic, immunologic, metabolic, lifestyle factors, the presence of 
illness and concomitant medications, among others, both at baseline and over time, with the risk of 
T1D stage progression will be assessed in Cox regression models over time. The effects of changes 
in longitudinal factors on risk of progression will be assessed using time-dependent covariates for these 
factors. Analyses will be conducted separately within the hydroxychloroquine and placebo groups, and 
differences between groups in covariate effects (group by covariate interactions) will be assessed. 
Models will then be assessed within the two groups combined, taking account of any group by covariate 
interactions. Subgroup analyses will be conducted comparing the effects of hydroxychloroquine versus 
placebo on the risk of progression to Stage 2 or 3 T1D with a test of the group by subgroup factor 
interaction in a Cox proportional hazard (PH) Model. Specifically, we will evaluate potential differential 
effects of treatment in relation to other factors at baseline, including: age (both continuous as well as 
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categorical: children < 8 years of age vs. ≥ 8 years, as well as < 8 years vs. 8 to 12 years vs. adolescents 
[13-17 years] vs. adults [≥ 18 years]), gender, race/ethnicity, whether or not they have a first degree 
relative (FDR) with T1D, and specific autoantibody status. Similar analyses will be conducted using the 
values of quantitative baseline metabolic and anthropometric factors such as weight, BMI, and the 
immunologic and metabolic factors described in Section 6 that include the autoantibody titers, basal C-
peptide, stimulated C-peptide (peak and AUC mean), and measures of insulin resistance modeled from 
the OGTT. These potential effect modifiers, or interaction effects, will be evaluated both using tests of 
interaction in the Cox regression model as well as stratified analyses. In addition, we will evaluate 
differences in time to T1D stage progression across all randomized participants using multivariable Cox 
regression models with tests of interaction and stratified analyses to assess potential differential effects, 
based on whether or not participants were previously treated for T1D prevention.
Toxicity is defined as adverse events that are classified as either possibly, probably, or definitely related 
to study treatment. The maximum grade for each type of toxicity will be recorded for each participant, 
and frequency tables will be reviewed to determine toxicity patterns. In addition, we will review all 
adverse event data that is graded as 3, 4, or 5 and classified as either “unrelated” or “unlikely to be 
related” to study treatment in the event of an actual relationship developing. The incidence of severe 
(grade 3+) adverse events or toxicities will be described for each treatment arm, but will also be 
compared between the arms. Fisher’s exact tests will be used to quantitatively compare the incidence 
of severe as well as specific toxicities of interest between the treatment arms, and we will graphically 
assess differences in maximum grades observed for toxicities between the arms. These analyses will 
be useful in identifying potential toxicity patterns of interest and in planning future trials. We will also 
assess tolerability of hydroxychloroquine through assessing the proportion of patients who discontinue 
treatment due to adverse reactions or even those who refuse further treatment for lesser toxicities that 
inhibit their willingness to continue participation on the trial. These tolerability measures will be 
assessed within each of the treatment arms and we will explore differences in these measures between 
the arms. All participants who have received at least one dose of any of the therapeutic agents in a 
treatment arm will be evaluable for toxicity and tolerability.
8.4.Study Power and Accrual Target 
This study has been designed to provide 83% power to detect a 50% risk reduction in the hazard rate 
for progression to Stage 2 or 3 T1D (abnormal glucose tolerance or diabetes diagnosis) using a two-
sided test at the 0.05 level after a minimum of two years of follow-up on all participants and an expected 
total study duration (accrual and follow-up) of six years. We note that if accrual is more rapid than 
expected, minimum follow-up on participants may need to be extended in order to meet the number of 
events required for the final analysis. 
A total of 201 participants will be randomized in a 2:1 allocation to treatment with hydroxychloroquine 
(n=135) vs. placebo (n=66). Randomization will be conducted using block randomization with variable 
block sizes with stratification on (1) whether or not participants have been previously treated for T1D 
prevention, and (2) age group (< 8 years old vs. 8 years old or older). 
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The assumptions underlying the estimated sample size for this design are: (1) 40% two-year event rate 
in the placebo group, (2) detectable hazard ratio of 0.50 (i.e. 22.5% two-year event rate) in the 
hydroxychloroquine group, (3) less than a 10% two-year dropout rate in both groups, and (4) survival 
curves which are consistent with exponential survival distributions. Even testing for variable proportions 
of each stratum and with slight variation in the expected hazard rates for the placebo group across the 
strata, our proposed sample size of 201 participants will provide at least 83% power to detect a hazard 
ratio of 0.50 in the time to T1D stage progression between the two treatment groups using a stratified 
Cox regression model75.
Under the above assumptions, the target total number of events is equal to 80; i.e., the final analysis 
will be conducted once 80 events (confirmed abnormal glucose tolerance, diabetes) have been 
observed. If the accrual rate is 50 patients per year, with the above assumptions it is estimated that the 
enrollment period will last approximately 4 years with a total study duration of approximately 6 years (4 
years of accrual + minimum of 2 years of follow-up) to provide a sufficient number of events to detect 
the assumed difference.
The final test of significance up to the six-year time point will employ group sequential critical values to 
protect against inflation in the type I error probability due to interim assessments of the emerging data 
for review by the DSMB (see Section 8.5). 
Note the accrual period and the study sample are only projections, since the actual accrual rate and 
the loss-to-follow-up rate are unknown. As the study progresses, more accurate projections of the study 
end date will be computed based on the observed rate of enrollment, the observed number of events, 
and the observed rate of loss-to-follow-up. These data will be provided to the DSMB and the TrialNet 
governing body, and if need be, this document will be amended.
8.5.Interim Monitoring Plan 
A formal interim analysis of efficacy will be conducted when 50% of events (i.e., information fraction = 
0.50) have been observed; i.e., we will conduct an interim analysis to compare the two treatment arms 
when 40 participants enrolled on this trial have a reported progression to Stage 2 or 3 T1D. To preserve 
the Type I error rate control for each of these comparisons on superiority, the Lan-DeMets error 
spending rate function with the O’Brien-Fleming boundaries is utilized. If these boundaries are crossed, 
then the TrialNet DSMB will determine if accrual to that arm should be suspended and/or if treatment 
of participants should be modified based on these results76. The interim and final analysis boundaries 
and characteristics were generated using the East 6 clinical trial software program (version 6.3, Cytel 
Inc). 
Information 
fraction
Cumulative events Alpha spent Efficacy  boundary (p-value)
0.50
40
0.0031
2.963 (or -2.963)
1.00
80
0.00153
1.969 (or -1.969)
The DSMB may terminate the trial prematurely if a statistically significant effect is observed and it is 
considered that all major trial objectives have been met. 
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In lieu of a formal futility rule at the interim analysis (i.e., 50% information), we will utilize conditional 
probability methods to assess the probability that hydroxychloroquine will delay time to T1D stage 
progression based on the data observed at that point if the observed hazard ratio is >1.05 (i.e., in favor 
of placebo). 
The DSMB will also consider early termination due to absence of a treatment effect (i.e., futility) based 
on computations of conditional power conducted both under the initial study design and under the 
current trend of the data77.
8.6.Withdrawal Criteria – Individual Participants
An intention-to-treat approach will be used in these analyses. Participants will not be replaced.  All data 
acquired prior to termination for the reasons outlined below will be included in the primary analysis 
unless a participant withdraws consent for the use of those data. Every effort will be made to conduct 
a final study visit with the participant, and participants will be followed clinically until, if applicable, all 
potentially study-related adverse events resolve. 

