Clinical trial NCT02387164
Conditions: Diabetes Mellitus, Type 1 Prediabetic State
Keywords: Type 1 diabetes Islet autoantibodies glutamate decarboxylase autoantibodies (GADA) Immune tolerance Prediabetes Glucose tolerance glutamate decarboxylase Prevention Children
Sponsor: Lund University
Start date: 2015-03-09; Completion date: 2019-10-07
Enrollment: 26 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this study is to evaluate if immune-tolerance with Alum-formulated GAD (Diamyd), in combination with high dose Vitamin D3, may delay or stop the autoimmune process leading to clinical type 1 diabetes in non-diabetic children with ongoing beta-cell autoimmunity as indicated by positive islet autoantibodies.

Detailed description:
The primary objective of this study is to evaluate if immune-tolerance with Alum-formulated GAD (Diamyd), combined with high dose Vitamin D3, may delay or stop the autoimmune process leading to clinical type 1 diabetes (diagnosed according to American Diabetes Association criteria) in non-diabetic 4-17.99 year old children with ongoing beta-cell autoimmunity as indicated by positive islet autoantibodies.

The secondary objective is to demonstrate that treatment with Diamyd is safe in children at risk for type 1 diabetes.

The children will be followed for 5 years in the study. Primary endpoint is proportion of subjects diagnosed with type 1 diabetes in each treatment arm. Secondary endpoints are 1) safety, 2) change in metabolic status from normal to impaired glucose metabolism in the group of children with normal glucose metabolism at baseline screening.

Arms: arm 1: Two doses à 20 microgram of subcutaneous alum-GAD (Diamyd), 30 Days apart. Vitamin D 2000 U/Daily with start 30 days before the first injection of Diamyd. Vitamin D treatment will continue throughout the whole study period of 5 years. arm 2: Two doses of subcutaneous placebo, 30 Days apart. Vitamin D 2000 U/Daily with start 30 days before the first injection of Diamyd. Vitamin D treatment will continue throughout the whole study period of 5 years
Interventions: intervention 1: Alum-GAD intervention 2: Vitamin D3
Intervention descriptions: intervention 1: Two doses à 20 microgram 30 days apart subcutaneously administrated intervention 2: 2000 Units (IE) (50 microgram) vitamin D3 daily

Protocol documents:
Prot_000.pdf: 
 
 
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DiAPREV-IT 2 
Diabetes Prevention – Immune Tolerance 2 
 
Clinical Trial Protocol 
 
 
A double-blind, randomized investigator-initiated study to determine the 
safety and the effect of Diamyd® in combination with Vitamin D on the 
progression to type 1 diabetes in children with multiple islet cell 
autoantibodies. 
 
 
Study No. DiAPREV/2014 
EudraCT Number: 2014 -003755-64 
Version 3, 2017-08-28 
 
Sponsor: 
Helena Elding Larsson 
Department of Clinical Sciences/Malmö, Jan Waldenstöms gata 35, 
60:11, Skåne University Hospital SUS, SE 205 02 Malmö, Sweden 
Phone:  +46 (0)40 337676 
 
Principal 
Investigator: 
Docent Helena Elding Larsson, MD, PhD 
Lund University 
Department of Clinical Sciences/Malmö 
Jan Waldenströms gata 35, 60:11 
Skåne University Hospital SUS 
205 02 Malmö 
Sweden 
 
Co-investigators: 
Professor Åke Lernmark PhD, Malmö 
Berglind Jonsdottir, MD 
Markus Lundgren, MD  
 
 
 
 
 
 
 
 
 
 
 
 


 
 
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TABLE OF CONTENTS 
TABLE OF CONTENTS ....................................................................................................................................... 2 
1 
INTRODUCTION AND RATIONALE ...................................................................................................... 4 
1.1 
BACKGROUND AND DIAGNOSIS ................................................................................................................... 4 
1.2 
RATIONALE FOR GAD65, VITAMIN D AND PREVENTION OF TYPE 1 DIABETES ........................................... 5 
1.2.1 
GAD65 preclinical and clinical intervention studies ..................................................................... 5 
1.2.2 
GAD65 and prevention of type 1 diabetes ..................................................................................... 7 
1.2.3 
Vitamin D supplementation ............................................................................................................ 7 
1.3 
OBJECTIVES AND ENDPOINTS ...................................................................................................................... 8 
1.3.1 
Objectives ....................................................................................................................................... 8 
1.3.2 
Endpoints ........................................................................................................................................ 8 
1.4 
STUDY POPULATION .................................................................................................................................. 10 
1.5 
SAMPLE SIZE ............................................................................................................................................. 11 
1.6 
DOSE AND FREQUENCY OF DOSES ............................................................................................................. 11 
1.7 
CHOICE OF PREPARATION AND ADMINISTRATION ..................................................................................... 11 
2 
RISK-BENEFIT ANALYSIS ..................................................................................................................... 12 
3 
STUDY DESIGN ......................................................................................................................................... 13 
3.1 
STUDY PROCEDURE ................................................................................................................................... 13 
3.2 
VITAMIN D SUPPLEMENT: ......................................................................................................................... 14 
3.3 
SAFETY PROCEDURES ................................................................................................................................ 17 
3.4 
DECISION CRITERIA .................................................................................................................................. 18 
4 
STUDY POPULATION ............................................................................................................................. 18 
4.1 
MAJOR INCLUSION CRITERIA .................................................................................................................... 18 
4.2 
MAJOR EXCLUSION CRITERIA ................................................................................................................... 18 
4.3 
PARTICIPANT WITHDRAWAL CRITERIA ...................................................................................................... 19 
5 
STUDY VISIT ASSESSMENTS................................................................................................................ 20 
5.1 
LABORATORY EXAMINATIONS .................................................................................................................. 20 
5.2 
METABOLIC STATUS.................................................................................................................................. 20 
5.3 
SAFETY ASSESSMENTS .............................................................................................................................. 21 
6 
SUBJECT MANAGEMENT ..................................................................................................................... 22 
6.1 
SCREENING VISIT AND ELIGIBILITY ASSESSMENT ...................................................................................... 22 
6.2 
DIAMYD® .................................................................................................................................................. 22 
7 
STUDY MEDICATION ............................................................................................................................. 23 
8 
ADVERSE EVENTS REPORTING AND SAFETY MONITORING .................................................. 24 
8.1 
DEFINITION OF ADVERSE EVENT ............................................................................................................... 24 
8.2 
DEFINITION OF SERIOUS ADVERSE EVENT ................................................................................................. 24 
8.3 
ADVERSE EVENTS REPORTING AND MONITORING...................................................................................... 24 
8.4 
TIMELINES FOR REPORTING SAE .............................................................................................................. 26 
8.5 
UNRESOLVED EVENTS .............................................................................................................................. 26 
8.6 
PREGNANCY: ............................................................................................................................................. 26 
9 
DATA HANDLING .................................................................................................................................... 26 
9.1 
CRF .......................................................................................................................................................... 26 
9.2 
BLOOD SAMPLES ....................................................................................................................................... 27 
10 
STATISTICAL ANALYSES ..................................................................................................................... 27 
10.1 
RANDOMIZATION OF PARTICIPANTS ....................................................................................................... 27 
10.2 
STATISTICAL CONSIDERATIONS AND ANALYTIC PLAN ........................................................................... 27 
10.3 
POWER CALCULATION ........................................................................................................................... 29 
10.4 
DROP OUT .............................................................................................................................................. 29 
11 
MONITORING AND QUALITY CONTROL......................................................................................... 29 


 
 
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11.1 
CRF ....................................................................................................................................................... 29 
11.2 
PATIENT JOURNALS ................................................................................................................................ 30 
11.3 
MONITORING AND QUALITY CONTROL .................................................................................................. 30 
11.4 
QUALITY ASSURANCE ........................................................................................................................... 30 
12 
REGULATORY AN D ETHICAL ASPECTS ......................................................................................... 30 
12.1 
PARTICIPANT CONFIDENTIALITY ............................................................................................................ 31 
12.2 
AMENDMENTS ....................................................................................................................................... 31 
12.3 
END OF TRIAL ........................................................................................................................................ 32 
12.4 
STUDY REPORT ...................................................................................................................................... 32 
12.5 
GCP ....................................................................................................................................................... 32 
13 
PUBLICATION OF RESULTS ................................................................................................................. 33 
14 
DOCUMENTATION, RECORD KEEPING AND ARCHIVING......................................................... 33 
REFERENCES ..................................................................................................................................................... 34 
15 
INVESTIGATOR SIGNATURE ............................................................................................................... 38 
APPENDIX 1, DECLARATION OF HELSINKI ............................................................................................. 39 
APPENDIX 2: POST DIAGNOSIS INTERVENTION .................................................................................... 45 
 
 
 
 
 


 
 
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1 INTRODUCTION AND RATIONALE 
 
 
1.1 
Background and diagnosis 
Type 1 diabetes (former known as insulin dependent diabetes mellitus), is one of the most 
common serious chronic diseases in children. The disease is caused by immune-mediated 
destruction of the pancreatic islet beta cells resulting in loss of insulin production. Children 
developing type 1 diabetes become dependent on lifelong treatment with insulin, with 
enormous consequences for the child, its family and society. Late complications are common 
and life expectancy shortened. Years to months before the classical clinical symptoms of type 
1 diabetes onset there has been an autoimmune sub-clinical destruction of beta cells. At the 
onset of clinical symptoms, only a small fraction of the beta cells are left. In young children 
the clinical onset of the disease is often dramatic, and sometimes life threatening. 
 
The incidence rate of type 1 diabetes is increasing worldwide. It is reaching epidemic 
levels in some countries. In Sweden alone, two children per day are diagnosed with type 1 
diabetes. Unfortunately the increase in type 1 diabetes is the greatest in children under five 
years of age.  
The etiology of type 1 diabetes is still unknown. Genetic factors, such as Human 
Leucocyte Antigen (HLA) genotypes, confer susceptibility to the disease, but other factors, 
both genetic and environmental, are needed to initiate the autoimmune process that results in 
beta cell death. The environmental factors that either trigger the development of the 
autoimmune process or accelerate the beta cell destruction process are still to be defined. 
About 50-60% of the genetic susceptibility is conferred by HLA-DQ 1,2. Several other 
non-HLA genetic factors are known to affect the risk, but account for only 10-15 % of the 
genetic susceptibility 3. HLA genotypes with increased risk of type 1 diabetes can be used to 
identify children at increased risk of the disease. HLA-DQA1*0301-B1*0302 (DQ8) and 
HLA-DQA1*0501-B1*0201 (DQ2) are the haplotypes associated with the highest risk for 
type 1 diabetes. About 90 % of children with type 1 diabetes have at least one of these HLA-
DQ haplotypes, compared with about 20 % in the general population 2,4. A child inheriting 
both of these two haplotypes, DQ2/8 has the highest risk for type 1 diabetes to represent about 
30% of type 1 diabetes children compared to only 3% of the healthy population 2. Still, the 
risk of developing the disease during a lifetime is only 6 % even with this genotype, 
emphasizing the importance of environmental triggering factors. 
The destruction of the pancreatic beta cells in type 1 diabetes is associated with 
cellular immune responses against the islet beta cells and reflected by the appearance of 
autoantibodies against beta cell antigens. At the clinical onset of type 1 diabetes about 95% 
have one or several of autoantibodies against glutamic acid decarboxylase (GADA), 
insulinoma-associated protein 2 (IA-2A), insulin (IAA) or zinc T8 (ZnT8A) 5,6. These 
autoantibodies precede the clinical onset of type 1 diabetes and are shown to be predictive 
markers of the disease.  


 
 
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Autoantibody positivity may appear early in life, and has been reported in children as 
young as 3 months 7,8. While a single autoantibody may be harmless and often represents non-
progressive beta cell autoimmunity, the appearance of multiple autoantibodies most often 
reflects a progressive process 9-13. The number of detectable autoantibodies is unequivocally 
related to the risk of type 1 diabetes, both in first-degree relatives and in the general 
population. In studies of family members of type 1 diabetes patients, 60-100 % of individuals 
with three autoantibodies developed clinical diabetes over the next 5-6 years, and population-
based studies indicate that the risk is similar in the general population 14-16. The autoantibody 
pattern differs between age groups. IAA is the most frequent autoantibody in the very young 
children 17 and therefore tend to be the first autoantibody to appear. GAD65Ab is the most 
frequent antibody in older patients, adolescents and young adults, whereas positive IAA or 
IA-2Ab decreases with increasing age at onset 18.  
 
 
1.2 
Rationale for GAD65, Vitamin D and prevention of type 1 diabetes  
 
 
1.2.1 GAD65 preclinical and clinical intervention studies 
GAD65 is present in neurons and pancreatic islet beta cells and is an enzyme in which 
converts glutamic acid to γ-aminobutyric acid (GABA) – a major inhibitory transmitter 
substance in the nervous system. GAD65 is encoded on chromosome 10p11 and is a protein 
of 585 amino acids. The role of GAD65 within the islet cells is still unknown, as is the reason 
why GAD65 is a major autoantigen in type 1 diabetes. However, studies of the spontaneously 
diabetic non-obese diabetic (NOD) mouse have indicated that administration of GAD65 can 
prevent autoimmune destruction of the pancreatic beta cells 19-24. These findings indicate that 
recombinant human GAD65 may be a preventive treatment of type 1 diabetes in individuals 
with positive GAD65 autoantibodies (GAD65Ab).  
Two formulations of recombinant human GAD65 have been evaluated in preclinical 
and clinical studies. The bulk Drug Substance (formulated in buffer from the manufacturing 
process) was used for initial preclinical safety studies, a skin Prick Test in type 1 diabetes 
teenagers and a Phase I clinical trial (formulated in phosphate buffered saline). An adjuvanted 
formulation based on Alhydrogel® has also been developed to provide the Drug Product 
alum-formulated GAD65 (Diamyd®) used for evaluation in Phase II and Phase III clinical 
trials. 
A preclinical safety evaluation program has been conducted by Diamyd Medical AB, 
Stockholm, Sweden to support the progression of Diamyd® into clinical development. This 
has included single- and repeat-dose toxicity, local tolerance, immuno-toxicity, neuro-
toxicity, reproduction-toxicity and investigation of the potential for effects on behaviour and 
cardiovascular/respiratory function. Evaluation of all preclinical safety studies performed to 
date have not provided concerns for clinical safety even at multiples of the highest clinically-
intended dose level, nor resulted in the observation of any target organs of toxicity. Likewise, 


 
 
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evaluation of the effects of Diamyd® in several different animal models of autoimmune 
disease did not indicate any potential for undesirable effects on the immune system. 
A dose-finding Phase IIa study in 47 patients with Latent Autoimmune Diabetes in 
Adults (LADA) has extended experience with the Alhydrogel-formulated Diamyd® 24. This 
randomized, double-blind and placebo-controlled Phase IIa study also demonstrated efficacy 
in preventing beta cell destruction in the group of patients receiving the 20 µg dose. There 
were no serious adverse events reported during the 6 month long main study period. The 
majority of reported adverse events were due to influenza-like symptoms. A minority of 
injections resulted in injection-site reactions, which were mild and occurred primarily on the 
day of the injections. These findings support the safety of immuno-modulation by treatment 
with alum-formulated GAD65. The 5-year final follow-up investigation did not reveal any 
study-related serious adverse events. 
In a Phase IIb, randomized, double-blind, placebo-controlled multicenter study in 160 
LADA-patients, the subjects received 20 µg of GAD65 or placebo on 2 occasions 4 weeks 
apart. The trial had a main study period of 18 months and was scheduled for un-blinding in 
June 2007. Unfortunately, as it was impossible to guarantee absolute identity of the contents 
of the injections administered to the patients the study was invalidated. However, no safety 
concerns were raised. None of the serious adverse events reported in the study were related to 
the treatment. 
In another Phase IIb clinical trial, the efficacy and safety of Diamyd® were investigated 
in children 10-18 years of age, recently diagnosed with type 1 diabetes. A total of 70 GAD65 
autoantibody-positive children with a recent diagnosis (<18 months duration) of type 1 
diabetes and remaining beta-cell capacity measured by C-peptide levels, were included in the 
study. The study was randomized, double-blind and placebo-controlled and 20 µg Diamyd® 
was given twice four weeks apart. The study has provided strong support for the clinical 
safety of Diamyd® and statistically significant and clinically relevant positive effect on the 
preservation of beta cell function (production of C-peptide) was seen after 30 months25. The 
frequency and pattern of adverse events did not differ significantly between the placebo and 
active treatment group. There were no treatment-related serious adverse events reported.  
A 3-armed Phase III, randomized double-blind, placebo-controlled multi-center 
intervention study has been completed. In this study a total of 334 children and adolescents in 
the EU, 10-20 years of age with recent onset of type 1 diabetes (within 3 months) was given 
placebo or 20 µg Diamyd® in a prime-and-boost regimen on days 1 and 30 to confirm 
previous Phase II results. Additionally, one arm of patients was given two additional single 
doses of 20 µg Diamyd® also on days 90 and 270, to evaluate optimization of the treatment. 
This study did not fulfil the efficacy goal at the 15 months follow-up, although a small 
positive effect was observed (16.4% treatment effect; p=0.10) 26. A US sister study with 
identical study design, was closed early due to the EU study outcome. The safety analyses 
performed by the DSMB with data from both the EU Phase III study and the parallel US 
Phase III study further supports the clinical safety of Diamyd® administration in children and 
adolescents with T1D. In addition, an investigator initiated Phase II intervention clinical trial 


 
 
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performed by TrialNet, in recent-onset type 1 diabetes patients, reported results in June 2011 
and this trial did not meet its efficacy endpoints 27. 
 