Withdrawal of consent by the participant

Withdrawal of the participant by the investigator 

Intercurrent illness or event that precludes further visits to the study site or ability to evaluate 
disease.
9. ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD CLINICAL PRACTICE
9.1.
Statement of Compliance
This study will be conducted in compliance with the protocol and consistent with current Good Clinical 
Practices (GCP), adopting the principles of the Declaration of Helsinki, and all applicable regulatory 
requirements (ICH E6, 45CFR46, and FDA 21CFR sections 11, 50, 56, 312).
Prior to study initiation, the protocol and the informed consent documents will be reviewed and 
approved by a Central Institutional Review Board (CIRB) or an appropriate Independent Ethics 
Committee/Research Ethics Board (IEC/REB) or Institutional Review Board (IRB).  Any amendments 
to the protocol or consent materials must also be approved before they are implemented.
9.2.
Participating Centers
Participating TrialNet clinical sites must have an appropriate assurance, such as a Federal-wide 
Assurance (FWA) or an Unaffiliated Investigators Agreement (UIA), with the Office for Human Research 
Protections (OHRP), since they are actively engaged in research and obtain informed consent.  The 
protocol and consent forms will be approved by a Central Institutional Review Board (CIRB) or 
Institutional Review Boards or Ethics Committees/Research Ethics Boards at each of the participating 
clinical sites.  HIPAA and applicable local regulations will be followed by each participating institution 
in accordance with that institution’s requirements.  The participating international sites will obtain 
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approval from their corresponding review boards in accordance with their local procedures and 
institutional requirements.
The investigator is required to keep accurate records to ensure the conduct of the study is fully 
documented.  The investigator is required to ensure that all source documents are legibly completed 
for every participant entered in the trial.
The investigational sites participating in this study will maintain the highest degree of confidentiality 
permitted for the clinical and research information obtained from participants in this study.  When a 
participant participates in this study at more than one TrialNet site, sharing of this information is 
required.  Sharing of information obtained during this study between the TrialNet Hub, TrialNet Clinical 
Centers and affiliates will occur whenever necessary to assure participant understanding and consent, 
safety, and adherence to protocol according to country-specific guidelines.
9.3.
Informed Consent
The process of assuring that individuals (and parent/guardian if less than 18 years of age) are making 
an informed decision about participating in this study includes both verbal and written communication.  
Written materials include a Volunteer Handbook, Volunteer Understanding Assessment, and written or 
electronic consent forms. The consent form describes the procedures, risks and benefits for the study. 
The consent forms will be reviewed with participants (and their parent/guardian in the case of 
participants under 18 years of age) and the participant will be given time to review the written or 
electronic consent form and ask questions.  An assent form has also been developed for participants 
less than 18 years of age (unless local IRB/REB requirements differ in procedure). 
As part of the informed consent process, prior to treatment, the participant and/or parent or guardian 
(if the participant is less than 18 years of age) will also be required to complete a short, written Volunteer 
Understanding Assessment that is designed to ensure that the participant understands the study, as 
well as what is being asked of him/her. The participant will have access to a copy of their consent/assent 
forms.  
The consent process will be conducted by qualified study personnel (the Trial Investigator or Study 
Coordinator and/or other designee).  All participants (or their legally acceptable representative) must 
read, sign and date a written orm prior to participation in the study, and/or undergoing any study-specific 
procedures.
Remote informed consent may be used as needed when a participant is unable to travel to a TrialNet 
site. When providing remote consent (via phone or video conference) the POC will review the checklist 
and long-form informed consent document with the subject/parent or guardian. After the subject/parent 
or guardian is provided the printed long form informed consent document, the POC will address any 
questions the subject/parent or guardian may have before the informed consent form is signed and any 
procedures are performed. The informed consent document may be provided to the subject/parent or 
guardian using any method that the complete content can be available in print. Signed and completed 
documentation must be available to the subject/parent or guardian and the study team, by any method 
that assures that the complete content can be available in print. 
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Written assent for participants <18 years old may be waived if enrolled using the remote informed 
consent process. Written assent will be obtained at the participant’s next in-person visit.  
When a participant turns 18 they are permitted to re-consent remotely should they be unable to return 
to the study site for an in-person visit. Re-assent when a participant reaches age 12 is waived until the 
participant returns for an in-person visit at the study site.
The informed consent form must be updated or revised whenever there is new, clinically significant 
information applicable to the safety of the participants, when indicated for a protocol amendment, and/or 
whenever any new information becomes available that may affect a participant’s participation in the 
study.  
Participants will be re-consented if they reach the age of 18 years while enrolled in the study.
9.4.
Study Participant Confidentiality
The investigational sites participating in this study will maintain the highest degree of confidentiality 
possible for the clinical and research information obtained from participants.  Participant-identifying 
clinical and research information will be shared between TrialNet sites to support participants and 
assure protocol compliance as applicable. As a part of the quality assurance and legal responsibilities 
of an investigation, the investigational site must permit authorized representatives of the sponsor(s) 
and regulatory agencies to examine (and when required by applicable law, to copy) records for the 
purposes of quality assurance reviews, audits, and evaluation of the study safety and progress. Unless 
required by the laws permitting copying of records, only the coded identity associated with documents 
or other participant data may be copied (obscuring any personally identifying information). Authorized 
representatives, as noted above, are bound to maintain the strict confidentiality of medical and research 
information that may identify individuals. The investigational site will normally be notified in advance of 
auditing visits. Study records with the study participant’s information for internal use at the clinical sites 
will be secured at the study site during the study. At the end of the study, all records will continue to be 
kept in a secure location. There are no plans to destroy the records. 
Study participant data, which is for reporting purposes, will be stored at the TrialNet Coordinating 
Center.  Case report forms sent to the Coordinating Center will identify participants by their unique 
TrialNet Identification Number.  The data entry system at the Coordinating Center is a secured, 
password-protected computer system.  At the end of the study, all study databases will be archived at 
the Coordinating Center, and the data collection forms will be electronically scanned and saved in 
electronic format for long-term storage.  
Stored samples including genetic samples could be utilized to learn more about causes of T1D, its 
complications (such as eye, nerve, and kidney damage) and other conditions for which individuals with 
diabetes are at increased risk, and how to improve treatment.  The results of these future analyses, 
and any mechanistic studies, will not be made known to the participant.
9.5.
Risks and Benefits
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The risks of this study are presented in this protocol, the prescribing information package insert, and 
informed consent form.  There is no guaranteed benefit to participants for their participation in the study.  
This study will examine whether intervention with hydroxychloroquine will delay or prevent the 
development of abnormal glucose tolerance, but there is no guarantee that this will occur.  However, 
all participants will benefit from close monitoring for the development of diabetes.  This close monitoring 
significantly reduces the morbidity typically associated with clinical onset of disease.
Special consideration regarding risks and benefits for children is described in section 7.1.
9.6.Ethics
The study protocol, along with the required informed consent forms, will be approved by a Central 
Institutional Review Board (CIRB), and/or each participating institution’s Institutional Review Board 
(IRB) or Ethics Committee/Research Ethics Board (EC/REB) at international sites prior to the initiation 
of any research procedures (at the site). In addition to details described in the sections above (informed 
consent, confidentiality, and risks and benefits), the investigators have reviewed and considered ethical 
ramifications in the design and development of this protocol. The investigators have made every effort 
to minimize and monitor risks and discomforts to participants throughout the course of the study. 
10.   STUDY ADMINISTRATION
10.1. Organizational Structure
This study is part of Type 1 Diabetes TrialNet, which is funded by the National Institutes of Health. 
Funding will cover the costs of administration and laboratory tests associated with this study. 
10.2. Groups and Committees
10.2.1.
Hydroxychloroquine Prevention Study Chair Committee
The Study Chair and TrialNet Executive Committee will receive periodic reports from the TNCC on the 
progress of the study.  These will include accrual rates and baseline demographic characteristics. 
Criteria and results of ongoing monitoring of the TrialNet labs in terms of reproducibility will also be 
provided on a routine basis and reported on during the Joint Study Chair Committee meetings, as 
scheduled. 
10.2.2. TrialNet Chairman’s Office and TNCC
The TrialNet Chairman’s Office, the TNCC, and the TrialNet Hub will collaboratively provide leadership 
to the TrialNet study group; this will include protocol and manual preparation, training for clinical sites, 
development of statistical design for each study, and analysis of study results.  The TNCC will also 
coordinate interactions among the participating TrialNet Clinical sites, laboratories (including TrialNet 
core Laboratories and other subcontract laboratories), NIDDK, and other sponsoring agencies.
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10.2.3. Clinical Sites
Principal Investigators at each participating TrialNet clinical site will oversee all operations at that site.  
The clinical sites will forward all laboratory and data collection form information to the TNCC for 
analysis.  Direct communication and site visits, as needed, will facilitate evaluation of the trial 
management.  
10.2.4. Eligibility and Events Committee
A TrialNet Eligibility and Events Committee will review all relevant information for each participant in 
which there is uncertainty related to eligibility or to diagnosis of diabetes according to criteria stated in 
section 9.1. The Committee will determine whether the diagnosis of diabetes or abnormal glucose 
tolerance in each of these participants is sufficiently sound so as to include that participant among the 
cases who have reached the primary outcome in the statistical analysis. The Committee will be masked 
to treatment assignment as it reviews each case.
10.2.5. Clinical Site Monitoring
In order to conduct this study with established research principles, site visits may be conducted during 
the study to evaluate study conduct and ensure participant safety.  All sites will be monitored by the 
TNCC and appropriate TrialNet committees for patient enrollment, compliance with protocol 
procedures, completeness and accuracy of data entry, the occurrence and reporting of adverse events 
(AEs) and serious adverse events (SAEs), site pharmacy accountability/operations, and to confirm the 
presence of appropriate IRB/REB regulatory approvals/documents.  
10.3. Medical Monitor and Data Safety and Monitoring Board (DSMB)
All adverse events will be recorded on source documents; adverse event forms will be sent to the local 
IRBs/REBs, per their reporting requirements, and, for AEs ≥ Grade 2, to the TNCC.  
An independent physician will be designated to serve as the medical monitor for this study who will 
maintain regular contact with the study and the Study Chair.  (S)he will review all adverse event reports, 
masked to treatment assignment, and will file event reports with regulatory authorities as appropriate.  
The DSMB will meet approximately every 3 months and as needed to review indicators of safety.  In 
addition, they will meet every 6 months to review the interim effectiveness and potential toxicity of the 
study treatments based on interim analyses of indicators of effectiveness and safety (separately by 
treatment group) prepared by the TNCC.  The DSMB will independently evaluate whether there are 
grounds to modify or discontinue the study.
10.4. Sample and Data Storage
Samples to be stored for research purposes will be located at the NIDDK Repository and at TrialNet 
Laboratory Sites.  While TrialNet is active, the use of the samples will be restricted to TrialNet 
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researchers, unless researchers from outside of TrialNet obtain approval from the TrialNet Steering 
Committee and the NIDDK to utilize the samples.  All samples will be coded with unique study numbers, 
but TrialNet researchers will be able to identify samples if it is necessary to contact participants for 
reasons of health or to notify them about future studies. Approval from the TrialNet Steering Committee 
and the NIDDK would be required before such linkage could occur.  Researchers from outside of 
TrialNet will not be permitted to identify samples. 
Data collected for this study will be sent to the TNCC.  De-identified data will be stored at the NIDDK 
Repository, under the supervision of the NIDDK/NIH, for use by researchers including those outside of 
TrialNet.
With permission of the participant, when TrialNet is completed, samples will continue to be stored at 
the NIDDK Repository.  Since the stored data will be fully de-identified upon the completion of TrialNet, 
it will no longer be possible to identify samples.  Thus, whereas a sample can be destroyed upon a 
participant’s request during the existence of TrialNet, it can no longer be destroyed once TrialNet is 
completed.  However, there will still be the potential to link data derived from the samples with data that 
was derived from TrialNet studies.  Once TrialNet is completed, researchers will only obtain access to 
samples through grant proposals approved by the NIDDK.  The NIDDK will convene an external panel 
of experts to review requests for access to samples.
10.5. Preservation of the Integrity of the Study
The scientific integrity of the trial dictates that results be reported on a study-wide basis; thus, an 
individual Center will not report the data collected from its site alone.  All presentations and publications 
using TrialNet trial data must protect the main objectives of the study.  Data that could be perceived as 
threatening the study outcome will not be presented prior to release of the primary study outcomes.  
Approval as to the timing of presentations of data and the meetings at which they might be presented 
will be granted by the TrialNet Publications and Executive Committees.  Study results should be 
discussed with the news media only upon authorization of TrialNet Executive Committee, and never 
before the results are presented.  Any written statements about this study that are shared with national 
media must be approved by TrialNet Executive Committee before release.
10.6. Participant Reimbursement and Compensation
Participants may be compensated for each visit attended in the study. In compliance with ICH Guidance 
E6, the amount and method of payments to participants shall be designed to avoid coercion or undue 
influence on the study participants. Payments to participants will be prorated and not wholly contingent 
on completion of the trial by the participant.
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APPENDIX 1: Hydroxychloroquine in At-Risk Individuals:   Study Flow Chart
TrialNet Pathway to Prevention Study Results 
Antibody results meet eligibility criteria1. Confirmation of 2 positive 
autoantibodies must occur within six months prior to randomization, but 
the confirmation does not have to involve the same 2 autoantibodies.
OGTT within 52 days of Baseline:
Fasting Plasma Glucose <110 mg/dL (6.1 mmol/L) AND
2 hour Plasma Glucose <140 mg/dL (7.8 mmol/L) AND
30, 60 and 90-minute glucose < 200 mg/dL (11.1 mmol/L)
Hydroxychloroquine Prevention Study Screening
Procedures
Consent to study participation is reviewed and signed. Laboratory 
assessments, History, Physical Examination, Volunteer Understanding 
Assessment, Study Education.
Autoantibodies and/or OGTT if not performed during Pathway to 
Prevention Monitoring Visit, or if repeat testing is needed to meet entry 
criteria windows.
Results to move on to 
Meets all study inclusion criteria
Hydroxychloroquine
Does not meet any exclusion criteria2, 3
Randomization
Hydroxychloroquine Prevention Study Randomization and Baseline 
Procedures
Confirmation 
of 
eligibility, 
randomization, 
baseline 
laboratory 
assessments, 
and 
administration 
of 
hydroxychloroquine 
study 
drug/placebo.
1If autoantibodies are not confirmed positive on the second test a tiebreaker draw will be required.  While 
TN18 (Abatacept Prevention) is open for enrollment, if the individual is ≥ 6 years of age and eligible for 
TN18, at least one of the antibodies must be mIAA.  
2If previous abnormal glucose tolerance, two most recent consecutive OGTTs must show normal glucose 
tolerance.  