All studies did confirm the previously determined favorable safety profile for Diamyd® 
and no neurological concerns have been identified. 
A Phase II clinical intervention study (DIABGAD-1) with Linköping University in 
Sweden as sponsor is ongoing. This study will examine the ability of Diamyd®, combined 
with relatively high doses of vitamin D and the anti-inflammatory drug ibuprofen, to preserve 
beta cell function in children and adolescents newly diagnosed with T1D (10-18 years of age) 
The study will also evaluate the effect of a double dose of Diamyd®.  
 
1.2.2 GAD65 and prevention of type 1 diabetes  
Since positive effects in preservation of residual beta cell function was shown in newly 
diagnosed children and in LADA patients, we initiated a prevention study, where we 
intervene earlier in the autoimmune process when a majority of beta cells are still producing 
insulin. The hypothesis was that treating children with an ongoing subclinical autoimmune 
process against the beta cells may delay the clinical disease, or even stop the beta cell 
destructive process. Before any large-scale prevention studies with Diamyd® in children at 
risk for type 1 diabetes was started, we wanted to evaluate the safety of Diamyd® treatment in 
a smaller group of GADA positive subjects. The secondary objective would be to evaluate 
indications of reduced incidence or delay of the clinical onset of type 1 diabetes.  The 
Diabetes Prevention – Immune Tolerance (DiAPREV-IT) study started in May 2009 and was 
fully recruited with 50 children 4-17.99 years of age in January 2012. All children were 
treated with 2 doses of 20 microgram Diamyd (n=25) or placebo (n=25). The children will be 
followed for 5 years. Up to date, no study related SAE have been reported. A total of 14/50 
children have developed type 1 diabetes during follow-up. Full un-blinded analysis will be 
performed in Jan 2017 after completing the 5 year follow-up. 
 
Since DiAPREV-IT was initiated as a small-scale study to primarily evaluate safety of 
Diamyd in non-diabetic children, a larger study will be important to be able to evaluate a 
possible preventive effect of the drug. In the current study, DiAPREV-IT 2, we will further 
investigate the preventive efficacy of Diamyd in children with multiple islet autoantibodies 
but not yet type 1 diabetes. The inclusion criteria and the outcome measurements will be 
identical between the two studies and the study protocol similar, with the following 
modifications. The first is to stratify randomization with glucose metabolism instead of 
number of islet autoantibody types and the second is to add supplementation with high dose 
Vitamin D to all participating children.  
 
1.2.3 Vitamin D supplementation  
Vitamin D have been shown to affect the maturation and activation of dendritic cells, 
leading to a suppressed T-cell activation28. Vitamin D-deficiency has been reported to be 
associated with increased incidence of type 1 diabetes. Previous studies have indicated that 


 
 
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Vitamin D supplementation from birth may protect the child from type 1 diabetes 29-31. 
Additionally, low levels of vitamin D have been reported in children with newly diagnosed 
type 1 diabetes compared to healthy controls 32. It is suggested that Vitamin D may have a 
direct effect on the beta cells, with improved insulin sensitivity33 and a protection against 
cellular stress 34. 
The rationale for the vitamin D treatment in this study is based on the above findings. 
The children will be treated for 30 days with vitamin D before the first injection with 
Diamyd® or placebo, to enhance the response to the first injection of the study substance and 
to make sure that all children are fully substituted with vitamin D at baseline. All children will 
be treated, unless Vitamin D levels exceed 125 nmol/L, to ensure that vitamin D levels in the 
treatment arm and the placebo arm are similar. Additionally, levels of Vitamin D will be 
measured and evaluated by a paediatrician after the first 30 days of treatment and then every 
6th month during follow-up. Treatment with Vitamin D will continue during the full study 
period, unless levels are above 125 nmol/L.  Calcium levels will for safety reasons be 
measured at baseline and every 3rd month during follow-up and treatment with Vitamin D will 
be omitted if hypercalcemia occurs. 
 
An additional reason for treatment is that levels in children with known multiple islet 
autoantibodies and a high risk of diabetes may vary, since we know from previous studies 
(DiPiS, TEDDY and DiAPREV-IT) that some, but not all, parents may choose to give their 
children Vitamin D as a supplement in an attempt to prevent type 1 diabetes. Therefore 
substitution with Vitamin D will minimize the risk of differing Vitamin D levels as a 
confounder. 
 
1.3 
Objectives and Endpoints 
 
1.3.1 Objectives 
The primary objective is to evaluate if Diamyd®, in children treated with relatively high dose 
vitamin D, may delay or stop the autoimmune process leading to clinical type 1 diabetes in 
children with ongoing persistent beta cell autoimmunity as indicated by multiple positive islet 
cell autoantibodies.  
The secondary objective is to demonstrate that Diamyd® is safe in children at risk for type 1 
diabetes.  
The subjects will be followed for 5 years.  
 
1.3.2 Endpoints 
 
1.3.2.1 
Primary endpoints 
The proportion of subjects diagnosed with clinical type 1 diabetes in the Diamyd® treated 
group, compared to the placebo treated group at five years after the first injection.  
 


 
 
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1.3.2.2 
Secondary endpoints 
To evaluate safety and the change in metabolic status from normal to impaired glucose 
metabolism in the group of children with normal glucose metabolism at baseline as well as the 
progression in metabolic status in the children with impaired glucose metabolism at baseline 
screening, in the non-diabetic children with multiple islet autoantibodies treated with 
Diamyd® compared to those treated with placebo. 
  
Variables to evaluate safety: 
 
• Injection site reactions 
• Occurrence of adverse events (AEs) 
• Laboratory measurements (biochemistry and haematology including complete blood 
count (CBC)), including Calcium and Vitamin D in serum 
• Urine analysis 
• Physical examinations, including neurological assessments 
• 
Epitope-specific GADA titer, isotypes and subtypes as well as antiidiotypic 
autoantibodies to GADA 
 
Metabolic status: 
 
• Change from normal to impaired glucose metabolism, defined as any of  
 
a)  F-glucose ≥ 6.1 mmol/L  
b) maximum p-glucose at 30, 60, 90 minutes ≥ 11.1 mmol/L in the OGTT 
c) 120 min p-glucose ≥ 7.8 mmol/L on OGTT  
d) HbA1c ≥ 39 mmol/mol.  
 
Impaired glucose metabolism has to be confirmed at a second visit. This endpoint will 
be used in the group of children with normal glucose metabolism at baseline screening 
 
• Progression of impaired glucose metabolism from one or several of the above 
variables to additional signs of reduced glucose metabolism, confirmed at a second 
visit. This endpoint will be used in the group of children with impaired glucose 
metabolism at baseline. 
 
1.3.2.3 
Exploratory endpoints: 
 
• Proportion of subjects diagnosed with clinical type 1 diabetes at 1, 2, 3 and 4 years of 
follow-up. 
• Time from baseline visit to clinical type 1 diabetes diagnosis 
• Change from baseline in the following key metabolic variables at various time points: 
HbA1c, First phase insulin response and K-value from IvGTT, AUC p-glucose and C-
peptide from OGTT, 120 minutes glucose and C-peptide after OGTT, fasting C-
peptide, insulin and glucose 
• Change in other metabolic variables from baseline: AUC C-peptide, glucose and 
insulin from IvGTT, AUC insulin from OGTT, change in max p-glucose on OGTT. 


 
 
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1.4 
Study population 
Children repeatedly positive for more than one islet cell autoantibody, as a sign of an ongoing 
autoimmune process against the beta cells, will be invited to participate in DIAPREV-IT 2. 
The invited children have been screened for autoantibodies in a population based follow-up 
study such as Diabetes Prediction in Skåne (DiPiS) study, the Environmental Determinants of 
Diabetes in the Young (TEDDY) study or TEDDY relatives, screened as a relative to a type 1 
diabetes patient in TrialNet or found positive for islet autoantibodies for example at screening 
at the clinic. All participating children have to be 4.00-17.99 years of age at screening and 
have autoantibodies to GAD65 and at least one more islet autoantibody (to IA-2A, IAA or 
ZnT8A). 
DiPiS is a prospective population-based study of diabetes in children 35-37. The aim of 
DiPiS is to determine the predictive value of genetic risk combined with islet cell 
autoantibody markers for type 1 diabetes and to identify factors before, during and after 
pregnancy that may trigger type 1 diabetes. In DiPiS, newborn children in the general 
population were screened during September 2000 to August 2004 for type 1 diabetes high-risk 
HLA genotypes. Children with increased risk of type 1 diabetes are thereafter followed from 2 
to 15 years of age. Children with multiple islet autoantibodies are followed every 3rd month in 
the study. The study is approved by The Ethics Committee at Lund University, Sweden. 
TEDDY is a prospective multicenter study sponsored by the National Institutes of 
Health. Skåne is participating as one of six centres in the world, along with Finland, Germany 
and the US states of Washington, Colorado and Georgia. Screening for the TEDDY began in 
September 2004 and was completed in February 2010. Children in the general population 
with diabetes high-risk HLA genotypes are followed from 3 months of age. The study was 
approved by the Regional Ethics Board at Lund University, Sweden. By now more than 2 000 
children in Skåne are followed in the TEDDY study until 15 years of age.  
 
Trial Net is a consortium providing screening for islet cell autoantibodies and HLA-
typing of relatives of patients with type 1 diabetes. Relatives who are positive for 
autoantibodies will be HLA-typed to rule out the protective genotype DQ6. The participants 
are thereafter followed with annual OGTT´s to investigate the natural history of development 
of diabetes. Trial Net in Skåne, Sweden, was approved by the Central Ethics Committee 
2009-08-27 (2009/470). The screening in Skåne started in March 2010. Participants in the 
Trial Net screening from 4 years of age who are positive for GAD65Ab and at least one more 
islet cell autoantibody and who have not the protective genotype DQ6 allele will be invited to 
participate in DiAPREV-IT 2. Between DiPiS, TEDDY and TrialNet we follow a large 
number of children positive for 2 or more islet cell autoantibodies who will be asked to 
participate in the study.  
 
In TEDDY relatives, relatives to TEDDY children are screened for autoantibodies and 
followed if positive. Children found positive for GADA and at least one more islet 
autoantibody will also be asked to participate in DiAPREV-IT 2. 
 


 
 
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1.5 
Sample size 
Up to 80 children will be asked to participate in DIAPREV-IT 2. By including children 
followed in DiPiS, TEDDY, TEDDY relatives and TrialNet, who fulfil the inclusion criteria, 
we expect that we will be able to identify at least 80 children for inclusion in DiAPREV-IT 2.  
Although the number of subjects is somewhat limited, we expect that DiAREV-IT 2 will have 
power to evaluate the safety and the possible preventive effect of Diamyd®.  
 
1.6 
Dose and frequency of doses 
In DIAPREV-IT 2 we will use the previously tested dose of 20 µg Diamyd® administered as a 
prime-and-boost at days 1 and 30. This dose regimen has been tested in several clinical trials 
in type 1 diabetes children and adolescents with no safety concerns. 
A repeat administration of the same dose at day 30 is the ‘prime-and-boost’ regimen 
conventionally used to initiate and then promote immune response for most vaccines. A 
dosing interval of 4 weeks is considered sufficient to initiate and then recall specific cellular 
and humoral responses to antigens.  
 
A relatively high dose of Vitamin D (2000 IU/day) will be given to all children in 
the study with start 30 +-7 days before the first subcutaneous injection of 20 µg Diamyd® or 
placebo. Samples for Vitamin D will be taken at screening and evaluated after analysis. If the 
levels at screening are above 125 nmol/L, the treatment with Vitamin D will be omitted.  
The Vitamin D treatment will be given to all participating children below 125 nmol/L, 
regardless of treatment group. Vitamin D levels will be determined and evaluated at 
screening, randomization and every 6th month during follow up to secure compliance and for 
safety reasons. Treatment with Vitamin D will continue during the full study period, unless 
levels are above 125 nmol/L.   
 
1.7 
Choice of preparation and administration 
Aluminum hydroxide is commercially called Alhydrogel®. The substance is commonly used 
in vaccines. Aluminium salts are well recognized and preferentially induce a humoral (Th2) 
rather than a cellular (Th1) immune response. Since the autoimmune process leading to type 1 
diabetes is thought to be a mainly cellular (Th1) immune response, Aluminium salts could 
steer the process against a humoral (Th2) response and minimize the likelihood of 
exacerbating cell-mediated beta cell destruction.  
The subcutaneous route of administration has been selected in previous studies of 
Diamyd®. Although intra-muscular route of administration is frequently used for vaccine 
administration, dermal tissues are more capable than muscle tissue to recognize and present 
antigens. In addition subcutaneous injections are less painful than intra-muscular injections. 
 
 


 
 
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2 RISK-BENEFIT ANALYSIS 
 
Type 1 diabetes is a chronic lifelong disease that results from the immunological 
destruction of the pancreatic islet beta cells.. By the time a type 1 diabetes patient is 
diagnosed, up to 85-90% of pancreatic islet beta cells have been destroyed by autoimmune 
responses against specific beta cell autoantigens. The beta cells express autoantigen-peptides 
on HLA Class I proteins on the cell surface, which makes it possible for cytotoxic CD8 
positive T cells to kill the beta cells. Over time this autoimmune response persists and the 
amount of endogenous insulin that is produced is reduced as the beta cells are killed. As this 
happens a type 1 diabetes patient is reliant on exogenous insulin and is at greater risk for both 
short and long term complications. In the more acute manifestation of type 1 diabetes, or the 
absence of treatment, the lack of insulin can result in diabetic ketoacidosis (DKA), a 
potentially life-threatening medical emergency. Insulin therapy dramatically reduces the 
possibility of death from DKA but even patients adequately treated with insulin are still at 
increased risk to a number of acute and long-term (chronic) complications. Acute 
complications include DKA and severe hypoglycaemia. Long-term complications of T1D 
include microvascular, macrovascular and neurologic complications that increase morbidity 
as well as mortality. Studies have shown that a higher level of retained residual insulin 
production is associated with a reduction in DKA and hypoglycemic risk as well as the 
development of long-term complications 38.  
Upon diagnosis of type 1 diabetes, the standard treatment is based on insulin 
replacement with injections or administered through insulin pump, and blood glucose 
monitoring through blood tests 5-10 times per day. Long acting insulin or basal dose in pump 
is complemented with doses to all meals. The doses are dependent on the glucose value, the 
carbohydrate content of the meal and on physical activity, among other. The treatment has 
immense implications on daily life. The continued beta cell destruction results in permanent 
loss of endogenous insulin secretion, as well as loss of any potential regeneration of islet beta 
cells. There is currently no approved method or medicament to halt or slow the immune 
destruction of remaining pancreatic islet beta cells. 
It is estimated that only 10-15% of the beta cells are still functioning at time of clinical 
symptoms and diagnosis of type 1 diabetes. Saving these remaining insulin producing beta 
cells may result in clinically meaningful benefits including improved metabolic control and 
lower risk for hypoglycaemia and chronic complications. However, to save beta cells even 
earlier, before the clinical onset of type 1 diabetes, may result in delayed or prevented clinical 
onset of clinical disease. This would be of benefit for the child, family and society.  
Diamyd® therapy aims at intervening in the autoimmune process in order to preserve 
beta cell function and endogenous insulin secretion by modulating the immune system in a 
discrete, antigen-specific fashion to stop the destruction of beta cells. Thus, the goal of 
Diamyd® therapy would be to dramatically slow or halt the ongoing autoimmune destruction 


 
 
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of pancreatic islet beta cells in order to preserve the largest possible amount of endogenous 
insulin production.  
 