Note, individuals are permitted to undergo OGTT and autoantibody testing to determine eligibility for 
hydroxychloroquine during a screening visit if not yet eligible through OGTT and autoantibody testing in 
TN01; Pathway to Prevention Monitoring Visit.  Data obtained from screening tests for this study may be 
used if needed for eligibility for other TN studies. 
3If participant is not eligible or unwilling to participate in this study but remains eligible for monitoring, the 
participant may be followed in TN01 Pathway to Prevention Study as applicable.
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APPENDIX 2: Schedule of Assessments
Double-Masked Treatment
Screening
Baseline
Month 1
Month 3
Month 6 & every 
subsequent 6 
months 1
Month 9 & every 
subsequent 6 
months 
Visit number
-1
0
1
2
3+
4+
Informed Consent
X
Eligibility including G6PD screening
X
Medical history
X
X
Complete Physical Examination 2
X
X (annual visits only)
Limited/Directed Examination 2
X
X
X (six month visits 
only)
CBC with differential and platelets
X
X
X
X
Chemistries and liver function tests
X
X
X
Urine pregnancy test (as appropriate)
X
X
X
X
HIV, Hep B and C serology
X
Adverse Events
X
X
X
X
Prior/ Concomitant Meds
X
X
X
X
X
Dosing & Drug Dispensing 3
X
X
X
OGTT 4
(X5)
X
HbA1c
X
X
Islet autoantibodies
(X5)
X
X
X
Mechanistic assessments 6
X
X
X
X
Ophthalmology examination 7
X7
X (annual visits only)
Study med accountability
X
X
Interim contact 8
X
X
1= Participants will be followed every six months until development of study endpoint, diabetes, or the end of the trial.
2= Interim medical history and directed/limited physical examination every six months. Complete examination at screening and annually thereafter.   Once 
Tanner Stage assessed as ≥ 3, no further genitalia examination required.  All visits include adverse event assessment and collection of concomitant 
medications.
3= Initial dosing and dispensation must occur during the baseline visit.
4= If OGTT consistent with abnormal glucose tolerance or DM, repeat as soon as possible, but no less than one day apart.  Aim to repeat within 1 month. 
Mechanistic samples, including CBC/differential, will be collected at the confirmatory OGTT visit. In order to maintain the masking of the study outcome, a 
random subset of participants with a normal OGTT will also be brought back for an additional OGTT after undergoing a regularly scheduled OGTT. Individuals 
who report signs or symptoms of diabetes may undergo further testing as indicated including OGTT evaluation.
5= If not done as part of the TrialNet Pathway to Prevention study, OGTT and Islet Autoantibodies may be done at the screening visit.  
6= May include samples for pharmacokinetic assessment, RNA, plasma, serum, DNA, measures of B and T cell number and function to understand the effect 
of therapy on the immune system and infectious disease.  The schedule for these assessments may vary as appropriate.  At no time will the blood draw 
volume exceed what is allowable according to the participant’s age and body weight (For participants <18 years, 5 mL/kg per visit, 9.5 mL/kg in an 8 week 
period). Responses to clinically indicated immunizations may be determined through analysis of pre- and post-dose samples.
7= Ophthalmology examination: baseline examination may occur any time from screening until three months after randomization. Subsequent examinations 
will occur annually for all participants, starting at the 12 month visit.
8 = All subjects may have interim contact with study personnel for formal inquiry about adverse events and presence or absence of diabetes related symptoms. 
This interim contact does not require an in-person visit. 
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Olsen NJ, Schleich MA, Karp DR. Multifaceted effects of hydroxychloroquine in human 
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arthritis. A randomized controlled trial. Annals of Internal Medicine. 1993;119(11):1067-1071.
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2013;15(9):360.
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effects of antimalarials in systemic lupus erythematosus: a systematic review. Annals of the 
Rheumatic Diseases. 2010;69(01):20-28.
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hydroxychloroquine treatment on pregnancy outcome in women with antiphospholipid 
antibodies. American Journal of Obstetrics and Gynecology. 2016;214(2):273.e271-273.e278.
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sera from patients with rheumatoid arthritis: association with HLA-DRB1 alleles. Annals of the 
Rheumatic Diseases. 2009;68(5):736-743.
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Yavuz S, Asfuroglu E, Bicakcigil M, Toker E. Hydroxychloroquine improves dry eye 
symptoms of patients with primary Sjogren's syndrome. Rheumatology International. 
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Cairoli E, Rebella M, Danese N, Garra V, Borba EF. Hydroxychloroquine reduces low-density 
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lipid-lowering effect. Lupus. 2012;21(11):1178-1182.
52.
Penn SK, Kao AH, Schott LL, et al. Hydroxychloroquine and glycemia in women with 
rheumatoid arthritis and systemic lupus erythematosus. The Journal of rheumatology. 
2010;37(6):1136-1142.
53.
Gerstein HC, Thorpe KE, Taylor DW, Haynes RB. The effectiveness of hydroxychloroquine in 
patients with type 2 diabetes mellitus who are refractory to sulfonylureas--a randomized trial. 
Diabetes Res Clin Pract. 2002;55(3):209-219.
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Chen Y-M, Lin C-H, Lan T-H, et al. Hydroxychloroquine reduces risk of incident diabetes 
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with rheumatoid arthritis. Jama. 2007;298(2):187-193.
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Solomon DH, Massarotti E, Garg R, Liu J, Canning C, Schneeweiss S. Association between 
disease-modifying antirheumatic drugs and diabetes risk in patients with rheumatoid arthritis 
and psoriasis. Jama. 2011;305(24):2525-2531.
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Wasko MC, McClure CK, Kelsey SF, Huber K, Orchard T, Toledo FG. Antidiabetogenic 
effects of hydroxychloroquine on insulin sensitivity and beta cell function: a randomised trial. 
Diabetologia. 2015;58(10):2336-2343.
58.
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Type 1 Diabetes Development. Diabetes Care. 2017.
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Ferrara CT, Geyer S, Evans-Molina C, et al. The Role of Age and Excess Body Mass Index in 
Progression to Type 1 Diabetes in At-Risk Adults. The Journal of Clinical Endocrinology & 
Metabolism. 2017:jc.2017-01490-jc.02017-01490.
60.
Tsakonas E, Fitzgerald AA, Fitzcharles MA, et al. Consequences of delayed therapy with 
second-line agents in rheumatoid arthritis: a 3 year followup on the hydroxychloroquine in 
early rheumatoid arthritis (HERA) study. The Journal of Rheumatology. 2000;27(3):623-629.
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Rantalaiho V, Korpela M, Laasonen L, et al. Early combination disease-modifying 
antirheumatic drug therapy and tight disease control improve long-term radiologic outcome in 
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patients with early rheumatoid arthritis: the 11-year results of the Finnish Rheumatoid Arthritis 
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rheumatoid arthritis: a randomized, double-blind, placebo controlled clinical trial--an Indian 
experience. Current Medical Research and Opinion. 2007;23(9):2227-2234.
63.
Costedoat-Chalumeau N, Galicier L, Aumaitre O, et al. Hydroxychloroquine in systemic lupus 
erythematosus: results of a French multicentre controlled trial (PLUS Study). Annals of 
Rheumatic Diseases. 2013;72(11):1786-1792.
64.
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and Rheumatism. 2006;54(11):3640-3647.
65.
Levy RA, Vilela VS, Cataldo MJ, et al. Hydroxychloroquine (HCQ) in lupus pregnancy: 
double-blind and placebo-controlled study. Lupus. 2001;10(6):401-404.
66.
Singal AK, Kormos-Hallberg C, Lee C, et al. Low-dose hydroxychloroquine is as effective as 
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Petersen CS, Thomsen K. High-dose hydroxychloroquine treatment of porphyria cutanea tarda. 
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Mercer E, Rekedal L, Garg R, Lu B, Massarotti EM, Solomon DH. Hydroxychloroquine 
improves insulin sensitivity in obese non-diabetic individuals. Arthritis Res Ther. 
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Achuthan S, Ahluwalia J, Shafiq N, et al. Hydroxychloroquine's Efficacy as an Antiplatelet 
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Wolfe F, Marmor MF. Rates and predictors of hydroxychloroquine retinal toxicity in patients 
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Marmor MF, Kellner U, Lai TY, Melles RB, Mieler WF. Recommendations on Screening for 
Chloroquine and Hydroxychloroquine Retinopathy (2016 Revision). Ophthalmology. 
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74.
Bosi E, Boulware DC, Becker DJ, et al. Impact of Age and Antibody Type on Progression 
From Single to Multiple Autoantibodies in Type 1 Diabetes Relatives. J Clin Endocrinol 
Metab. 2017;102(8):2881-2886.
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-       Study #: 20180480        Effective Date: 7/14/2020

SAP_001.pdf: 
1 
Version  09SEPT2021 
TN-22 Statistical Analysis Plan 
 
 
TRIAL FULL TITLE 
HYDROXYCHLOROQUINE FOR PREVENTION OF 
ABNORMAL GLUCOSE TOLERANCE AND DIABETES 
IN INDIVIDUALS AT-RISK FOR TYPE 1 DIABETES 
MELLITUS (Protocol TN-22) 
EUDRACT NUMBER 
 2018-000659-42 
Phase 
Phase IIb 
SAP VERSION 
 Version  
ISRCTN NUMBER 
SAP VERSION DATE 
September 26, 2022  
TRIAL STATISTICIAN 
Lu You 
TRIAL CHIEF 
INVESTIGATOR 
Dr. Polly Bingley and Dr. Jane Buckner 
SAP AUTHOR 
Jeffrey Krischer 


2 
Version  09SEPT2021 
 
Contents 
Table of Contents ...................................................................................... Error! Bookmark not defined. 
1. 
Abbreviations and Definitions .......................................................................................................... 3 
2. 
Introduction ..................................................................................................................................... 3 
3. 
Primary Analysis ............................................................................................................................. 4 
4. 
Secondary Outcomes and Analyses ............................................................................................... 4 
5. 
Study Power and Sample Size ........................................................................................................ 6 
6. 
Interim Monitoring Plan ................................................................................................................... 7 
7. 
General Considerations .................................................................................................................. 7 
8. 
Safety Analyses ............................................................................................................................... 8 
9. 
Reporting Conventions .................................................................................................................... 9 
10. 
Per Protocol Analysis ...................................................................................................................... 9 
11. 
Technical Details ............................................................................................................................. 9 
12. 
References .................................................................................................................................... 10 
 


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1. 
Abbreviations and Definitions 
 
 
AE 
Adverse Event 
ADA 
American Diabetes Association 
AGT 
Abnormal Glucose Tolerance  
AUC 
Area Under the Curve 
BMI 
Body Mass Index 
DSMB 
Data Safety Monitoring Committee 
ITT 
Intent to Treat 
OGTT 
Oral Glucose Tolerance Test 
PH 
Proportional hazards 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
T1DM 
Type 1 Diabetes Mellitus 
 
 
2. 
Introduction 
 
Preface 
 
While the mechanism of action of hydroxychloroquine is poorly understood, it appears to act on 
autoimmune responses; thought to target B cell responses. In particular, hydroxychloroquine 
has been shown to decrease cellular activation, including hindering of antigen presentation to 
CD4+ T cells and reducing the expression of the CD4+ T cell activation marker CD15430,31. 
Hydroxychloroquine is also thought to have a number of other immune actions that may lead to 
decreased autoantibody production, including (i) alkalinization of intracellular vacuoles leading 
to inhibition of proteolysis, chemotaxis, phagocytosis, and antigen processing32, (ii) reduction 
of macrophage-mediated cytokine production33 and (iii) inhibition of function of toll-like 
receptors34.  
 