In order to preserve residual endogenous insulin production a treatment to reduce or 
stop the autoimmune responses against remaining functional beta cells must be initiated in the 
period when a significant number of these cells still exist. Therefore, this study is proposed to 
include non-diabetic children with multiple islet antibodies, i.e. at high risk for the clinical 
onset of type 1 diabetes.  
The dose of 20 μg of Diamyd®, administered as a prime dose with a booster dose 30 
days later or as a prime and boost on days 1 and 30 followed by one or two additional single 
doses (days 90 and 270) have been shown to be safe in man (children and adolescents) with 
few and mild adverse reactions.  All clinical studies performed with Diamyd® to date 
indicates a favorable safety profile for Diamyd® and no neurological concerns have been 
identified (see section 1.2 above). 
Risk-benefit Vitamin D:  As reported previously in the rationale, Vitamin D may protect 
or delay the clinical onset of type 1 diabetes in children with multiple islet autoantibodies. 
Vitamin D in a dose of 2000 IU/day in children has been reported safe 39. Additionally, a dose 
of up to 7000 IU/day to children from 5 years of age with HIV did not rise any safety 
concerns40. The dose can be compared to the supplementation dose of 400 IU/day (800 IU/day 
to children with darker skin) that are recommended to children up to 6 years of age to avoid 
vitamin D deficiency. For safety reasons, we will follow D-vitamin serum levels of both 
vitamin D and Calcium every 6th month. A paediatrician will dismiss the Vitamin D 
supplementation if Vitamin D levels are above 125 nmol/L. 
 
3 STUDY DESIGN 
 
3.1 
Study procedure 
Individual subjects will be exposed to the study drug Diamyd®/Placebo for a maximum of 1 
month in the primary study. All participants will be monitored by study personnel at the study 
clinic for at least 30 minutes after administration of each study drug injection. In addition, the 
participants will be offered to stay for an additional 2 hours, or contact the physician/study 
nurse by phone.  
 
We intend to treat 50% of the double antibody positive children with Diamyd® and the 
remaining 50% with placebo (Alhydrogel®). The randomisation will be stratified according to 
glucose metabolism at baseline screening. The tests and examinations scheduled for each visit 
during the first year are presented in Table 1. The participants will be followed for 5 years 
after the first injection with a first analysis carried out 3 years after the first injection.  All 
patients will be supplemented with Vitamin D, regardless of randomization. If levels taken at 
screening or during follow-up are found to exceed 125 nmol/L, the treatment will be omitted 
as soon as the results come. 


 
 
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The rational to stratify this new study by glucose metabolism is based on findings from 
baseline data from the first cohort of 50 participants in DiAPREV-IT. The major finding in 
this analysis was that 43% of the first cohort of 50 children enrolled in the study, with GADA 
and at least one more islet autoantibody (inclusion criteria), had impaired glucose metabolism 
already at baseline, based on 2-OGTT plasma glucose and FPIR tests, despite normal k-
values, HbA1c and fasting glucose and C-peptide levels. Hence, two distinct groups were 
identified in the study population at baseline; one group with impaired glucose metabolism 
(43 %, n=20) and one group with normal glucose metabolism (57%, n=27) 41. In our 
expanded analyses of baseline data we additionally included p-glucose >11.1 mmol/L at 30, 
60 and 90 min on OGTT in the diagnosis of impaired glucose metabolism (manuscript data). 
A total of 52 % had impaired glucose metabolism at baseline screening when including 30, 
60, 90 and 120 min values from OGTT and 12/14 children who, up to date, have developed 
diabetes in the study were in this group (submitted manuscript).   
 
3.2 
Vitamin D Supplement:  
In DiAPREV-IT 2 we will supplement all included children with Vitamin D. The children 
will get a prescription of vitamin D drops, or equivalent, by a paediatrician in a daily dose of 
2000 IU. Samples for Vitamin D will be taken at screening and evaluated after analysis. If the 
levels at screening are above 125 nmol/L, the treatment with Vitamin D will be omitted as 
soon as the results come. Blood draws for Vitamin D will be taken at visit 0 (baseline), at visit 
1 (after 30 days of treatment) at visit 4 (6 months from visit 1) and thereafter every 6 months. 
Should the blood concentration during Vitamin D treatment exceed 125 nmol/L or if the child 
would develop hypercalcemia, the Vitamin D treatment will be stopped. The subject will 
however continue in the study and complete all the other study related evaluations. Vitamin D 
levels will continue to be evaluated every 6th months and all results will be evaluated by the 
investigator. Vitamin D treatment will be started again if the levels fall <100 nmol/L.  
 
3.4  Post diagnosis intervention protocol (PDIP) 
Pending study drug shelf life, children diagnosed with type 1 diabetes within the study period 
may be offered to continue in the trial in a post-diagnosis intervention protocol (PDIP) to 
receive additional injections in the following manner: 
 
• Within 4 months since diabetes diagnosis, participants will receive one injection 
of Diamyd® 20 µg on Day 1 in the Post Diagnosis Follow-up, followed by a 
second injection of Diamyd® on Day 30, in a prime and boost fashion (a total 
received dose of 40 µg Diamyd® for participants who pre-diagnosis was 
randomized to receive placebo and a total received dose of 80 µg Diamyd® for 
participants who pre-diagnosis was randomized to receive Diamyd®)  
 
All children that are enrolled in the PDIP will be discontinued from the original prevention 
protocol to be followed thoroughly for safety and efficacy according to the post-diagnosis 


 
 
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intervention protocol for 15 months following the first injection of Diamyd® in the PDIP 
(Appendix 2). 


 
 
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Table 1. Examinations for each visit 
Event 
Visit 0 
Information 
and consent 
 
 
 
Visit 1 
Day 1 
 
30 +/- 7  days 
from  visit 0  
Visit 2 
Month 1 
 
30 +/-7 days 
from visit 1 
 
Visit 3 
Month 3 
 
3 months 
+/- 14 days 
from visit 1 
Visit 4 
Month 6 
 
6 months 
+/- 14 days 
from visit 1 
Visit 5 
Month 9 
 
9 months  
+/- 14 days  
from visit 1 
Visit 6 
Month 12 
 
12 months  
+/- 14 days  
from visit 1 
5 Years Follow up 
Every 3rd  or 6th 
month 
Every visit  
+/- 14  days 
from visit 1 
Informed Consent 
X 
 
 
 
 
 
 
 
Randomization 
 
X 
 
 
 
 
 
 
Diamyd/Placebo 
Administrationa 
 
X 
X 
 
 
 
 
 
Start of Vitamin D 
X 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
General Physical Exam 
X 
X 
X 
X 
X 
 
X 
Every 6th  month 
Concomitant Medication 
X 
X 
X 
X 
X 
 
X 
Every 6th  month 
Weight, Height 
X 
X 
X 
X 
X 
 
X 
Every 6th  month 
Vital signs (BP) 
X 
X 
X 
X 
X 
 
X 
Every 6th  month 
Blood Sampling:  
 
 
 
 
 
 
 
Every 6th  month 
 Hematology, chemistry, 
including calcium 
X 
X 
X 
X 
X 
X 
X 
Every 3rd   month 
25 OH-vitamin D3 
X 
X 
 
 
X 
 
X 
Every 6th month 
Cellular analyses 
X 
 
X 
 
X 
 
X 
Every 6th  month 
GADA, IA-2A, ZnT8A, 
IAA 
X 
X 
X 
X 
X 
X 
X 
Every 3rd month 
 C-peptide  
 
X 
Fasting + 
stimulated 
X 
Fasting + 
stimulated 
X 
Random 
X 
Random 
X 
Fasting + 
stimulated 
X 
Random 
X 
Fasting + 
stimulated 
Every 6th month 
Fasting + stimulated 
 HbA1c 
X 
X 
X. 
X 
X 
X 
X 
Every 3rd month 
 Plasma Glucose 
X 
X 
X 
X 
X 
X 
X 
Every 3rd month 
 OGTT/IvGTT 
IvGTT 
OGTT 
 
 
 
OGTT 
 
IvGTT 
Every 6th month 
IvGTT/OGTT 
TPOAb, ThglAb, tTGAb 
X 
 
 
 
 
 
X 
Every 12th month 
Urine Analysis 
X 
X 
X 
X 
X 
 
X 
Every 6th  month 
Neurological Assessment 
X 
 
X 
X 
X 
 
X 
Every 6th  month 
Injection Site Inspection 
 
X 
X 
X 
X 
 
 
 
Adverse Events  
 
X 
X 
X 
X 
 
X 
Every 6th  month 
Diary 
 
X 
X 
X 
X 
 
X 
Every 6th  month 
Questionnaire to child 
about the study 
procedures 
 
X 
 
 
 
 
 
After 2 years and at the 
final visit. 


 
 
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Abbreviations: BP=Blood Pressure, OGTT=oral glucose tolerance test, IvGTT=Intravenous glucose tolerance test 
Possibility for telephone contact with physician/study personnel in between the visits. 
 
 
At all clinical visits the responsible paediatrician will fill out the Case Report Form 
(CRF) and also establish patient journals. After the first 6 months the clinical visits will be 
performed every 6th month with glucose tolerance tests, while blood sampling for 
autoantibodies, HbA1c, plasma glucose will be performed every 3rd month with the possibility 
of telephone contact with study personnel.  
A glucose tolerance test will be performed at baseline and every 6th month; OGTT at 
baseline and every half year visit and IvGTT at the baseline visit and at every full year visit 
from the first vaccination. The estimated time for those visits is 2,5-3,5 hours.  
 
 
3.3 
Safety procedures 
The investigator (in this section referred to as sponsor) reserves the right to discontinue 
further treatment within the study at any time for safety reasons or other reasons jeopardizing 
the justification of the study. If the study is prematurely terminated or suspended, the sponsor 
should promptly inform the subjects and assure appropriate therapy and follow-up. The 
sponsor will notify Regulatory Authorities of any plans to terminate the study, and notify the 
appropriate Regional Ethics Board. An independent Data Safety Monitoring Board (DSMB) 
is appointed. The DSMB will review the data and safety throughout the study period. The 
DSMB are responsible for safeguarding the interests of the trial participants and assessing the 
safety of the interventions during the trial. The DSMB will also be responsible for 
recommending the sponsor to discontinue further treatment within the study if the data would 
point towards 1) that children treated with Diamyd® develop type 1 diabetes at a higher 
frequency than the placebo group children (in reality the ability to terminate the study for this 
reason only applies during the active Diamyd®/placebo treatment phase of the trial) 2) that 
treatment with Vitamin D would cause serious unexpected adverse events (then the treatment 
with Vitamin D can discontinued in all children, while the follow-up in the study will 
continue as planned). Once all patients are recruited and have completed all injections the trial 
cannot be stopped because it has reached an observation only period.  
The Sponsor reserve the right to discontinue the study at any time for safety reasons or 
other reasons jeopardizing the justification of the study. Such a termination will be 
implemented in a time frame that is compatible with the participant’s wellbeing. 
If the study is prematurely terminated or suspended, the investigator should promptly 
inform the participant’s and assure appropriate therapy and follow-up. The Sponsor will 
notify the Regulatory Authorities and the Ethics Committee of any plans to terminate the 
study. 
 
The objectives of the study is to to evaluate if there is an effect of the treatment in 
preventing beta-cell destruction and clinical onset of type 1 diabetes and to test that the 


 
 
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treatment is safe in children at risk for type 1 diabetes. The subjects will be followed for 5 
years with a first analysis carried out 3 years after the first injection, but the study will be kept 
blinded for the entire study period to the parents and participating children, investigators and 
all study personell. The study will be unblinded groupwise to the biostatistician responsible 
for statistical analyses and if DSMB request unblinded data groupwise. Safety assessment will 
be conducted periodically during the study and will be reviewed by the DSMB.  
 
3.4 
Decision Criteria 
The child is included in the study when the informed consent form has been signed at the 
information visit (Visit 0). No children will be enrolled until approvals from the appropriate 
Ethics Committees and Regulatory Authorities have been obtained. Prior to the study starting, 
it will be confirmed that all regulatory and ethical requirements for starting the study are met.  
 
4 STUDY POPULATION 
Children participating in DiPiS, TEDDY, TrialNet, TEDDY relatives or found to have 
autoantibodies in other settings fulfilling the inclusion criteria will be asked to participate. Up 
to 80 children are intended to be included in the study during the study years.  
 
 
4.1 
Major Inclusion criteria 
 
1. Children from four (4) to 17,99 years of age. 
2. Positive GADA and at least one additional type 1 diabetes-associated autoantibody 
(IA-2A, ZnT8R/W/QA or IAA). 
3. Written informed consent from the child and the child’s parents or legal acceptable 
representative(s) according to local regulations. 
 
4.2 
Major Exclusion criteria 
 
1. Ongoing treatment with immunosuppressant therapy (topical or inhaled steroids are 
accepted). 
2. Diabetes. 
3. Treatment with any oral or injected anti-diabetic medications. 
4. Significantly abnormal hematology results at screening. 
5. Clinically significant history of acute reaction to vaccines or other drugs. 
6. Treatment with any vaccine, other than influenza, within one month prior to the first 
dose of the study drug or planned treatment with vaccine up to two months after the 
last injection with the study drug.  
7. A history of epilepsy, serious head trauma or cerebrovascular accident, or clinical 
features of continuous motor unit activity in proximal muscles. 


 
 
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8. Participation in other clinical trials with a new chemical entity within the previous 3 
months. 
9. History of hypercalcemia. 
10. Unwilling to abstain from other medication with Vitamin D during the study period. 
11. Significant illness other than diabetes within 2 weeks prior to first dosing. 
12. Known human deficiency virus (HIV) or hepatitis. 
13. Presence of associated serious disease or condition, including active skin infections 
that preclude subcutaneous injection, which in the opinion of the investigators makes 
the patient non-eligible for the study. 
14. Diabetes-protective HLA-DQ6-allele. 
15. Females who are lactating or pregnant (for females who have started menstruating 
the possibility of pregnancy must be excluded by urine βHCG onsite within 24 hours 
prior to the study drug administration) 
16. Males or females not willing to use adequate contraception, if sexually active, until 1 
year after the last Diamyd administration. Adequate contraception is as follows:  
 
For females of childbearing potential: 
 
a. oral (except low-dose gestagen (lynestrenol and norestisteron)), injectable, 
or implanted hormonal contraceptives (females) 
b. intrauterine device (females) 
c. intrauterine system (for example, progestin-releasing coil) (females) 
d. vasectomized male (with appropriate postvasectomy documentation of the 
absence of sperm in the ejaculate)  
 
For males of childbearing potential: 
 
a. condom (male) 
 
4.3 
Participant withdrawal criteria 
The participant and the guardian will receive oral and written information about the study, 
which includes information about the right to withdraw from the trial at any time without 
prejudice to future treatment. In addition, the participant may be withdrawn at the 
investigator’s discretion at any time if regarded in the child’s best interest. Reasonable efforts 
should be made to contact any participant lost to follow-up during the course of the trial in 
order to complete assessments and retrieve any outstanding data. A subject that has 
withdrawn from the study may not be further evaluated in the study. However, the data 
obtained up to the date of withdrawal may be used in the study analyses. 
 
Vitamin D treatment should not be continued if the patient after inclusion in the study 
develops 
 
- 
Symptoms of hypercalcemia such as tiredness, euphoria, drowsiness, nausea, weight 
loss, thirst, polyuria, nefrocalcinosis, renal failure  
- 
ECG changes, arrhythmia  


 
 
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- 
Pancreatitis 
 
Diamyd should not be given to the patient if the patient after inclusion in the study has got 
- 
- brain damage, epilepsy, head trauma, neurological disease 
- 
- other severe autoimmune disease that in the opinion of the investigator makes the 
subject non-eligible for the continuing study treatment  
- 
- immune-suppressive treatment 
- 
- cancer, cancer treatment 
- 
- drug/alcohol abuse 
- 
or if the patient has become pregnant or is no longer willing to use safe contraceptives 
during the study 
 
 
 
 
5 STUDY VISIT ASSESSMENTS 
 
5.1 
Laboratory examinations 
At each visit, blood will be drawn for efficacy and safety evaluations after application of 
EMLA cream to reduce pain. Blood samples during the first study year will be analyzed for 
hematological parameters, i.e. Hemoglobin, White Blood Cells, and Thrombocytes, and 
chemical variables, i.e. sodium, potassium, creatinin, calcium, vitamin D, liver functioning 
tests and specific diabetes-related parameters such as plasma-glucose, insulin, C-peptide, 
HbA1c, GADA, IA-2A, ZnT8A, IAA, CBC and analyses of lymphocytes by flow cytometry. 
To evaluate other autoimmunity than diabetes, autoantibodies to Thyroid peroxidase (TPO), 
Thyroglobulin (Thgl) and Tissue transglutaminase (tTg) will be analysed at visit 0 and yearly. 
The analyses will be performed at the Clinical Chemisty Department, University Hospital 
MAS, Malmö (hematology, chemical variables, insulin, C-peptide, HbA1c) and at Clinical 
Research Center, Skåne University Hospital SUS, Malmö (autoantibodies, lymphocyte 
analyses). Plasma-glucose will be analyzed locally with Hemocue. All analyses are 
standardized according to international recommendations. Blood volume taken will not 
exceed 100 ml per visit.  
 