  Purpose of the analyses 
 
Analyses of study data will be conducted to address all objectives and other interrelationships 
among elements of study data of interest to the investigators and of relevance to the objectives 
of the study. Analyses by sex, age, and race/ethnicity are also planned. 
 
All primary analyses will be conducted under the intention-to-treat principle whereby all outcome 
data in all randomized subjects will be included, regardless of treatment compliance. 
 
Primary Outcome 
 
The primary outcome is the elapsed time from random treatment assignment to the 
development of confirmed abnormal glucose tolerance or diabetes among those enrolled in the 
primary analysis cohort consisting of subjects with insulin autoimmunity and absence of 
metabolic abnormalities (normal OGTT).    
 
The study endpoint is realized with either OGTT criteria for abnormal glucose tolerance or 
diabetes or clinical criteria for diabetes.   
 
OGTT criteria for abnormal glucose tolerance or diabetes:  


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The presence of an OGTT consistent with abnormal glucose tolerance or diabetes on two 
sequential dates.  A subject with abnormal glucose tolerance or diabetes on an OGTT should 
undergo a repeat OGTT as soon as possible, but no less than one day apart.  Aim to repeat 
within one month.  The time of abnormal glucose tolerance or diabetes will then be taken as 
the date of the confirmatory abnormal OGTT. The definition of abnormal glucose tolerance or 
diabetes is: 
a. Fasting plasma glucose ≥ 110 mg/dL (6.1 mmol/L) and < 126 mg/dL (7 mmol/L) or 
b. 2 hour plasma glucose ≥ 140 mg/dL (7.8 mmol/L) and < 200 (11.1 mmol/L, or 
c. 30, 60, 90 minute plasma glucose during OGTT ≥ 200 mg/dL (11.1 mmol/L) 
 
Onset of Diabetes:  
Criteria for diabetes onset (T1DM) are based on glucose testing, or the presence of 
unequivocal hyperglycemia with acute metabolic decompensation (diabetic ketoacidosis). One 
of the following criteria must be met on two occasions as soon as possible but no less than 
one day apart for diabetes to be defined: 1. Symptoms of diabetes plus casual plasma glucose 
concentration ≥ 200 mg/dL (11.1 mmol/L). Casual is defined as any time of day without regard 
to time since last meal. The classic symptoms of diabetes include polyuria, polydipsia, and 
unexplained weight loss. 2. Fasting plasma glucose ≥ 126 mg/dL (7 mmol/L). Fasting is 
defined as no caloric intake for at least 8 hours. 3. 2 hour plasma glucose ≥ 200 mg/dL (11.1 
mmol/L). The test should be performed using a glucose load containing the equivalent of 
1.75g/kg body weight to a maximum of 75g anhydrous glucose dissolved in water. It is 
preferred that at least one of the two testing occasions involve an OGTT. Cases identified will 
be confirmed as having diabetes if the glucose values to make these determinations were 
obtained in a TrialNet laboratory as part of an OGTT. Cases diagnosed with diabetes by 
symptoms and casual glucose ≥ 200mg/dL or by other criteria than the above will be 
adjudicated by the TrialNet Endpoint Adjudication Committee. 
 
The date of arriving at the study endpoint is the date of the second occasion that meets either of 
the above criteria or the date in which the participant presented in a state of DKA.  
 
3. 
Primary Analysis 
 
The study design is a randomized double-blind placebo-controlled trial. The primary objective 
of the study is to assess the effect of hydroxychloroquine versus placebo on the risk of 
developing abnormal glucose tolerance (AGT) as measured from an OGTT. 
 
The cumulative incidence of AGT over time since randomization within each treatment group 
will be estimated from a Kaplan-Meier estimate of the "AGT-free" survival function.  Time-to-
AGT was discretized to 6-month times in keeping with the OGTT schedule. The difference 
between groups in the cumulative incidence functions, and the associated hazard functions, 
will be tested at the 0.05 level, two-sided, using the Cox regression including age as a 
covariate (1, 5).  The critical value for the test statistic, and confidence intervals in this primary 
analysis will be determined by the group-sequential procedure.  
 
Data from the TrialNet Pathway to Prevention Study may be used to determine participant 
eligibility. Outcomes from other studies may be used in meta-analyses, but not in the evaluation 
of this study’s efficacy. 
 
 
4. 
Secondary Outcomes and Analyses 


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Version  09SEPT2021 
 
4.1 Subgroup analyses.  
 
Subgroup analyses will be conducted comparing the effects of hydroxychloroquine versus 
placebo on the risk of AGT with a test of the group by subgroup factor interaction in a Cox 
proportional hazard (PH) Model. Subgroups of the population by age (classified 
appropriately), gender, race/ethnicity (white, nonwhite and Hispanic yes/no), and specific 
antibody status at baseline. Differences in the treatment effect between subgroups will be 
tested using Likelihood Ratio Test (LRT) in a Cox PH model (1).   
 
4.2 Covariate analyses.  
 
Similar analyses will be conducted using the values of quantitative baseline factors. They 
include age, weight, BMI, HbA1c, HLA (DR3/4 vs. others), autoantibody titers, basal C-
peptide, stimulated C-peptide ( peak and AUC), glucose (fasting, 2-hour, AUC and 
measures of insulin secretory rate (0-30 min) and sensitivity (ISI) (6,7) modeled from the 
OGTT) at study entry. The dependence of the treatment effect on the quantitative levels of 
a covariate will also be assessed by a covariate by treatment group interaction in a PH 
model. The ISI can also inform on the treatment effect independent of C-peptide. 
 
The association of demographic, genetic, immunologic, metabolic, and lifestyle factors, the 
presence of illness and concomitant meds, both at baseline and over time, with the risk of 
AGT onset will be assessed in Cox PH Models over time. The effects of changes in 
longitudinal factors on AGT risk will be assessed using time-dependent covariates for these 
factors.  
 