Urine will be analysed according to glucosuria, ketones, leucocytes and protein. During 
the 5 years follow up blood samples will be analyzed for plasma-glucose, HbA1c, GADA, IA-
2A, IAA and ZnT8A.  
 
 
5.2 
Metabolic status 
HbA1c and plasma-glucose will be measured every third month to be able to diagnose type 1 
diabetes at an early stage. An increase in HbA1c values, even if within normal ranges, have 
been shown to precede clinical onset of type 1 diabetes in islet cell autoantibody-positive 
subjects 42. Fasting and stimulated C-peptide levels will be measured biannually. An OGTT 
and IvGTT will be performed at baseline. During the 5 years follow-up, IvGTT will annually 


 
 
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be performed with analyses of insulin and C-peptide and first-phase insulin response (FPIR) 
and K-value. In addition an OGTT will be performed at visit 4 and every 6th month after 
baseline and thereafter annually in between the IvGTT. If a participant, who previously had a 
normal glucose metabolism develop impaired glucose metabolism, defined as any of a) F-
glucose ≥ 6.1 mmol/L, b) maximum p-glucose 30, 60, 90 minutes ≥ 11.1 mmol/L in the 
OGTT, c) 120 min p-glucose ≥ 7.8 mmol/L on OGTT, d) HbA1c ≥ 39, a confirmatory OGTT 
will be performed within a month. Metabolic status will be evaluated from the baseline OGTT 
and IvGTT. 
 
5.3 
Safety assessments 
The following parameters will be assessed during the study: 
1. Laboratory analyses (chemistry, hematology, and urine analyses). 
2. GADA titers, isotype, epitope and affinity, anti-idiotypic antibodies.  
3. Clinical neurological examination*. 
4. Vital signs and physical examination. 
5. Occurrence of adverse events/serious adverse events with specific questions about 
treatment specific reactions, such as fever, muscle pain, discomfort, headache. 
6. Injection site discomfort or local reaction. 
7. Participant diary. 
 
The results of these measures will be evaluated for clinically significant values. 
 
*The patients will undergo a standardized clinical neurological examination. The neurological 
tests are performed in order to detect possible mild signs of neuromuscular disease such as 
disturbance of strength, balance, and coordination. The tests and the results may be modified 
and adapted to age in children younger than 6 years. The overall neurological status will also 
be assessed, since some children may refuse to perform all parts of the described neurological 
status and other kind of neurological assessment will be made during the examination of those 
children. 
 
The neurological examination includes:  
 
• Extremity reflexes 
• Romberg (balance and coordination) 
• Walk on a line, 2 meters (balance and coordination) 
• Jumping on 1 leg 10 times, left and right (balance and coordination) 
• Finger-nose (coordination) 
• Mimic (cranial nerves) 
• Babinski reflex (central function) 
• Muscle strength (shake hands) biceps, triceps, distal extensors, and flexors 
• Overall neurological status 


 
 
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These examinations may also be repeated between scheduled visits at the discretion of the 
investigator. Screening for neurological disease with electroencephalogram (EEG) is not 
included due to low sensitivity and specificity. However, if any signs of neurological 
dysfunction are detected, the patient should be referred to a paediatric neurologist for further 
evaluation. 
 
 
6 SUBJECT MANAGEMENT 
 
6.1 
Screening visit and eligibility assessment 
Children participating in DiPiS, TEDDY, TEDDY relatives or TrialNet and who have more 
than one positive islet cell autoantibody are followed at one of our sites in Malmö, 
Helsingborg or Kristianstad every 3rd month.  
  
The children who are eligible to participate in DIAPREV-IT 2 and their parents will be 
informed about the study by the study personnel. Both oral and written information about the 
study will be given. If the children and parents agree to participate, a written consent will be 
signed.  
 
Blood samples for islet cell autoantibodies, HbA1c and plasma-glucose are taken at the 
visit, all according to the study protocols. HLA-genotypes of all children in DiPiS and 
TEDDY were determined in their cord blood samples, while children followed in TrialNet are 
tested for HLA to rule out the protective HLA-DQ6. Children not tested for HLA in studies 
will be tested at screening for DiAPREV-IT 2.  
 
6.2 
Diamyd® 
The Diamyd Drug Product is composed of the recombinant human GAD65 (rhGAD65) 
protein formulated in a sterile, non-pyrogenic phosphate buffered saline containing the 
aluminum hydroxide adjuvant, Alhydrogel®. The active ingredient, rhGAD65, is 
manufactured via a process involving expression in an insect cell line. The highly purified and 
unmodified form of rhGAD65 is currently indistinguishable from the native human protein. 
To maximize immunogenicity and bias towards a humoral rather than a cellular immune 
response, rhGAD65 is formulated with the adjuvant alum (Alhydrogel®). All study drugs will 
be kept between 2 and 8º C in a refrigerator, with limited access. The study nurses will 
administer the study medicine. After administration the used vial should be placed back into 
the vial box and kept between 2 and 8◦C in a refrigerator, with limited access until the site is 
instructed to return the study medication. Diamyd Medical AB, Stockholm, Sweden will 
supply Diamyd® for the present investigation (Supplier). 
 
The Study Product is supplied in a 3-mL glass vial with either 20 µg Diamyd alum-
formulated vaccine/mL or placebo. The dose volume is 0.5 mL each for subcutaneous 
administration. The vials have a slight overage (up to 0.8 mL) to allow withdrawal of 0.5 mL. 


 
 
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The study medication will be available in individually treatment numbered packages. The 
treatment numbered packages will be a box containing 1 or 2 glass vials with medication for 
one or two injections (as suggested by the packaging company), which corresponds to the 
study randomization list. Since each patient will receive 2 injections, each treatment number 
on the study randomization list will be used on 2 vials.  
 
Each treatment numbered box will be identified by an injection number (either 1 or 2 
for the injections in the original protocol and 3 or 4 for the injections in the post diagnosis 
protocol). 
 
The Supplier will ship the study medication to a central distributor, who will ship the 
medication to study sites according to local laws and regulations. The procedures for 
shipment, ordering, and storage will be outlined in a Study Drug Accountability Document. 
Both the distributor and the investigator must keep record of all drugs received, used and 
returned. Both distributor and study sites are obliged to properly measure and record the 
storage temperature. 
 
When the study is completed all unused investigational products must be returned to the 
central facility and subsequently to the study drug Supplier unless the Supplier has approved 
other arrangements. Any destruction of investigational products must be performed in 
accordance with the documented approved procedure from the Supplier. 
 
7 STUDY MEDICATION 
 
The following medication supplies will be used in the study: 
 
 
A. 
Study medication:  
GAD-Alum (Diamyd) subcutaneous injection or placebo  
Dosage and interval: One injection of 20 µg Diamyd or placebo 
will be administered 1 month apart  
 
IMP supplier: 
 
      Diamyd Medical AB, Stockholm, Sweden. 
 
B. 
Drug 
Vitamin D (Calciferol) in oral solution or eqvivalent 
Dosage and interval: 2000 IE daily for 5 years 
 
Drug supplier 
Commercially available 
 
 


 
 
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8 ADVERSE EVENTS REPORTING AND SAFETY 
MONITORING 
 
 
8.1 
Definition of adverse event 
In this clinical trial, an adverse event (AE) is any unfavorable and unintended clinical sign or 
symptom, any illness or disease, which develops or worsens in intensity during the course of 
the trial. It also includes an abnormal laboratory finding, if i.e., the abnormality results in trial 
withdrawal, is serious, is associated with clinical signs or symptoms, or is considered being of 
clinical relevance. 
It could also include accidents and reasons for changes in medication (drug and/or 
dose), any medical/nursing/pharmacy consultation and admission to hospital/surgical 
operations. 
Note that hospital admission and/or surgical operations for illness, which existed before 
the study drug was given or the subject was enrolled in the clinical trial and did not worsen 
during the study, are not AEs. As type 1 diabetes is the study outcome, this will not be 
reported as an AE. Neither will mild injection site reactions.  
 
8.2 
Definition of serious adverse event 
A serious adverse event (SAE) is defined as an AE that: 
1. results in death 
2. is immediately life-threatening  
3. requires in-patient hospitalization or requires prolongation of existing hospitalization 
4. results in persistent or significant disability/incapacity 
5. is a congenital anomaly/birth defect 
6. is an important medical event 
 
Important medical events are those, which may not be immediately life-threatening or result 
in death or hospitalization, but may jeopardize the individual or may require intervention to 
prevent one of the other outcomes listed above.  
 
8.3 
Adverse events reporting and monitoring 
All participants are carefully monitored for occurrence of any AE. At each clinical visit after 
the inclusion in the study AE will be collected by the investigators via non-leading questions 
as ‘Have you had any new or worsening health problems since the previous visit/last contact?’ 
as well as reporting events directly observed or spontaneously volunteered by the participant. 
The investigator will also ask the participants direct questions about treatment specific 
reactions, such as such as fever, muscle pain, discomfort and headache. In addition, any local 
reactions from the injection site will be asked for as well as recorded in the participant diary. 


 
 
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Early AEs after the vaccination will be documented in the participant diary. The family will 
also be encouraged to contact the responsible paediatrician if an AE, suspected to be related to 
the vaccine, occur between day 1 and 30. Any abnormal findings in physical examination, 
vital signs, and clinical laboratory tests must also be reported as AE. All reported AE are to be 
recorded in the CRF. 
 
The participants will receive adequate therapy for concomitant diseases at the discretion 
of the investigator. Any concomitant medication must be recorded in the CRF. Note: if a new 
medication is introduced or an existing medication is changed due to a new medical condition 
or worsening of a pre-existing medical condition, the condition must be reported as an AE.   
 
Symptoms and signs of development of type 1 diabetes (increased fasting glucose or 
postprandial glucose, urine-glucose, hypoglycemia in between meals, increased thirst and 
urination in combination with high plasma-glucose, weight loss in combination with high 
plasma-glucose) will be recorded separately. 
 
Following parameters of the AE should be recorded: 
Description, date of onset and date of resolution, intensity (mild, moderate, severe), action 
taken regarding the study drug, treatment of event, causality (unlikely, possibly, probably), 
outcome and sequel, seriousness (death, life-threatening, inpatient hospitalization or 
prolongation of hospitalization, result in persistent disability, congenital /birth defect, 
important medical event). 
Intensity: 
Mild: awareness of signs or symptoms, but easily tolerated (acceptable) 
Moderate: discomfort to interfere with usual activity (disturbing) 
Severe: incapacity to work or to do usual activity (unacceptable) 
Causality: 
 
Relationship to study medication will be assessed for the two treatments (GAD-Alum, and 
Vitamin D) separately. 
 
Unlikely: Time relationship non-existent or doubtful and/or other factor(s) certain or probable 
to have been causative. 
Possibly: Time relationship exists. Other possible causative factor(s) may exist (e.g., 
concurrent disease or concomitant medication). Improvement on dechallenges or dose 
reduction may or may not have been seen.   
Probably: Time relationship exists. No other possible causative factor(s) may exist (not 
reasonably explained by the patient’s known clinical state or concomitant medication). 
Improvement on dechallenges or dose reduction (if performed) has occurred.  Recurrence of 
symptoms on rechallenge (if performed) has occurred. A specific laboratory investigation (if 
performed) has confirmed the relationship. 
 


 
 
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8.4 
Timelines for reporting SAE 
The investigator should report an SAE within 24 hours of the investigator becoming aware of 
the SAE. All SAE must be reported to the DSMB and to Diamyd Medical AB, whether or not 
considered attributable to the study drugs. 
 
Reporting of Suspected Unexpected Serious Adverse Reactions (SUSARs) will be 
collected and reported to the competent authorities and relevant ethics committees in 
accordance with Directive 2001/20/EC or as per national regulatory requirements in 
participating countries. 
 
All AEs/SAEs must be followed until final outcome is known or the condition is stable. 
 
8.5 
Unresolved Events 
If an AE/SAE is present when the patient has completed the study the course of the event 
must be followed until final outcome is known or the condition is stable. 
 
8.6 
Pregnancy: 
Pregnant and lactating women will not be included in the study. Menarchal females must have 
a negative urine pregnancy test prior to randomization and a negative urine pregnancy test at 
each study visit with study drug administration, prior to injection of study drug. Patients will 
be required to use an adequate form of birth control during the study as specified in exclusion 
criterion #14 above (section 4.2). At Visit 1 and 2 the need for birth control will be re-
assessed.  Patients and their partners will be strongly advised to avoid pregnancy for 1 year 
following the last dose of Diamyd and instructed to use adequate birth control. 
 
A pregnancy occurring during the trial must be recorded on the Pregnancy Report Form 
and no further doses of Diamyd will be given. If the pregnancy is verified prior to any of the 
injections, no further injection shall be given. Any pregnancy must be followed until delivery 
or to the end of pregnancy. 
 
 
9 DATA HANDLING 
 
9.1 
CRF 
The online CRF will be completed at all visits by the local responsible pediatrician. The 
original CRF will be kept locally and under lock and key (printed, signed versions). Only 
study personnel will have access to the CRF. The online CRF directly transfer data to the 
database. Personal code number of the participants will be available in the study database 
(BC/GENE), where all parameters from the visits and CRF will be inserted. All laboratory 
parameters will also be kept locally and in the database. Login and Password will be limited 
to the principal investigator (Helena Elding Larsson), those personally working with the study 
and the DSMB. 
 


 
 
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9.2 
Blood samples 
All blood samples will be kept in -80 freezers at CRC, Malmö, Lund University, Sweden. The 
samples will be registered in the Region Skåne Biobank 136 in the Biobank Department 
BD31 under sample collection DIAPREV-IT 2. The samples will be analyzed regularly for 
islet cell autoantibodies and chemical analyses.  
 
 
10 STATISTICAL ANALYSES 
Children screened in DiPiS, TEDDY, TEDDY relatives or Trial Net will be invited from 4 
years of age if repeatedly positive for GAD65Ab and at least one additional type 1 diabetes-
associated autoantibody. About 10 % of the autoantibody positive children in DiPiS are 
expected to decline to participate. We expect that 50 % of children with more than one 
positive islet cell autoantibody will develop type 1 diabetes within 5 years.  
 
10.1 Randomization of participants 
Eligible participants will be randomized to active treatment or placebo, with 50 % of the 
participants in each of the two treatment arms. The distributor of Diamyd® (Penn 
Pharmaceuticals Ltd) will do the randomization for the study. Treatment will be stratified at 
randomization for the following groups:  
 
A) Impaired glucose metabolism defined as low first phase insulin response on 
IvGTT (≤30) and/or an increased 30, 60, 90 min glucose values (≥ 11.1 
mmol/L) and/or 120 minutes glucose value (≥7.8 mmol/L) on OGTT, and/or 
HbA1c ≥ 39  and/or fasting glucose ≥6.1 mmol/L  
B) Normal glucose metabolism  
 
An independent statistician will perform the randomization procedures. The randomization 
code for each child will be preserved in envelopes in a locked safe. The treating physician 
might break the code in cases of emergency. Further the DSMB will have access to a 
randomization list. 
 
10.2 Statistical considerations and analytic plan 
Analysis of study data will be conducted to address both safety and efficacy of the treatment. 
A Statistical Analytic Plan (SAP) will be developed before the statistical analyses. 
 
The primary objective is to evaluate if Diamyd may delay or stop the autoimmune 
process leading to clinical type 1 diabetes in children with ongoing and persistent beta cell 
autoimmunity as indicated by multiple positive islet cell autoantibodies. The secondary 
objective is to demonstrate that Diamyd is safe in children at risk for type 1 diabetes. The 
subjects will be followed for 5 years. The study will be kept blinded for parents and 
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Data from the 80 children in DiAPREV-IT 2 will be analysed when the full cohort have 
been followed for 5 years.  
 
The analysis of efficacy will be carried out five (5) years after the first injection. The 
primary endpoint is the proportion of children developing clinical typ 1 diabetes during the 
five year follow-up in the two treatment groups.  
 
Children will be staged for glucose metabolism at baseline screening. As secondary endpoints 
we will use  
1) progression from normal to impaired glucose metabolism, defined as any of  a) F-
glucose ≥ 6.1 mmol/L, b) maximum p-glucose at 30, 60, 90 minutes ≥ 11.1 mmol/L in 
the OGTT, c) 120 min p-glucose ≥ 7.8 mmol/L on OGTT, d) HbA1c ≥ 39 mmol/mol. 
Impaired glucose metabolism has to be confirmed at a second visit. This endpoint will 
be used in the group of children with normal glucose metabolism at baseline screening 
2) Progression of impaired glucose metabolism from one or several of the above 
variables to additional signs of reduced glucose metabolism, confirmed at a second 
visit. This endpoint will be used in the group of children with impaired glucose 
metabolism at baseline. 
 