Using a step-up procedure additional covariates will be tested and included in the model 
only if they improve the log-likelihood at 0.10 level (2-sided). This will be accomplished 
with the treatment assignment variable included but the inclusion/exclusion of the 
candidate covariates will be completely independent of the treatment variable’s impact on 
the model.  The LRT associated with treatment variable in the full, adjusted model will be 
used for the test of treatment effect described in the previous paragraph. Thus, the 
adjustment of the significance level, as with multiple testing, is unnecessary. 
 
 
4.3 Longitudinal analyses. 
 
Longitudinal analyses will assess the effects of hydroxychloroquine versus placebo 
treatment on immunologic and metabolic markers over time up to the onset of AGT. 
Differences between groups in the mean levels of quantitative factors over time will be 
assessed using a normal errors linear model for repeated measures. Differences between 
groups in the prevalence of qualitative factors over time will be assessed using generalized 
estimating equations for categorical measures. Generalized estimating equations may also 
be employed for the analysis of quantitative factors if the assumption of multivariate normal 
random errors is violated.   
 
 
 
4.4 Diabetes.  
 
Additionally, as noted in section 4.9.2 of the protocol, subjects will be able to be followed for 
the occurrence of diabetes.  The treatment arms will be compared on the corresponding 


6 
Version  09SEPT2021 
incidence rates of Type 1 diabetes using the log-rank statistic. Subgroup analyses 
analogous to those described for the AGT endpoint will be conducted on the endpoint of 
Type 1 diabetes.   
 
Additional, pre-specified secondary analyses are identified as follows: 
 
1. Evidence that the HR is not constant over the period of follow up will be assessed and 
the risk of diabetes will be assessed in a PH model. The primary test for treatment effect 
is based on the Cox model, which assumes a proportional hazard between treatment 
groups. If there is a true treatment effect but it is not proportional over the follow-up 
period the test will have substantially less statistical power than stated in the statistical 
section of the protocol. Therefore, to establish a reasonable guide to pursue the 
possibility of a non-proportional treatment effect we will use the guideline requiring a 
significance level of 0.10 or less of the LRT from the standard Cox model.  Graphical 
diagnostics using Schoenfeld residuals will be employed to explore evidence for a 
monotonically decreasing effect of treatment over the follow-up p e r i o d .  Also, plotting 
the Kaplan-Meier time-to-AGT rates by treatment group on the log-log scale will provide 
visual assessment of any diminishing effect of treatment (equal distance separation of 
curves indicates proportional hazard).  We will characterize any such decreasing effect 
of treatment by model parameterization with monotonically deceasing benefit over the 
follow-up time.  Initially, a treatment interaction with log-transformed time-on-study will be 
fit to the data and the log likelihood improvement in the fit noted. Other time transforms 
will be explored only to characterize mathematically the rate of the diminishing effect. Of 
particular interest will be characterizing the point in follow-up where the hazard ratio is 1. 
Any variations in proportional hazards other than a monotonically decreasing effect will 
not be of interest because of the possibility that it is simply random error. 
 
 
5. 
Study Power and Sample Size 
 
This study has been designed to provide 83% power to detect a 50% risk reduction in the hazard 
rate for progression to Stage 2 or 3 T1D (abnormal glucose tolerance or diabetes diagnosis) using 
a two- sided test at the 0.05 level after a minimum of two years of follow-up on all participants and 
an expected total study duration (accrual and follow-up) of six years. We note that if accrual is 
more rapid than expected, minimum follow-up on participants may need to be extended in order 
to meet the number of events required for the final analysis. 
 
A total of 201 participants will be randomized in a 2:1 allocation to treatment with 
hydroxychloroquine (n=135) vs. placebo (n=66). Randomization will be conducted using block 
randomization with variable block sizes with stratification on (1) whether or not participants have 
been previously treated for T1D prevention, and (2) age group (< 8 years old vs. 8 years old or 
older). 
  
The assumptions underlying the estimated sample size for this design are: (1) 40% two-year 
event rate in the placebo group, (2) detectable hazard ratio of 0.50 (i.e. 22.5% two-year event 
rate) in the hydroxychloroquine group, (3) less than a 10% two-year dropout rate in both groups, 
and (4) survival curves which are consistent with exponential survival distributions. Even testing 
for variable proportions of each stratum and with slight variation in the expected hazard rates for 
the placebo group across the strata, our proposed sample size of 201 participants will provide at 
least 83% power to detect a hazard ratio of 0.50 in the time to T1D stage progression between 
the two treatment groups using a stratified Cox regression model75. 


7 
Version  09SEPT2021 
 
Under the above assumptions, the target total number of events is equal to 80; i.e., the final 
analysis will be conducted once 80 events (confirmed abnormal glucose tolerance, diabetes) have 
been observed. If the accrual rate is 50 patients per year, with the above assumptions it is 
estimated that the enrollment period will last approximately 4 years with a total study duration of 
approximately 6 years (4 years of accrual + minimum of 2 years of follow-up) to provide a 
sufficient number of events to detect the assumed difference. 
 
The final test of significance up to the six-year time point will employ group sequential critical 
values to protect against inflation in the type I error probability due to interim assessments of the 
emerging data for review by the DSMB (see Section 8.5). 
 
Note the accrual period and the study sample are only projections, since the actual accrual rate 
and the loss-to-follow-up rate are unknown. As the study progresses, more accurate projections 
of the study end date will be computed based on the observed rate of enrollment, the observed 
number of events, and the observed rate of loss-to-follow-up. These data will be provided to the 
DSMB and the TrialNet governing body, and if need be, this document will be amended. 
 
6. 
Interim Monitoring Plan 
 
A formal interim analysis of efficacy will be conducted when 50% of events (i.e., information 
fraction = 0.50) have been observed; i.e., we will conduct an interim analysis to compare the two 
treatment arms when 40 participants enrolled on this trial have a reported progression to Stage 2 
or 3 T1D. To preserve the Type I error rate control for each of these comparisons on superiority, 
the Lan-DeMets error spending rate function with the O’Brien-Fleming boundaries is utilized. If 
these boundaries are crossed, then the TrialNet DSMB will determine if accrual to that arm should 
be suspended and/or if treatment of participants should be modified based on these results76. 
The interim and final analysis boundaries and characteristics were generated using the East 6 
clinical trial software program (version 6.3, Cytel Inc). 
 
Information 
fraction  
Cumulative events Alpha spent Efficacy boundary (p-value) 
0.50 
40 
0.0031 
2.963 (or -2.963) 
1.00 
80 
0.00153 
1.969 (or -1.969) 
 
The DSMB may terminate the trial prematurely if a statistically significant effect is observed and it 
is considered that all major trial objectives have been met. 
  