The exploratory endpoints are safety as well as the proportion of subjects in the two treatment 
groups who develop type 1 diabetes, according to the criteria below, after 1, 2, 3, 4 and 5 
years as well as time to clinical type 1 diabetes onset from baseline. As exploratory endpoints 
we will use change from basline of 1) first-phase insulin response and K-value on IvGTT 
from baseline, 2) fasting, maximum and 2 hours C-peptide levels on OGTT as well as AUC, 
3) fasting, maximum and 2 hours plasma-glucose and AUC glucose from OGTT and 4) 
HbA1c. Change in these variables will be compared across the two treatment groups after 1, 
2, 3, 4 and 5 years.  
 
The diagnosis of diabetes will follow the criteria developed by the World Health 
Organization (WHO) and the American Diabetes Association (ADA), also recommended by 
the International Society for Pediatric and Adolescent Diabetes (ISPAD): 
1) A casual venous plasma glucose of ≥ 11.1 mmol/L (or capillary plasma glucose ≥ 12.2 
mmol/L) and symptoms of diabetes (polyuria, polydipsia, unexplained weight loss) 
or 
2) A venous fasting glucose of ≥ 7 mmol/L. 
or  
3) A venous plasma glucose of ≥ 11.1 mmol/L (or capillary plasma glucose ≥ 12.2 
mmol/L) 2 hours after glucose load in an oral glucose tolerance test (OGTT . 
 
In the absence of unequivocal hyperglycaemia, the result must be confirmed on a subsequent 
day either by repeated sampling as above or by an OGTT. Since the children in DIAPREV-IT 
2 will be closely followed, we expect to diagnose diabetes at an early stage, when the first 


 
 
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sign of diabetes usually is an increased postprandial plasma-glucose value or 2 hours after 
OGTT (values ≥ 11.1). Therefore, criteria 1 and/or 3, confirmed the subsequent day, will be 
used for diagnosis of diabetes in DIAPREV-IT 2. 
 
Children with positive islet cell autoantibodies who develop type 1 diabetes have been 
reported to have gradually deteriorating glucose tolerance with declining C-peptide levels two 
hours after the glucose load in OGTT over a period of at least 2 years before onset of disease, 
despite the fact that fasting C-peptide levels remained stable 43. Another early sign of 
progression towards type 1 diabetes is a reduced first-phase insulin response at IvGTT 9,43. 
Therefore, analyses of change in C-peptide levels and plasma-glucose values two hours after 
OGTT as well as first-phase insulin response in the annual IvGTT will be compared between 
the treatment groups, as a measure of efficacy.  
Analyses will be performed by using parametric statistics. If criteria of normality of 
variables are not met (e.g. Kolmogorov-Smirnov tests) non-parametric statistics of Wilcoxon 
type will be used. Four-fields tables will be analysed using the Fisher´s exact test. Time to 
diabetes will be analysed using life-table analyses, such as Kaplan-Meier and Cox regression. 
Analyses of efficacy will be performed as well on per-protocol (PP) as on intention-to-treat 
(ITT) basis. In ITT the last observation carried forward will be applied. 
 
10.3 Power calculation 
In this study we will include 80 children, 40 in each of the study arms.  
We expect that 50 % of the untreated children with multiple autoantibodies will develop type 
1 diabetes within 5 years. This frequency has previously been reported equal among relatives 
to diabetes patients and in the general population. If 20 % of the treated children will develop 
type 1 diabetes within the same period of time, we will have a power of 82 % with alfa=5 % 
with a group of 40+40=80 children. P value <0.05 will be used as significance level.  
 
As exploratory values of effect, first phase insulin response, fasting and stimulated C-
peptide levels, plasma-glucose and HbA1c, are used. These variables will be analysed to 
reveal differences of change from baseline between the untreated and treated group of 
children. 
 
10.4 Drop out 
Participating children may drop out from the study due to change in residence. The parents 
can also withdraw the informed consent whenever they want within the study period.  
 
11 MONITORING AND QUALITY CONTROL 
 
11.1 CRF 
All data obtained at the visits will be reported in study specific online CRF’s. The CRF’s will 
be developed by the sponsor prior to the start of the study. All investigators who are 
authorized to fill out the CRF’s will be listed with name and signature in the ‘Investigator 
signature list’ prior to the study start. 


 
 
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The online CRF enables tracking of changes and a full log of persons that have been 
editing data. The results from the CRF are forwarded directly into the database (BC/OS)  
The results from the CRF will be inserted in a database (BC/GENE). The printed and signed 
CRF will be kept locally at a safe place, to enable monitoring and quality control. 
 
11.2 Patient journals 
Patient journals will be established at each of the participant’s visits.  
 
11.3 Monitoring and Quality control 
The monitoring of the study is made to ensure the scientific integrity, the data quality, the 
safety and integrity of the participating subjects and that the study is compliant with the 
current version of the Declaration of Helsinki (Appendix 1 in the protocol).  
The Sponsor will delegate monitoring and quality control of the study to the 
Competence Centre for Clinical Research (RSKC) in Lund. RSKC is a center independent 
and detached from the study and will be responsible for on site monitoring of the study 
before, during and after the study. RSKC will control that the study is performed according to 
the rules of Good Clinical Practice (GCP).  
To assure the accuracy and completeness of the data recorded in the trial, the monitor will 
compare CRFs with medical records and other relevant documentation during the on-site 
monitoring visits. The monitor must therefore be allowed direct access to all source data 
according to ICH GCP to confirm that required protocol procedures are being followed and 
check consistency between patient record and CRF data. Incorrect or missing entries into the 
CRFs will be queried and must be corrected. Study monitoring will not jeopardise patient 
confidentiality.   
 
 
11.4 Quality Assurance  
 
During or after the study are completed, regulatory authorities, Diamyd Medical, assigned 
CRO or other involved party may wish to carry out an audit. These representatives must have 
the same access to study data and patient source data as the monitor. 
 
 
12 REGULATORY AN D ETHICAL ASPECTS 
 
Any regulatory requirements must have been met before starting the study. The Sponsor will 
apply for the regulatory approval to the appropriate authorities. 
 
Study sites, facilities, laboratories and all data (including source data) and 
documentation must be made available for inspection by the authorities. 
 
The principles of informed consent as stated in the current revision of the Declaration of 
Helsinki (Edinburgh 2000) will be implemented in this study. The study will be conducted in 


 
 
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compliance with ICH GCP and local laws and regulations relevant to the use of new 
therapeutic agents.  
All participating children and their parents/guardians are to provide written informed 
consent in accordance with the Declaration of Helsinki and the applicable national laws. 
The participant and parent/guardian will sign and date the Informed Consent Form 
(ICF) before entering the trial, i.e., before the first injection of substance is given. The 
investigator will explain the nature, purpose, and risks of the trial and provide the participant 
and the parents with a copy of the Patient Information Sheet. The participant and the 
parent/guardian will be given sufficient time to consider the trial’s implications before 
deciding whether to participate. The participant and parent/guardian will be provided with a 
copy of the signed consent form. The investigator will document the process of consent. The 
investigator is responsible for obtaining the participant’s and/or guardian’s freely given 
written consent, including date, and thereafter sign and date the consent form by her/him 
before any study related procedure is performed.  
It is the responsibility of the investigator to apply for and obtain written approval from 
the relevant Ethics Committee before the start of the study.  
All participants are covered by the patient insurance in Region Skåne, Sweden. 
 
12.1 Participant confidentiality 
 
The investigator must ensure that the participant’s confidentiality will be maintained. 
CRFs or other documents submitted should only identify patients by their initials and study 
number. The investigator should keep a separate log of patient codes and names. 
The investigator is required to record safety data, concomitant medication and patient 
progress in the patient's file/notes/medical record. 
The patient's medical records will be reviewed by the study monitor to verify adequate 
source documentation, accuracy and completeness of Case Report Forms. The review will be 
conducted with strict adherence to professional standards of confidentiality. 
The investigator must keep a screening log, recording all children who were screened, 
whether they were enrolled or not, and a separate Patient Identification List showing code 
numbers, names, and dates of birth to allow unambiguous identification of each patient 
included in the study. 
 
12.2 Amendments 
If a substantial protocol amendment is necessary, this will be signed and submitted by the 
Sponsor for ethical and regulatory approval. The approval from the Ethics Committee and 
Competent Authority should be obtained before any implementation of the amendment is 
done. When the change or deviation is to eliminate or reduce risk to humans, the amendment 
may be implemented before review of approval by the Ethics Committee and Competent 
Authority. The sponsor should notify the Ethics Committee and Competent Authority of the 
change or deviation in writing within 10 working days after implementation. 


 
 
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Minor amendments which do not affect the safety or conduct of the study from the 
child’s and parents/guardians viewpoint, and which do not significantly reduce the scientific 
value of the protocol, and which do not require a significant change to be made to the consent 
form and/or the information sheet, will not be submitted for formal ethics and regulatory 
review. These will be sent to the Ethics committee and Competent Authority on an 
‘information only’ basis. 
 
Should amendment directly affect the participant’s participation in the trial e.g., a 
change in any procedure, or if new data is obtained during the trial that may influence the 
standpoint to participate in the study, the patient information will be amended to incorporate 
this modification and the participant/parent must agree to sign this amended form verifying 
that they re-consent to continue their participation in the trial. 
 
12.3 End of trial 
The end of the trial is defined as the last visit of the last patient included in the trial and all 
data have been collected.  
 
12.4 Study report 
A clinical study report will be prepared covering clinical and statistical aspects and 
summarizing all findings of the clinical study. The content has to be treated as strictly 
confidential.  
 
12.5 GCP 
The study will be managed and conducted according to the latest international (ICH) 
guidelines for Good Clinical Practice 
 
 
 
 
 
 


 
 
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13 PUBLICATION OF RESULTS 
 
It is intended that the results from the study will be published in referee reviewed scientific 
medical journals.  
 
 
14 DOCUMENTATION, RECORD KEEPING AND ARCHIVING 
 
Every participant will be given a specific study-ID. The information from the visits will be 
recorded in Case Report Form (CRF). The information will thereafter be recorded in the 
database of DIAPREV-IT (BCOS). The study journal will be kept safely at each study site. 
The source documents will be made available to the DSMB and authority auditors upon 
request. 
The investigator will arrange for the retention of the Investigator File for at least 10 
years after the completion or discontinuation of the trial. Patient hospital files and other 
source data must be kept for the maximum period of time permitted by the hospital, institution 
or private practice, but not less than 10 years.  
 
 
 
 


 
 
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Zuccotti GV, Gomez Gila A, Rica I, Barrio R, Clemente M, Lopez Garcia MJ, 
Rodriguez M, Gonzalez I, Lopez JP, Oyarzabal M, Reeser HM, Nuboer R, Stouthart 
P, Bratina N, Bratanic N, de Kerdanet M, Weill J, Ser N, Barat P, Bertrand AM, Carel 


 
 
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JC, Reynaud R, Coutant R, Baron S. GAD65 antigen therapy in recently diagnosed 
type 1 diabetes mellitus. The New England journal of medicine. Feb 2 
2012;366(5):433-442. 
27. 
Wherrett DK, Bundy B, Becker DJ, DiMeglio LA, Gitelman SE, Goland R, Gottlieb 
PA, Greenbaum CJ, Herold KC, Marks JB, Monzavi R, Moran A, Orban T, Palmer JP, 
Raskin P, Rodriguez H, Schatz D, Wilson DM, Krischer JP, Skyler JS, Type 1 
Diabetes TrialNet GADSG. Antigen-based therapy with glutamic acid decarboxylase 
(GAD) vaccine in patients with recent-onset type 1 diabetes: a randomised double-
blind trial. Lancet. Jul 23 2011;378(9788):319-327. 
28. 
Piemonti L, Monti P, Sironi M, Fraticelli P, Leone BE, Dal Cin E, Allavena P, Di 
Carlo V. Vitamin D3 affects differentiation, maturation, and function of human 
monocyte-derived dendritic cells. J Immunol. May 1 2000;164(9):4443-4451. 
29. 
Vitamin D supplement in early childhood and risk for Type I (insulin-dependent) 
diabetes mellitus. The EURODIAB Substudy 2 Study Group. Diabetologia. Jan 
1999;42(1):51-54. 
30. 
Hypponen E, Laara E, Reunanen A, Jarvelin MR, Virtanen SM. Intake of vitamin D 
and risk of type 1 diabetes: a birth-cohort study. Lancet. Nov 3 2001;358(9292):1500-
1503. 
31. 
Zipitis CS, Akobeng AK. Vitamin D supplementation in early childhood and risk of 
type 1 diabetes: a systematic review and meta-analysis. Archives of disease in 
childhood. Jun 2008;93(6):512-517. 
32. 
Baumgartl HJ, Standl E, Schmidt-Gayk H, Kolb HJ, Janka HU, Ziegler AG. Changes 
of vitamin D3 serum concentrations at the onset of immune-mediated type 1 (insulin-
dependent) diabetes mellitus. Diabetes research. Mar 1991;16(3):145-148. 
33. 
Teegarden D, Donkin SS. Vitamin D: emerging new roles in insulin sensitivity. 
Nutrition research reviews. Jun 2009;22(1):82-92. 
34. 
Mathieu C, Gysemans C, Giulietti A, Bouillon R. Vitamin D and diabetes. 
Diabetologia. Jul 2005;48(7):1247-1257. 
35. 
Larsson K, Elding-Larsson H, Cederwall E, Kockum K, Neiderud J, Sjoblad S, 
Lindberg B, Lernmark B, Cilio C, Ivarsson SA, Lernmark Å. Genetic and perinatal 
factors as risk for childhood type 1 diabetes. Diabetes Metab Res Rev. Sep 8 
2004;20:429-437. 
36. 
Lernmark B, Elding-Larsson H, Hansson G, Lindberg B, Lynch K, Sjöblad S. Parent 
responses to participation in genetic screening for diabetes risk. Pediatr Diabetes. Dec 
2004;5(4):174-181. 
37. 
Larsson HE, Lynch K, Lernmark B, Nilsson A, Hansson G, Almgren P, Lernmark Å, 
Ivarsson SA. Diabetes-associated HLA genotypes affect birthweight in the general 
population. Diabetologia. Aug 2005;48(8):1484-1491. 
38. 
Steffes MW, Sibley S, Jackson M, Thomas W. Beta-cell function and the development 
of diabetes-related complications in the diabetes control and complications trial. 
Diabetes care. Mar 2003;26(3):832-836. 
39. 
Maalouf J, Nabulsi M, Vieth R, Kimball S, El-Rassi R, Mahfoud Z, El-Hajj Fuleihan 
G. Short- and long-term safety of weekly high-dose vitamin D3 supplementation in 
school children. The Journal of clinical endocrinology and metabolism. Jul 
2008;93(7):2693-2701. 
40. 
Stallings VA, Schall JI, Hediger ML, Zemel BS, Tuluc F, Dougherty KA, Samuel JL, 
Rutstein RM. High-Dose Vitamin D3 Supplementation in Children and Young Adults 
with HIV: A Randomized, Placebo-Controlled Trial. The Pediatric infectious disease 
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41. 
Andersson C, Carlsson A, Cilio C, Cedervall E, Ivarsson SA, Jonsdottir B, Jonsson B, 
Larsson K, Neiderud J, Lernmark A, Elding Larsson H, Di A-ITSG. Glucose tolerance 
and beta-cell function in islet autoantibody-positive children recruited to a secondary 
prevention study. Pediatric diabetes. Aug 2013;14(5):341-349. 
42. 
Stene LC, Barriga K, Hoffman M, Kean J, Klingensmith G, Norris JM, Erlich HA, 
Eisenbarth GS, Rewers M. Normal but increasing hemoglobin A1c levels predict 
progression from islet autoimmunity to overt type 1 diabetes: Diabetes Autoimmunity 
Study in the Young (DAISY). Pediatr Diabetes. Oct 2006;7(5):247-253. 
43. 
Sosenko JM, Palmer JP, Greenbaum CJ, Mahon J, Cowie C, Krischer JP, Chase HP, 
White NH, Buckingham B, Herold KC, Cuthbertson D, Skyler JS. Patterns of 
metabolic progression to type 1 diabetes in the Diabetes Prevention Trial-Type 1. 
Diabetes care. Mar 2006;29(3):643-649. 
 
 


 
 
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15 INVESTIGATOR SIGNATURE 
 
Investigator’s Statement: 
 
I have read and understand the foregoing protocol with the title:  
 
“A double-blind, randomized investigator-initiated study to determine the safety and the 
effect of Diamyd® in combination with Vitamin D on the progression to type 1 diabetes in 
children with multiple islet cell autoantibodies.” 
 
Trial DIAPREV/2014 and agree to conduct the trial, in compliance with ICH notes on Good 
Clinical Practice (CPMP/ICH/135/95), designated Standard Operating Procedures, National 
Laws and regulations and within the principles of the current revision of Declaration of 
Helsinki (Edinburgh 2000). 
 
Coordinating Investigator’s Name:   
Helena Elding Larsson 
 
Coordinating Investigator’s Title:   
MD, PhD, Docent (Associate Professor) 
 
Coordinating Investigator’s Signature: 
 
 
…………..………………………………………… 
 
Date:  ………………………….. 
 


 
 
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APPENDIX 1, DECLARATION OF HELSINKI  
 
Ethical Principles for Medical Research Involving Human Subjects 
 
A. INTRODUCTION 
 
1. The World Medical Association has developed the Declaration of Helsinki as a 
statement of ethical principles to provide guidance to physicians and other 
participants in medical research involving human subjects.  Medical research 
involving human subjects includes research on identifiable human material or 
identifiable data. 
 
2. It is the duty of the physician to promote and safeguard the health of the 
people.  The physician's knowledge and conscience are dedicated to the 
fulfillment of this duty. 
 
3. The Declaration of Geneva of the World Medical Association binds the 
physician with the words, "The health of my patient will be my first 
consideration," and the International Code of Medical Ethics declares that, "A 
physician shall act only in the patient's interest when providing medical care 
which might have the effect of weakening the physical and mental condition 
of the patient." 
 
4. Medical progress is based on research which ultimately must rest in part on 
experimentation involving human subjects. 
 
5. In medical research on human subjects, considerations related to the well-
being of the human subject should take precedence over the interests of 
science and society. 
 
6. The primary purpose of medical research involving human subjects is to 
improve prophylactic, diagnostic and therapeutic procedures and the 
understanding of the aetiology and pathogenesis of disease.  Even the best 
proven prophylactic, diagnostic, and therapeutic methods must continuously 
be challenged through research for their effectiveness, efficiency, accessibility 
and quality.   
 
7. In current medical practice and in medical research, most prophylactic, 
diagnostic and therapeutic procedures involve risks and burdens. 
 


 
 
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8. Medical research is subject to ethical standards that promote respect for all 
human beings and protect their health and rights.  Some research populations 
are vulnerable and need special protection.  The particular needs of the 
economically and medically disadvantaged must be recognized.  Special 
attention is also required for those who cannot give or refuse consent for 
themselves, for those who may be subject to giving consent under duress, for 
those who will not benefit personally from the research and for those for 
whom the research is combined with care. 
 
9. Research Investigators should be aware of the ethical, legal and regulatory 
requirements for research on human subjects in their own countries as well 
as applicable international requirements.  No national ethical, legal or 
regulatory requirement should be allowed to reduce or eliminate any of the 
protections for human subjects set forth in this Declaration. 
 
B. BASIC PRINCIPLES FOR ALL MEDICAL RESEARCH 
 
10. It is the duty of the physician in medical research to protect the life, health, 
privacy, and dignity of the human subject. 
 
11. Medical research involving human subjects must conform to generally 
accepted scientific principles, be based on a thorough knowledge of the 
scientific literature, other relevant sources of information, and on adequate 
laboratory and, where appropriate, animal experimentation. 
 
12. Appropriate caution must be exercised in the conduct of research, which may 
affect the environment, and the welfare of animals used for research must be 
respected. 
 
13. The design and performance of each experimental procedure involving 
human subjects should be clearly formulated in an experimental protocol.  
This protocol should be submitted for consideration, comment, guidance, and 
where appropriate, approval to a specially appointed ethical review 
committee, which must be independent of the investigator, the sponsor or any 
other kind of undue influence. This independent committee should be in 
conformity with the laws and regulations of the country in which the research 
experiment is performed.  The committee has the right to monitor ongoing 
trials. The researcher has the obligation to provide monitoring information to 
the committee, especially any serious adverse events. The researcher should 
also submit to the committee, for review, information regarding funding, 


 
 
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sponsors, institutional affiliations, other potential conflicts of interest and 
incentives for subjects. 
 
14. The research protocol should always contain a statement of the ethical 
considerations involved and should indicate that there is compliance with the 
principles enunciated in this Declaration. 
 
15. Medical research involving human subjects should be conducted only by 
scientifically qualified persons and under the supervision of a clinically 
competent medical person.  The responsibility for the human subject must 
always rest with a medically qualified person and never rest on the subject of 
the research, even though the subject has given consent. 
 
16. Every medical research project involving human subjects should be preceded 
by careful assessment of predictable risks and burdens in comparison with 
foreseeable benefits to the subject or to others.  This does not preclude the 
participation of healthy volunteers in medical research.  The design of all 
studies should be publicly available. 
 
17. Physicians should abstain from engaging in research projects involving 
human subjects unless they are confident that the risks involved have been 
adequately assessed and can be satisfactorily managed. Physicians should 
cease any investigation if the risks are found to outweigh the potential 
benefits or if there is conclusive proof of positive and beneficial results. 
 
18. Medical research involving human subjects should only be conducted if the 
importance of the objective outweighs the inherent risks and burdens to the 
subject.  This is especially important when the human subjects are healthy 
volunteers. 
 
19. Medical research is only justified if there is a reasonable likelihood that the 
populations in which the research is carried out stand to benefit from the 
results of the research. 
 
20. The subjects must be volunteers and informed participants in the research 
project. 
 
21. The right of research subjects to safeguard their integrity must always be 
respected.  Every precaution should be taken to respect the privacy of the 
subject, the confidentiality of the patient's information and to minimize the 


 
 
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impact of the study on the subject's physical and mental integrity and on the 
personality of the subject. 
 
22. In any research on human beings, each potential subject must be adequately 
informed of the aims, methods, sources of funding, any possible conflicts of 
interest, institutional affiliations of the researcher, the anticipated benefits 
and potential risks of the study and the discomfort it may entail.  The subject 
should be informed of the right to abstain from participation in the study or to 
withdraw consent to participate at any time without reprisal. After ensuring 
that the subject has understood the information, the physician should then 
obtain the subject's freely-given informed consent, preferably in writing. If 
the consent cannot be obtained in writing, the non-written consent must be 
formally documented and witnessed. 
 
23. When obtaining informed consent for the research project the physician 
should be particularly cautious if the subject is in a dependent relationship 
with the physician or may consent under duress.  In that case the informed 
consent should be obtained by a well-informed physician who is not engaged 
in the investigation and who is completely independent of this relationship. 
 
24. For a research subject who is legally incompetent, physically or mentally 
incapable of giving consent or is a legally incompetent minor, the investigator 
must obtain informed consent from the legally authorized representative in 
accordance with applicable law.  These groups should not be included in 
research unless the research is necessary to promote the health of the 
population represented and this research cannot instead be performed on 
legally competent persons. 
 
25. When a subject deemed legally incompetent, such as a minor child, is able to 
give assent to decisions about participation in research, the investigator must 
obtain that assent in addition to the consent of the legally authorized 
representative. 
 
26. Research on individuals from whom it is not possible to obtain consent, 
including proxy or advance consent, should be done only if the 
physical/mental condition that prevents obtaining informed consent is a 
necessary characteristic of the research population.  The specific reasons for 
involving research subjects with a condition that renders them unable to give 
informed consent should be stated in the experimental protocol for 
consideration and approval of the review committee.  The protocol should 


 
 
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state that consent to remain in the research should be obtained as soon as 
possible from the individual or a legally authorized surrogate. 
 
27. Both authors and publishers have ethical obligations.  In publication of the 
results of research, the investigators are obliged to preserve the accuracy of 
the results.  Negative as well as positive results should be published or 
otherwise publicly available.  Sources of funding, institutional affiliations and 
any possible conflicts of interest should be declared in the publication.  
Reports of experimentation not in accordance with the principles laid down in 
this Declaration should not be accepted for publication. 
 
C. ADDITIONAL PRINCIPLES FOR MEDICAL RESEARCH COMBINED WITH 
MEDICAL CARE 
 
28. The physician may combine medical research with medical care, only to the 
extent that the research is justified by its potential prophylactic, diagnostic or 
therapeutic value.  When medical research is combined with medical care, 
additional standards apply to protect the patients who are research subjects. 
 
29. The benefits, risks, burdens and effectiveness of a new method should be 
tested against those of the best current prophylactic, diagnostic, and 
therapeutic methods.  This does not exclude the use of placebo, or no 
treatment, in studies where no proven prophylactic, diagnostic or therapeutic 
method exists.  (Note 1) 
 
 
30. At the conclusion of the study, every patient entered into the study should be 
assured of access to the best proven prophylactic, diagnostic and therapeutic 
methods identified by the study. (Note 2) 
 
31. The physician should fully inform the patient which aspects of the care are 
related to the research. The refusal of a patient to participate in a study must 
never interfere with the patient-physician relationship. 
 
32. In the treatment of a patient, where proven prophylactic, diagnostic and 
therapeutic methods do not exist or have been ineffective, the physician, with 
informed consent from the patient, must be free to use unproven or new 
prophylactic, diagnostic and therapeutic measures, if in the physician's 
judgment it offers hope of saving life, re-establishing health or alleviating 
suffering.  Where possible, these measures should be made the object of 
research, designed to evaluate their safety and efficacy.  In all cases, new 


 
 
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information should be recorded and, where appropriate, published.  The 
other relevant guidelines of this Declaration should be followed. 
 
Note 1 (Paragraph 29) 
 
The WMA hereby reaffirms its position that extreme care must be taken in 
making use of a placebo-controlled trial and that in general this methodology 
should only be used in the absence of existing proven therapy. However, a 
placebo-controlled trial may be ethically acceptable, even if proven therapy is 
available, under the following circumstances: 
 
- Where for compelling and scientifically sound methodological reasons its use is 
necessary to determine the efficacy or safety of a prophylactic, diagnostic or 
therapeutic method; or 
 
- Where a prophylactic, diagnostic or therapeutic method is being investigated 
for a minor condition and the patients who receive placebo will not be subject to 
any additional risk of serious or irreversible harm. 
 
All other provisions of the Declaration of Helsinki must be adhered to, especially 
the need for appropriate ethical and scientific review. 
 
Note 2 (Paragraph 30) 
 
The WMA hereby reaffirms its position that it is necessary during the study planning 
process to identify post-trial access by study participants to prophylactic, diagnostic 
and therapeutic procedures identified as beneficial in the study or access to other 
appropriate care. Post-trial access arrangements or other care must be described in the 
study protocol so the ethical review committee may consider such arrangements 
during its review. 


 
 
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APPENDIX 2: POST DIAGNOSIS INTERVENTION  
 
POST DIAGNOSIS INTERVENTION AND FOLLOW-UP 
Post-diagnosis intervention and follow-up of children participating in DiAPREV-IT 2, who 
develop type 1 diabetes during the study period. 
 
Background: 
Pending study drug shelf life, children developing type 1-diabetes within the study period 
may be offered to continue in the trial in a post-diagnosis intervention protocol (PDIP) where 
all participants diagnosed with type 1 diabetes will receive additional injections in the 
following manner: 
 
Within 4 months since diabetes diagnosis, participants will receive one injection of Diamyd® 
20 µg on Day 1 in the Post Diagnosis Follow-up, followed by a second injection of Diamyd® 
on Day 30, in a prime and boost fashion (a total received dose of 40 µg Diamyd® for 
participants who pre-diagnosis was randomized to receive placebo and a total received dose of 
80 µg Diamyd® for participants who pre-diagnosis was randomized to receive Diamyd®).  
 
 
 
All children that are included in the post diagnosis follow-up will be discontinued from the 
original prevention protocol to be followed thoroughly for safety and efficacy according to the 
post-diagnosis intervention protocol for 15 months following the first injection of Diamyd® in 
the PDIP.  
 
Post-diagnosis intervention protocol: 
At the time of clinical onset of type 1 diabetes, treatment with Diamyd® will be offered. The 
first injection will be administered within 4 months after diabetes diagnosis. The child will be 
followed according to the “Post-diagnosis intervention protocol” below. 
 
Post-diagnosis intervention protocol: 
 
Post-diagnosis visit P1 (within four months of diagnosis):  
Fasting C-peptide and p-glucose 
Insulin regimen and HbA1c 
Mixed meal tolerance test 
Hematology 
Chemistry 
Islet autoantibody analyses (GADA, IA-2A, IAA, ZnT8A) 
T-cell analyses 
Physical exam 


 
 
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Injection one of Diamyd® 
Adverse event report 
Diary 
Post-diagnosis visit P2 (30 days ± 7 days from post-diagnosis visit 1) 
Hematology 
Insulin regimen and HbA1c 
Chemistry 
Physical exam including neurological assessment 
Injection of Diamyd® or placebo pending assigned treatment group in DIAPREV-IT 
Adverse event report 
Diary 
Post-diagnosis visit P3 (3 months ±14 days from post-diagnosis visit 1)  
Fasting C-peptide and p-glucose 
Mixed meal tolerance test 
Hematology 
Insulin regimen and HbA1c 
Chemistry 
T-cell analyses 
Physical exam, including neurological assessment 
Adverse event report 
Diary 
Post-diagnosis visit P4 (9 months ±14 days from post-diagnosis visit 1) 
Fasting C-peptide, insulin and p-glucose 
Mixed meal tolerance test 
Hematology 
Insulin regimen and HbA1c 
Chemistry 
T-cell analyses 
Physical exam, including neurological assessment 
Adverse event report 
Diary 
Post-diagnosis visit P5 (15 months’ ±14 days from post-diagnosis visit 1) 
Fasting C-peptide and p-glucose 
Mixed meal tolerance test 
Hematology 
Insulin regimen and HbA1c 
Chemistry 
T-cell analyses 
Physical exam, including neurological assessment 
Adverse event report 
Diary 


 
 
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Visit P1 
Day 1 
Within 4 
month of 
diagnosis  
Visit P2 
Month 1 
(Day 30) 
30 +/-7 days 
from visit A1 
 
Visit P3 
Month 3 
3 months 
+/- 14 7 days 
from visit P1 
Visit P4 
Month 9 
9 months  
+/- 14 days  
from visit P1 
Visit P5 
Month 15 
15 months  
+/- 14 days  
from visit 
P1 
Study Drug 
Administration 
X 
X 
 
 
 
Medical History 
X 
X 
X 
X 
X 
General Physical Exam 
X 
X 
X 
X 
X 
Concomitant Medication 
X 
X 
X 
X 
X 
Weight, Height 
X 
X 
X 
X 
X 
Vital signs (BP) 
X 
X 
X 
X 
X 
Blood Sampling:  
 
 
 
 
 
 Hematology,  Chemistry 
X 
X 
X. 
X 
X 
Cellular analyses 
X 
X 
X 
X 
X 
GADAb, IA-2Ab, ZnT8Ab, 
IAA 
X 
X 
X 
X 
X 
 HbA1c, insulin regimen 
X 
X 
X 
X 
X 
 Plasma Glucose 
X 
 
X 
X 
X 
 MMTT (Mixed Meal 
Tolerance Test) 
 including C-peptide  
 p-glucose 
X 
 
 
X 
X 
X 
Neurological Assessment 
X 
X 
X 
X 
X 
Adverse Events  
X 
X 
X 
X 
X 
Diary 
X 
X 
X 
X 
 
Questionnaire to child 
about the study 
procedures 
 
 
 
 
At the final 
visit 


 
 
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APPENDIX 3  
PROTOCOL ADDENDUM 2017-08-25 
 
As described in the original protocol, the aim of DiAPREV-IT 2 is to test if immune tolerance 
with two doses of Diamyd® prevent or delay type 1 diabetes in children with multiple islet 
autoantibodies. 
The primary objective is to evaluate if Diamyd®, in children treated with relatively high dose 
vitamin D, may delay or stop the autoimmune process leading to clinical type 1 diabetes in 
children with ongoing persistent beta cell autoimmunity as indicated by multiple positive islet 
cell autoantibodies.  
The secondary objective is to demonstrate that Diamyd® is safe in children at risk for type 1 
diabetes.  
Primary and secondary endpoints are described in detail in the protocol.  
 
During 2017 we unblinded and analyzed data from the first study of Diamyd® in children with 
multiple islet autoantibodies Diaprev/2008, EuCT 2008-007484-16. In this group of 50 
children we did not find any effect on delaying or preventing type 1 diabetes, while GADA 
increased. This has been reported, through a written report, to the MPA during June 2017.  
 