In lieu of a formal futility rule at the interim analysis (i.e., 50% information), we will utilize 
conditional probability methods to assess the probability that hydroxychloroquine will delay time to 
T1D stage progression based on the data observed at that point if the observed hazard ratio is 
>1.05 (i.e., in favor of placebo). 
 
The DSMB will also consider early termination due to absence of a treatment effect (i.e., futility) 
based on computations of conditional power conducted both under the initial study design and 
under the current trend of the data77. 
7. General Considerations 
 
1. Analysis Populations 


8 
Version  09SEPT2021 
 
The Intention to Treat Population (ITT) 
The intention to treat population comprises all randomized (as planned) subjects. 
 
Full Analysis Population 
The Full Analysis Set (FAS) will comprise all subjects who received any study drug and who 
participated in at least one post-baseline assessment. These will be analyzed as 
randomized. FAS will be the primary efficacy population. So, FAS is a subset of ITT. 
 
Per Protocol Population 
The Per Protocol Set (PPS) will comprise all subjects who did not substantially deviate 
(defined as greater than or equal to 75% protocol compliance) from the protocol as to be 
determined on a per-subject basis before data base lock and unblinding.   
 
 
Safety Population 
All subjects who received any study treatment (including control) but excluding subjects who 
drop out prior to receiving any treatment. 
 
2. Timing of Analyses 
 
The final analysis will come after the total number of events (confirmed abnormal glucose 
tolerance) in the placebo group is between 64 and 70. This will result in detectable hazard 
ratios of 0.496 to 0.512 to  achieve the planned 80% statistical power when testing at the 0.05 
level (2-sided) for the primary analysis. 
 
3. Missing Data 
 
In general, missing values will be assumed to be missing completely at random (MCAR) 
unless empirical evidence to the contrary can be established internal to the study.  The 
methodology employed in analyzing time-to-T1D utilizes whatever follow-up has been 
recorded for each subject (i.e., maximum utilization of follow-up time).  Presuming no 
evidence against MCAR no methods will be employed to impute additional follow-up of 
subjects that drop out (i.e., lost to follow-up).  All secondary endpoints will use the complete-
case analysis approach which limits the analytical cohort to those subjects that have the 
secondary endpoint of interest measured and recorded.  In modeling to adjust for risk factors 
associated with the endpoint (i.e. covariates), missing values will be assigned the mean from 
the known covariate cohort.  This simple rule will be employed only if the percent missing is 
less than 10% for the analytical cohort.  If the missing is 10% to 20% a separate indicator for 
missing will be included in the modeling.  If the missing in exceeds 20% the covariate will be 
removed from consideration. 
 
8. Safety Analyses 
Safety will be evaluated with summary of adverse events for the safety population. 
The following parameters will be assessed during the study: 
 
Adverse Events 
The summary statistics will be produced in accordance with Section 8. Treatment emergent 
adverse events (AEs) are those events that occur after the baseline assessment. Only Grade 
2 or greater adverse events were reported in this study. The incidence of the following AEs 
will be reported: 
 


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A tabular summary of AE will present: Number of subjects with any AE; Number of SAEs 
with outcome death; Number subjects with SAE; Number subjects with AEs leading to 
discontinuation of study drug, even if by protocol; Number of subjects with AEs leading to 
discontinuation of study; Total number of AEs; Total number of SAEs [TABLE]. 
 
The Adverse Events summary tables will include number of adverse events, the number 
of subjects in each treatment group in whom the event occurred, and the incidence of 
occurrence and should be grouped by system organ class, preferred terms and/or other 
interested variables (e.g., relatedness, intensity and seriousness). [TABLE] 
 
When calculating the incidence of adverse events, or any sub-classification thereof by 
treatment, time period, severity, etc., each subject will only be counted once and any repetitions 
of adverse events will be ignored; the denominator will be the total population size. 
Deaths, Serious Adverse Events and other Significant Adverse Events 
 
All formal testing of adverse effects will be based on the subject as the experimental unit. Thus, 
for comparing incidence of AE within system organ by treatment group, a one-sided Fisher’s 
exact test will be conducted at 0.05 level (higher incidence in experimentally treated group is the 
alternative hypothesis). Also, highest AE grade will be determined for each subject and 
compared by treatment group using a 2 sample Wilcoxon test (one-sided at 0.05).  No 
correction for multiple testing will be employed in order that the statistical power is maintained. 
 
9. Reporting Conventions 
P-values ≥0.01 will be reported to 2 decimal places; p-values less than 0.01 and >0.001 will be 
reported to 3 decimal places; p-values less than 0.001 will be reported as “<0.001”. The mean, 
standard deviation, and any other statistics other than quantiles, will be reported to one decimal 
place greater than the original data. Quantiles, such as median, or minimum and maximum will 
use the same number of decimal places as the original data. Estimated parameters, not on the 
same scale as raw observations (e.g. regression coefficients) will be reported to 3 significant 
figures. 
 
10. Per Protocol Analysis 
 
Quantifying the evidence of any dose response relationship is part of a complete analysis of any 
well-run and completed clinical trial. This is especially true when the trial is negative when 
testing the primary outcome. To explore whether there is evidence that protocol treatment 
deviations may have played a role in the negative outcome, we plan to assess the treatment 
hazard ratio by the degree of compliance (percent missed infusions)  to the protocol scheduled 
dose in a quantitative manner. 
 
Using the Cox model we will assess the evidence for an effect of treatment compliance 
including the entire cohort. The number of infusions of treatment and the treatment dose (in a 
separate model) will be introduced into the model as a continuous variable (0=received no 
therapy; 1 = received the full prescribed dose) along with the interaction term with treatment 
group to determine its effect on risk.  The significance level of the interaction term (compliance 
value if hydroxychloroquine subject; 0 otherwise) will be considered important if it is 
approximately 0.05 or less one-sided and the coefficient is negative (indicating lower risk with 
greater compliance).  The procedure for including covariates, such as age, will follow the set up 
procedure as described above under Primary and Secondary analyses. 
 
11. Technical Details 


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Version  09SEPT2021 
The analysis will be performed in R, S-Plus or SAS. The distributional assumptions as well as 
other assumptions underpinning the planned analyses will be checked.  
 
 
 
 
 
 
 
12. References 
 
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4. JM Lachin.  Futility interim monitoring with control of type I and II error probabilities using 
the interim Z-value or confidence limit.  Clinical Trials 2009; 6 (6) 565-573 
5. Diggle PJ, Heagerty PJ, Liang K-Y, Zeger SL: Analysis of longitudinal data. Oxford, UK, 
Oxford Clarendon Press, 1994 
6. Ursini et al. (2015) Medicine 94 DOI: 10.1097/MD.0000000000000888 
7. Matsuda and DeFronzo Diabetes Care 22:1462–1470, 1999