Since the cohort of children, inclusion and exclusion criteria and dose of Diamyd® are all 
equal in the current study, with the exception that all children (unrelated to study arm) are 
treated with Vitamin D in the current protocol, the rational for the current study has changed 
on basis of the findings in the Diaprev/2008 study. We do not find it probable that the current 
study will delay or prevent type 1 diabetes, but it may have effects on the immune system that 
can be explored in mechanistic studies.  
 
Therefore, from 2017-08-25, we propose the following addendum to the protocol: 
 
• Inclusion is stopped – a total of 26 children have up to date been included and given 
study drug. 
• Included children are followed up to two years (24 months) after the first dose of 
Diamyd®, according to revised protocol below. 
• Children developing diabetes are not offered Diamyd® after diagnosis. 
• Children already participating in the post diagnosis protocol are followed for 9 months 
according to revised post diagnosis protocol below. 
• Main focus on analyses after the closure of the study will be secondary variables and 
mechanistic studies. 
• All included study subjects will be informed and reconsent to the change in follow-up, 
if currently still followed in the study.  
 
 


 
 
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Schedule for the remaining two year follow-up: 
 
 
 
Event 
Visit 0 
Information 
and consent 
 
 
 
Visit 1 
Day 1 
 
30 +/- 7  days 
from  visit 0  
Visit 2 
Month 1 
 
30 +/-7 days 
from visit 1 
 
Visit 3 
Month 3 
 
3 months 
+/- 20 days 
from visit 1 
Visit 4 
Month 6 
 
6 months 
+/-20 days 
from visit 1 
Visit 5 
Month 9 
 
9 months  
+/- 20 days  
from visit 1 
Visit 6 
Month 12 
 
12 months  
+/- 20 days  
from visit 1 
2 Years Follow up 
Every 3rd  or 6th 
month 
Every visit  
+/- 20 days 
from visit 1 
Informed Consent 
X 
 
 
 
 
 
 
 
Randomization 
 
X 
 
 
 
 
 
 
Diamyd/Placebo 
Administrationa 
 
X 
X 
 
 
 
 
 
Start of Vitamin D 
X 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
General Physical Exam 
X 
X 
X 
X 
X 
 
X 
Last visit 
Concomitant Medication 
X 
X 
X 
X 
X 
 
X 
 
Weight, Height 
X 
X 
X 
X 
X 
 
X 
Every 6th  month 
Vital signs (BP) 
X 
X 
X 
X 
X 
 
X 
Every 6th  month 
Blood Sampling:  
 
 
 
 
 
 
 
Every 6th  month 
 Hematology, chemistry, 
including calcium 
X 
X 
X 
X 
X 
X 
X 
Every 3rd   month 
25 OH-vitamin D3 
X 
X 
 
 
X 
 
X 
Every 6th month 
Cellular analyses 
X 
 
X 
 
X 
 
X 
Every 6th  month 
GADA, IA-2A, ZnT8A, 
IAA 
X 
X 
X 
X 
X 
X 
X 
Every 3rd month 
 C-peptide  
 
X 
Fasting + 
stimulated 
X 
Fasting + 
stimulated 
X 
Random 
X 
Random 
X 
Fasting + 
stimulated 
X 
Random 
X 
Fasting + 
stimulated 
Every 6th month 
Fasting + stimulated 
 HbA1c 
X 
X 
X. 
X 
X 
X 
X 
Every 3rd month 
 Plasma Glucose 
X 
X 
X 
X 
X 
X 
X 
Every 3rd month 
 OGTT/IvGTT 
IvGTT 
OGTT 
 
 
 
OGTT 
 
IvGTT 
Every 6th month 
IvGTT/OGTT 
TPOAb, ThglAb, tTGAb 
X 
 
 
 
 
 
X 
Every 12th month 
Urine Analysis 
X 
X 
X 
X 
X 
 
X 
Every 6th  month 
Neurological Assessment 
X 
 
X 
X 
X 
 
X 
Last visit 
Injection Site Inspection 
 
X 
X 
X 
X 
 
 
 


 
 
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Schedule for the remaining post diagnosis protocol: 
 
 
 
 
 
Adverse Events  
 
X 
X 
X 
X 
 
X 
Last visit 
Diary 
 
X 
X 
X 
X 
 
X 
Last visit 
Questionnaire to child 
about the study 
procedures 
 
X 
 
 
 
 
 
Last visit 
 
 
Visit P1 
Day 1 
Within 4 
month of 
diagnosis  
Visit P2 
Month 1 
(Day 30) 
30 +/-7 days 
from visit A1 
 
Visit P3 
Month 3 
3 months 
+/- 20 days 
from visit P1 
Visit P4 
Month 9 
9 months  
+/- 20 days  
from visit P1 
Study Drug 
Administration 
X 
X 
 
 
Medical History 
X 
X 
X 
X 
General Physical Exam 
X 
X 
X 
X 
Concomitant Medication 
X 
X 
X 
X 
Weight, Height 
X 
X 
X 
X 
Vital signs (BP) 
X 
X 
X 
X 
Blood Sampling:  
 
 
 
 
 Hematology,  Chemistry 
X 
X 
X. 
X 
Cellular analyses 
X 
X 
X 
X 
GADAb, IA-2Ab, ZnT8Ab, 
IAA 
X 
X 
X 
X 
 HbA1c, insulin regimen 
X 
X 
X 
X 
 Plasma Glucose 
X 
 
X 
X 
 MMTT (Mixed Meal 
Tolerance Test) 
 including C-peptide  
 p-glucose 
X 
 
 
X 
X 
Neurological Assessment 
X 
X 
X 
X 
Adverse Events  
X 
X 
X 
X 
Diary 
X 
X 
X 
X 
Questionnaire to child 
about the study 
procedures 
 
 
 
At the last 
visit 

SAP_001.pdf: 
 
 
 
 
 
 
 
 
 
 
A double-blind, randomized investigator-initiated study to determine the safety and the 
effect of Diamyd® in combination with Vitamin D on the progression to type 1 diabetes in 
children with multiple islet cell autoantibodies. 
 
Study code: DiAPREV -IT 2 
 
Phase IIa study 
 
 
 
STATISTICAL ANALYSIS PLAN 
 
 


Statistical Analysis Plan 
            Page 2 (13) 
 
Sponsor: Diamyd Medical AB 
Final 5.5.2020 
Study: DiAPREV -IT 2 
 
 
 
 
Signatures: 
 
 
Statistical Analysis Plan was prepared by: 
 
 
 
Jouni Junnila, MSc 
Date 
Study Statistician 
 
4Pharma Ltd 
 
 
Statistical Analysis Plan was reviewed/approved by: 
 
 
 
 
Tommi Pesonen, MSc 
Date 
Senior Statistician 
 
4Pharma Ltd 
 
 
 
 
Ulf Hannelius, PhD, MBA 
Date  
CEO, Diamyd Medical AB 
 
 
 
 
 
Helena Elding Larsson, MD, PhD 
Date 
Principal Investigator 
 
 
 
 


Statistical Analysis Plan 
            Page 3 (13) 
 
Sponsor: Diamyd Medical AB 
Final 5.5.2020 
Study: DiAPREV -IT 2 
 
 
 
 
Table of Contents 
 
1 
Abbreviations................................................................................................................................. 4 
2 
Study objective(s) .......................................................................................................................... 4 
3 
Design and type of the study ......................................................................................................... 4 
3.1 
Randomization and blinding ................................................................................................... 4 
4 
Study variables .............................................................................................................................. 5 
5 
Sample size considerations ............................................................................................................ 5 
6 
Statistical hypotheses ..................................................................................................................... 5 
7 
Analysis sets .................................................................................................................................. 6 
7.1 
Intention to Treat Population (ITT) ........................................................................................ 6 
7.2 
Full Analysis Population ......................................................................................................... 6 
7.3 
Per protocol (PP) Population .................................................................................................. 6 
7.4 
Safety Population .................................................................................................................... 6 
8 
General statistical considerations .................................................................................................. 6 
9 
Demographic and other baseline characteristics ........................................................................... 6 
10 
Concomitant medication/treatment ............................................................................................ 6 
11 
Extent of exposure and compliance ........................................................................................... 7 
12 
Analysis of efficacy .................................................................................................................... 7 
12.1 
Primary efficacy variables ................................................................................................ 7 
12.2 
Secondary efficacy variables ............................................................................................ 7 
12.3 
Subgroup analysis ............................................................................................................ 7 
12.4 
Exploratory efficacy variables ......................................................................................... 8 
13 
Analysis of safety ....................................................................................................................... 8 
13.1 
Adverse events ................................................................................................................. 8 
13.2 
Laboratory safety variables .............................................................................................. 8 
13.3 
Physical and neurological examination, vital signs ......................................................... 8 
13.4 
GADA titers, isotype, epitope and affinity, anti-idiotypic antibodies ............................. 9 
13.5 
Autoimmunity other than diabetes ................................................................................... 9 
13.6 
Injection site reactions ...................................................................................................... 9 
14 
Completion and premature discontinuation ............................................................................... 9 
15 
Post diagnosis intervention and follow-up ................................................................................. 9 
16 
Deviations from the analyses planned in the study protocol ...................................................... 9 
17 
Execution of statistical analyses ................................................................................................. 9 
18 
Hardware and software ............................................................................................................... 9 
19 
References .................................................................................................................................. 9 
20 
Appendices ................................................................................................................................. 9 
20.1 
Schedule of assessments ................................................................................................. 10 
20.2 
Table and figure plan ...................................................................................................... 11 
 


Statistical Analysis Plan 
            Page 4 (13) 
 
Sponsor: Diamyd Medical AB 
Final 5.5.2020 
Study: DiAPREV -IT 2 
 
1 
Abbreviations 
AE 
 
Adverse Event 
AUC  
Area Under the Curve 
BDR  
Blind Data Review 
DSMB  
Data Safety Monitoring Board 
FPIR  
First-phase insulin response 
HbA1c  
Hemoglobin A1c 
ITT 
 
Intent to Treat 
IvGTT  
Intravenous Glucose Tolerance Test 
OGTT  
Oral Glucose Tolerance Test 
PP 
 
Per-protocol 
SAE 
 
Serious Adverse Event 
SAP 
 
Statistical Analysis Plan 
 
2 
Study objective(s) 
The purpose of the study is to assess the safety and efficacy of Diamyd® 20 µg + vitamin D (2000 
IU/day) in comparison with placebo + vitamin D (2000 IU/day) in non-diabetic children with multiple 
islet autoantibodies. 
The primary objective of the study is to evaluate if Diamyd®, in children treated with relatively high 
doses of vitamin D, may delay or stop the autoimmune process leading to clinical type 1 diabetes in 
children with ongoing persistent beta cell autoimmunity as indicated by multiple positive islet cell 
autoantibodies. 
The secondary objective is to demonstrate that Diamyd® is safe in children at risk for type 1 diabetes.  
3 
Design and type of the study 
This is a phase IIa, double-blind, randomized, placebo-controlled, parallel group single centre study 
in children with multiple islet cell autoantibodies with a maximum of two-year follow-up. 
3.1 
Randomization and blinding 
Eligible participants were randomized to active treatment or placebo, with 1:1 ratio. This 
randomization was stratified by glucose metabolism at baseline applying the following 
categorisation: 
A) Impaired glucose metabolism defined as low first phase insulin response on IvGTT (≤30) 
and/or an increased 30, 60, 90 min glucose values (≥ 11.1 mmol/L) and/or 120 minutes 
glucose value (≥7.8 mmol/L) on OGTT, and/or HbA1c ≥ 39 and/or fasting glucose ≥6.1 
mmol/L 
B) Normal glucose metabolism 


Statistical Analysis Plan 
            Page 5 (13) 
 
Sponsor: Diamyd Medical AB 
Final 5.5.2020 
Study: DiAPREV -IT 2 
 
An independent statistician performed the randomization procedure. The randomization code for each 
child was preserved in envelopes in a locked safe. The treating physician had the possibility to break 
the code in cases of emergency. Further the Data Safety Monitoring Board (DSMB) had the 
possibility to request access to the randomization list. 
4 
Study variables 
The following variables will be evaluated. 
The demographic and baseline variables: 
Age, Gender, Height, Weight, HLA genotypes, First degree relative or from general population, 
Autoantibodies, Other autoimmune diseases (celiac disease, thyroid disease).  
The primary efficacy variable: 
Type I diabetes status after 2-year follow-up 
The secondary efficacy variables: 
Proportion of children who progress from normal to impaired glucose metabolism and proportion of 
children who progress from impaired to more severely impaired glucose metabolism 
The exploratory efficacy variables: 
C-peptide, oGTT/IvGTT, plasma glucose, HbA1c 
The safety variable: 
Adverse events (AE), safety laboratory, physical and neurological examination, vital signs, GADA 
titers, isotype, epitope and affinity, anti-idiotypic antibodies, injection site reactions  
5 
Sample size considerations 
The planned sample size of the study was 80 children, 40 in each of the study arms. This sample size 
was based on the expectation that 50% of the untreated children with multiple autoantibodies will 
develop type 1 diabetes within 5 years. This frequency had previously been reported equal among 
relatives to diabetes patients and in the general population. If 20% of the treated children would 
develop type 1 diabetes within the same period of time, there would have been a power of 82% with 
alpha=5% with a group of 40+40=80 children. P value <0.05 was to be used as the significance level. 
However, the study was stopped prematurely ending up with a total sample size of 29 children. The 
study was stopped due to negative results received from another similar study (DiAPREV/2008). In 
that study no effect on delaying or preventing type 1 diabetes with Diamyd®-treatment was shown, 
while GADA increased in the study patients. Since the cohort of children, inclusion and exclusion 
criteria and dose of Diamyd® were all equal in DiAPREV/2008 and DiAPREV-IT 2, it is not probable 
that the current study would show that Diamyd® would delay or prevent type 1 diabetes, but it may 
have effects on the immune system that can be explored in mechanistic studies. 
6 
Statistical hypotheses 
As the study was stopped prematurely with limited number of children within both treatment arms, 
no formal statistical hypotheses are defined. 


Statistical Analysis Plan 
            Page 6 (13) 
 
Sponsor: Diamyd Medical AB 
Final 5.5.2020 
Study: DiAPREV -IT 2 
 
7 
Analysis sets 
The database will be reviewed in a blinded manner prior to database lock. Patients are assigned to the 
respective analysis sets depending on the observed protocol violations. 
7.1 
Intention to Treat Population (ITT) 
The ITT-population comprises all randomized (as planned) subjects. 
7.2 
Full Analysis Population 
The Full Analysis Set (FAS) is a subset of ITT and comprises of all subjects who received any study 
drug and who participated in at least one post-baseline assessment. FAS is the primary efficacy 
population. 
7.3 
Per protocol (PP) Population 
The PP population comprises of all subjects who did not substantially deviate from the protocol as to 
be determined on a per-subject basis. The PP population is used for sensitivity purposes. 
7.4 
Safety Population 
The Safety Population comprises of all subjects who received at least one administration of the 
study drug at any dosage. 
8 
General statistical considerations 
Descriptive statistics include at least the number of participants, mean, standard deviation (SD), 
minimum, median and maximum for continuous variables, and frequencies and percentages for 
categorical variables. Line listings of all participants are provided as well. 
Missing values will not be imputed in the analyses. 
The table and figure plan is presented in Appendix 18.2. 
9 
Demographic and other baseline characteristics 
All background and other baseline characteristics will be tabulated with descriptive statistics. Medical 
history will be summarized with frequency distributions including both event and subject counts. 
Tabulations will be conducted by treatment group. No statistical tests will be carried out for any 
baseline measures between the treatments. 
10 Concomitant medication/treatment 
Concomitant medication and treatments will be summarized overall and by treatment group using 
ATC classification. 


Statistical Analysis Plan 
            Page 7 (13) 
 
Sponsor: Diamyd Medical AB 
Final 5.5.2020 
Study: DiAPREV -IT 2 
 
11 Extent of exposure and compliance 
The extent of exposure and treatment compliance will be summarized descriptively overall and by 
treatment group. 
12 Analysis of efficacy 
 
Descriptive statistics of all the efficacy data will be presented by visit and treatment group. ITT-
population will serve as the primary efficacy population. 
12.1 
Primary efficacy variables 
The primary efficacy variable is the type 1 diabetes status (with vs without type 1 diabetes) at the end 
of the two-year follow-up. The proportion of children developing clinical type 1 diabetes during the 
two-year follow-up will be summarized with frequency tables. The proportions of children with type 
1 diabetes will also be summarized for other study visits by treatment group. The analysis will be 
conducted for both ITT and PP populations. 
In addition, time to the development of type 1 diabetes up to 2 years will be graphically illustrated by 
Kaplan-Meier curves by treatment. 
12.2 
Secondary efficacy variables 
The secondary efficacy variables are the proportion of children who have progression from normal 
to impaired glucose metabolism (within children with normal glucose metabolism at screening) and 
the proportion of children who have progression from already impaired glucose metabolism from one 
or several criteria to additional signs of reduced glucose metabolism (within children with impaired 
glucose metabolism at screening).  
Impaired glucose metabolism is defined as acceptance of at least one of the following criterion; a) F-
glucose ≥ 6.1 mmol/L, b) maximum p-glucose at 30, 60, 90 minutes ≥ 11.1 mmol/L in the OGTT, c) 
120 min p-glucose ≥ 7.8 mmol/L on OGTT, d) HbA1c ≥ 39 mmol/mol.  
Both the secondary efficacy variables will be summarized descriptively by visit and treatment group. 
12.3 
Subgroup analysis 
Subgroup analysis will be conducted for the primary and secondary efficacy variables, so that the 
above-mentioned analyses will also be assessed by HLA genotype (DR3-DQ2, present/absent). The 
DR3-DQ2 subgroup will be derived from the database so that patients that have HLA-DQ haplotype 
of DQ2 (DQ2/8 or DQ2/x) are classified as DR3-DQ2 (present) and the ones without DQ2 (DQ2.2/8; 
DQ8/8 or DQ8/x) are classified as DR3-DQ2 (absent). 
 
 


Statistical Analysis Plan 
            Page 8 (13) 
 
Sponsor: Diamyd Medical AB 
Final 5.5.2020 
Study: DiAPREV -IT 2 
 
12.4 
Exploratory efficacy variables 
The exploratory efficacy variables include  
1) first-phase insulin response (FPIR) and K-value from IvGTT  
2) fasting, maximum, 2 hours and AUC0-120min C-peptide levels from OGTT,  
3) fasting, maximum, 2 hours and AUC0-120min plasma-glucose from OGTT,  
4) HbA1c (mmol/mol), 
5) Fasting and AUC0-120min insulin levels from OGTT, 
6) AUC90min of C-peptide, glucose and insulin from IvGTT 
All the exploratory efficacy variables will be summarized descriptively including both the absolute 
and change from baseline values by visit and treatment group if feasible. In addition, the endpoints 
will be illustrated graphically if feasible. 
The AUC values for C-peptide, plasma glucose and insulin will be calculated applying linear 
trapezoidal rule. In case of single missing time points for the C-peptide/plasma-glucose/insulin are 
detected, it will be assumed that missing values fall linearly between existing data points. In case the 
0 min timepoint is missing, the corresponding AUC is set to missing. 
13 Analysis of safety 
All safety analyses will be conducted for the Safety population. 
13.1 
Adverse events 
All adverse events (AEs) will be tabulated (according to the MedDRA dictionary) by treatment group, 
system organ class (SOC), preferred term (PT), severity and causality. Both participant and event 
counts will be calculated. For participants with more than one occurrence of the same preferred term, 
the strongest causality and severity will be used when participant counts are reported.  
In addition, serious adverse events (SAE) and adverse events leading to discontinuation will be 
reported separately. 
13.2 
Laboratory safety variables 
Descriptive statistics of laboratory safety variables (hematology, chemistry and urine analyses) will 
be presented by visit and treatment group, together with the changes from baseline (pre-dose) values.  
13.3 
Physical and neurological examination, vital signs 
Vital signs (blood pressure, heart rate) will be summarized descriptively by visit and treatment group, 
together with changes from baseline (pre-dose). The abnormal findings in physical and neurological 
examinations will be presented as frequencies and percentages by visit and treatment group.   


Statistical Analysis Plan 
            Page 9 (13) 
 
Sponsor: Diamyd Medical AB 
Final 5.5.2020 
Study: DiAPREV -IT 2 
 
13.4 
GADA titers, isotype, epitope and affinity, anti-idiotypic antibodies 
GADA titers, isotype, epitope and affinity, anti-idiotypic antibodies (GADA (U), IA-2A (U), IAA ny 
(U/mL), ZnT8ArgA (U), ZnT8TrpA (U), ZnT8GluA (U)) will be summarized descriptively by visit 
and treatment group. In addition, GADA titers will be illustrated graphically. 
13.5 
Autoimmunity other than diabetes 
Autoantibodies to Thyroid peroxidase (TPO), Thyroglobulin (Thgl) and Tissue transglutaminase (tTg), 
(Thyroid peroxidase Ab (U), Thyroglobulin Ab (U), tTG IgA (U) and tTG IgG (U) together with TSH 
(mU/L) and Free T4 (pmol/L), will be summarized descriptively at visit 0, 12 months and 24 months by 
treatment group. 
13.6 
Injection site reactions 
The number of injection site reaction by severity will be summarized by visit and treatment group. 
14 Completion and premature discontinuation 
Completion and premature discontinuation will be listed. The reasons for premature discontinuation 
will be presented. 
15 Post diagnosis intervention and follow-up 
Study participants who developed type 1 diabetes during the study period were offered to participate 
in a post diagnosis intervention and follow up protocol. The collected post diagnosis data from the 
included participants will be summarized using data listings. 
16 Deviations from the analyses planned in the study protocol 
Due to the premature stop of the study, all the formal statistical analyses were removed from the 
statistical analysis plan compared to the protocol. 
17 Execution of statistical analyses 
Statistical analyses will be performed by 4Pharma Ltd. 
18 Hardware and software 
Statistical analysis, tables and patient data listings will be performed with SAS® version 9.4 for 
Windows (SAS Institute Inc., Cary, NC, USA). Pharmacokinetic parameters (AUC0-120min for C-
peptide, glucose and insulin) will be calculated using Phoenix WinNonlin software (version 8.1; 
Certara USA, Inc; Princeton; NJ; USA). 
19 
References 
Clinical Study Protocol, Version 3 (28.08.2017)  
20 
Appendices


 
Statistical Analysis Plan 
            Page 10 (13) 
 
Sponsor: Diamyd Medical AB 
Final 5.5.2020 
Study: DiAPREV-IT 2 
 
 
20.1 
Schedule of assessments 
 
Event  
Visit 0  
Information 
and consent  
Visit 1  
Day 1  
30 +/- 7 days 
from visit 0  
Visit 2  
Month 1  
30 +/-7 days 
from visit 1  
Visit 3  
Month 3  
3 months  
+/- 14 days 
from visit 1 
Visit 4  
Month 6  
6 months  
+/- 14 days  
from visit 1 
Visit 5 Month 
9  
9 months  
+/- 14 days  
from visit 1 
Visit 6  
Month 12  
12 months  
+/- 14 days  
from visit 1 
5 Years Follow up  
Every 3rd or 6th month  
Every visit  
+/- 14 days  
from visit 1 
Informed Consent 
X
Randomization 
X
Diamyd/Placebo 
Administration 
 
X 
X 
 
 
 
 
 
Start of Vitamin D 
X
Medical History 
X
General Physical Exam 
X
X
X
X
X
X
Last visit
Concomitant Medication 
X
X
X
X
X
X
Every 6th month
Weight, Height 
X
X
X
X
X
X
Every 6th month
Vital signs (BP) 
X
X
X
X
X
X
Every 6th month
Blood Sampling:
Hematology, chemistry, 
including calcium 
X 
X 
X 
X 
X 
X 
X 
Every 3rd month 
25 OH-vitamin D3 
X
X
X
X
Every 6th month
Cellular analyses 
X
X
X
X
Every 6th month
GADA, IA-2A, ZnT8A, 
IAA 
X 
X 
X 
X 
X 
X 
X 
Every 3rd month 
C-peptide  
X 
Fasting + 
stimulated
X 
Fasting + 
stimulated
X 
Random 
X 
Random 
X 
Fasting + 
stimulated
X 
Random 
X 
Fasting + 
stimulated
Every 6th month 
Fasting + stimulated 
HbA1c 
X
X
X.
X
X
X
X
Every 3rd month
Plasma Glucose 
X
X
X
X
X
X
X
Every 3rd month
OGTT/IvGTT  
IvGTT 
OGTT 
 
 
OGTT 
 
IvGTT 
Every 6th month 
IvGTT/OGTT
TPOAb, ThglAb, tTGAb 
X
X
Every 12th month
Urine Analysis 
X
X
X
X
X
X
Every 6th month
Neurological Assessment 
X
X
X
X
X
Last visit
Injection Site Inspection 
X
X
X
X
Adverse Events 
X
X
X
X
X
Last visit
Diary 
X
X
X
X
X
Last visit
Questionnaire to child 
about the study procedures 
 
X 
 
 
 
 
 
Last visit 



 
Statistical Analysis Plan 
            Page 11 (13) 
 
Sponsor: Diamyd Medical AB 
Final 5.5.2020 
Study: DiAPREV-IT 2 
 
 
20.2 
Table and figure plan 
14.1 
Demographic and Baseline Characteristics 
Table 14.1.1  
Disposition of patients (Total population) 
Table 14.1.2 
Analysis sets (Total population) 
Table 14.1.3 
Descriptive statistics of demographic variables (ITT) 
Table 14.1.4 
Medical history (ITT) 
Table 14.1.5 
Current medical conditions at baseline (ITT) 
Table 14.1.6 
Concomitant medication (ITT) 
14.2 
Efficacy 
14.2.1 
Primary efficacy analyses
Table 14.2.1.1  
Type 1 diabetes status by treatment and visit (ITT) 
Table 14.2.1.2 
Type 1 diabetes status by treatment and visit within subgroup (ITT) 
Table 14.2.1.3 
Time to development of type 1 diabetes (ITT) 
Table 14.2.1.4 
Time to development of type 1 diabetes by subgroup (ITT) 
14.2.2 
Secondary efficacy analyses 
Table 14.2.2.1  
Number of children developing impaired glucose metabolism (ITT)
Table 14.2.2.2 
Number of children developing impaired glucose metabolism by subgroup (ITT)
Table 14.2.2.3 
Number of children having progressive impaired glucose metabolism (ITT)
Table 14.2.2.4 
Number of children having progressive impaired glucose metabolism by subgroup (ITT)
14.2.3 
Exploratory efficacy analyses 
Table 14.2.3.1 
Descriptive statistics of first-phase insulin response (FPIR) and K-value from IvGTT by treatment and 
visit (ITT)
Table 14.2.3.2 
Change from baseline in first-phase insulin response (FPIR) and K-value from IvGTT by treatment and 
visit (ITT)
Table 14.2.3.3 
C-peptide concentrations from OGTT by treatment, visit and timepoint (ITT) 
Table 14.2.3.4 
Maximum and AUC0-120min C-peptide values from OGTT by treatment and visit (ITT) 
Table 14.2.3.5 
Change from baseline in fasting, maximum and 2 hours C-peptide (OGTT) by treatment, visit and 
timepoint (ITT)
Table 14.2.3.6 
P-glucose concentrations from OGTT by treatment, visit and timepoint (ITT) 
Table 14.2.3.7 
Maximum and AUC0-120min P-glucose values from OGTT by treatment and visit (ITT) 
Table 14.2.3.8 
Change from baseline in fasting, maximum and 2 hours P-glucose (OGTT) by treatment, visit and 
timepoint (ITT)
Table 14.2.3.9 
HbA1c concentrations by treatment, visit and timepoint (ITT) 
Table 14.2.3.10 
Change from baseline in HbA1c concentration by treatment, visit and timepoint (ITT) 
Table 14.2.3.11 
Insulin concentrations from OGTT by treatment, visit and timepoint (ITT) 
Table 14.2.3.12 
Maximum and AUC0-120min Insulin values from OGTT by treatment and visit (ITT) 
Table 14.2.3.13 
Change from baseline in fasting and maximum Insulin concentration (OGTT) by treatment, visit and 
timepoint (ITT)
Table 14.2.3.14 
C-peptide concentrations from IvGTT by treatment, visit and timepoint (ITT) 
Table 14.2.3.15 
AUC0-90min C-peptide values from IvGTT by treatment and visit (ITT) 
Table 14.2.3.16 
Glucose concentrations from IvGTT by treatment, visit and timepoint (ITT) 
Table 14.2.3.17 
AUC0-90min Glucose values from IvGTT by treatment and visit (ITT) 
Table 14.2.3.18 
Insulin concentrations from IvGTT by treatment, visit and timepoint (ITT) 
Table 14.2.3.19 
AUC0-90min Insulin values from IvGTT by treatment and visit (ITT) 
 
 


 
Statistical Analysis Plan 
            Page 12 (13) 
 
Sponsor: Diamyd Medical AB 
Final 5.5.2020 
Study: DiAPREV-IT 2 
 
 
14.3 
Safety 
14.3.1  
Adverse events 
Table 14.3.1.1  
Summary of adverse events (Safety)
Table 14.3.1.2  
Adverse events by SOC and PT (Safety)  
Table 14.3.1.3  
Adverse events by SOC, PT and severity (Safety)  
Table 14.3.1.4 
Adverse events by SOC, PT and causality (Safety)  
Table 14.3.1.5 
Serious adverse events (Safety)  
Table 14.3.1.6 
Adverse events leading to discontinuation (Safety)  
14.3.2 
Laboratory safety variables 
Table 14.3.2.1  
Summary of safety laboratory - Hematology (Safety) 
Table 14.3.2.2
Summary of safety laboratory - Clinical chemistry (Safety) 
Table 14.3.2.3
Summary of safety laboratory - Urinanalysis (Safety) 
14.3.3 
Physical and neurological examination, vital signs 
Table 14.3.3.1  
Physical examination by treatment and visit (Safety)
Table 14.3.3.2 
Neurological examination by treatment and visit (Safety) 
Table 14.3.3.3 
Vital signs (blood pressure, heart rate) by treatment and visit (Safety) 
Table 14.3.3.4 
Change from baseline in Vital signs (blood pressure, heart rate) by treatment and visit (Safety) 
14.3.4 
GADA titers, isotype, epitope and affinity, anti-idiotypic antibodies 
Table 14.3.4.1  
Descriptive statistics of GADA (U) by treatment and visit
Table 14.3.4.2 
Descriptive statistics of IA-2A (U), IAA ny (U/mL) by treatment and visit
Table 14.3.4.3 
Descriptive statistics of ZnT8ArgA (U), ZnT8TrpA (U), ZnT8GluA (U) by treatment and visit
14.3.5 
Autoimmunity other than diabetes 
Table 14.3.5.1  
Descriptive statistics of autoimmunity related autoantibodies by treatment and visit
14.3.6 
Injection Site Reactions (Safety) 
Table 14.3.6.1  
Injection site reactions (erythmea, oedema, haematoma, tenderness, pain, itching), pre-injection by visit 
and treatment (Safety)
Table 14.3.6.2  
Injection site reactions (erythmea, oedema, haematoma, tenderness, pain, itching), post-injection by visit 
and treatment (Safety)
14.3.7 
Questionnaire to child about study procedures 
Table 14.3.7.1  
Frequency table of child's assessment of study procedures (Safety)
14.2 
Figures 
Figure 14.2.1  
Kaplan-Meier curves of time to development of type 1 diabetes (ITT)
Figure 14.2.2 
C-peptide concentrations (OGTT - ITT) 
Figure 14.2.3 
P-Glucose concentrations (OGTT - ITT) 
Figure 14.2.4 
Insulin concentrations (OGTT - ITT) 
Figure 14.2.5 
C-peptide concentrations (IvGTT - ITT) 
Figure 14.2.6 
Glucose concentrations (IvGTT - ITT) 
Figure 14.2.7 
Insulin concentrations (IvGTT - ITT) 
Figure 14.2.8 
HbA1c concentrations (ITT) 
 
 


 
Statistical Analysis Plan 
            Page 13 (13) 
 
Sponsor: Diamyd Medical AB 
Final 5.5.2020 
Study: DiAPREV-IT 2 
 
 
16.2 
Patient Data Listings 
Listing 16.2.1  
Protocol Deviations
Listing 16.2.2 
Demographic data 
Listing 16.2.3 
Medical history  
Listing 16.2.4 
Concomitant medications 
16.2.5 
Individual Efficacy Response data 
Listing 16.2.5.2 
Glucose data (OGTT, IvGTT) 
Listing 16.2.5.3 
C-peptide data (OGTT, IvGTT) 
Listing 16.2.5.4 
Insulin data (OGTT, IvGTT)
Listing 16.2.5.5 
HbA1c data
Listing 16.2.6 
Adverse events
Listing 16.2.7 
Individual laboratory measurements by patient 
Listing 16.2.8 
Physical, neurological examination, vital signs 
Listing 16.2.9 
Injection site reactions
Listing 16.2.10 
Other safety variables 
16.2.11 
Post diagnosis data
Listing 16.2.11.1 
C-peptide, glucose, HbA1c
Listing 16.2.11.2 
MMTT-data
Listing 16.2.11.3 
Safety laboratory
Listing 16.2.11.4 
Physical, neurological examination, vital signs
Listing 16.2.11.5 
Adverse events