Conditions: Diabetes Mellitus, Type 2 Hypercholesterolemia
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 12
Healthy volunteers: no

Brief summary:
Objective: To investigate the effect of 5 weeks dapagliflozin 10 mg once daily treatment on glucose and lipid fluxes in patients with type 2 diabetes.

Study design: Single center single arm (mechanistic) intervention trial.

Study Population: Male or postmenopausal female patients with type 2 diabetes BMI \> 25 kg/m2and more than 12 weeks a stable dose of metformin treatment \> 1500mg, HbA1C ≥6.5% - \<8.5%, Fasting Plasma Glucose (FPG) \<13.2 mmol/l, LDL cholesterol \>2.5 mmol/l, willing to switch to rosuvastatin 10mg once daily for 4 weeks, and then receive 10 mg dapagliflozin once daily orally, for 5 weeks.

Treatment: After a statin washout fase of 4 weeks, baseline cholesterol synthesis will be measured (2H3 Leucine, 2H2O deuterated water). Then, treatment with rosuvastatin 10mg for 4 weeks will be initiated after which, patients will undergo glucose (2H2enriched glucose) and lipid flux (2H3 Leucine, 2H2O deuterated water and oral 1,2,3,4-13C16 - palmitate enrichment measurements) followed by 5 weeks treatment with dapagliflozin 10mg once daily. In the final week glucose/lipid flux measurements will be repeated.

Sample Size: 12 DM2 subjects.

Outcome measures: The primary endpoint is effect of 5 weeks Sodium-Glucose Linked co-transporter (SGLT) 2 inhibition on LDL cholesterol synthesis in patients with DM2. Secondary endpoints are effect of SGLT2 inhibition on triglyceride and cholesterol fluxes as well as (hepatic and peripheral) insulin sensitivity and energy expenditure. Finally, effect of SGLT2 inhibition on dietary intake, liver fat content (MRI liver) and fecal microbiome will be studied at these timepoints.

Detailed description:
Background: Type 2 diabetes is associated with an increased cardiovascular risk. Besides metformin, a new treatment strategy is oral SGLT2 inhibition (dapagliflozin), Although the recently published, first-in-class cardiovascular outcome trial (EMPA-REG OUTCOME) has suggested a beneficial effect on all cause cardiovascular mortality upon SGLT2 inhibition, a known (class) side effect in worsening of dyslipidemia in all DM2 patients. The investigators thus aim to dissect the effect of SGLT2 inhibition (Dapagliflozin 10mg once daily for 5 weeks) on glucose and lipid fluxes in uncomplicated DM2 subjects.

Objective: To investigate the effect of 5 weeks dapagliflozin 10 mg once daily treatment on glucose and lipid fluxes in patients with type 2 diabetes.

Study design: Single center single arm (mechanistic) intervention trial.

Study Population: Male or postmenopausal female patients with type 2 diabetes BMI \> 25 kg/m2and more than 12 weeks a stable dose of metformin treatment \> 1500mg, HbA1C ≥6.5% - \<8.5%, FPG\<13.2 mmol/l, LDL cholesterol \>2.5 mmol/l, willing to switch to rosuvastatin 10mg once daily for 4 weeks, and then receive 10 mg dapagliflozin once daily orally, for 5 weeks.

Treatment: After a statin washout fase of 4 weeks, baseline cholesterol synthesis will be measured (2H3 Leucine, 2H2O deuterated water). Then, treatment with rosuvastatin 10mg for 4 weeks will be initiated after which, patients will undergo glucose (2H2enriched glucose) and lipid flux (2H3 Leucine, 2H2O deuterated water and oral 1,2,3,4-13C16 - palmitate enrichment measurements) followed by 5 weeks treatment with dapagliflozin 10mg once daily. In the final week glucose/lipid flux measurements will be repeated.

Outcome measures: The primary endpoint is effect of 5 weeks SLGT2 inhibition on LDL cholesterol synthesis in patients with DM2. Secondary endpoints are effect of SGLT2 inhibition on triglyceride and cholesterol fluxes as well as (hepatic and peripheral) insulin sensitivity and energy expenditure. Finally, effect of SGLT2 inhibition on dietary intake, liver fat content (MRI liver) and fecal microbiome will be studied at these timepoints.

Sample Size: Based on published data, the investigators expect 10% higher plasma LDL level (from 3.1 ± 0.8 to 1.7 ± 0.4 mmol/l ) upon SGLT2 inhibition in DM2. DM2 subjects have concomitant LDL- ApoB synthesis (1.8 ± 0.4 gram/day) after 4 weeks of rosuvastatin 10mg. Assuming an increase in LDL-apoB synthesis of 0.3 gram/day with SD of 0.4 and using single-sided test (with alfa of 0.05 and 85% power), the sample size needs to be 11 DM2 subjects on dapagliflozin 10mg treatment. Taking a 10% patient dropout rate, the aim is to include 12 DM2 subjects in total.

Nature and extent of the burden and risks associated with participation, benefit and group relatedness: The risk for patients to participate in this study is minimal. All of the stable isotopes are GMP produced and analyses techniques have been previously used and published by the investigators. Also, REE and liver MRI measurements are not associated with adverse events. Both rosuvastatin and dapagliflozin have been approved by FDA/EMA and are widely prescribed. In total 470 ml blood (100 ml per lipidflux day, 90 ml per clamp day) will be drawn over period of 13 weeks (divided over 5 visits).

Arms:
arm 1: Rosuvastatin 10mg once daily for 9 weeks, with 5 weeks of once daily Dapagliflozin 10mg added

Interventions: intervention 1: Dapagliflozin intervention 2: Rosuvastatin
Intervention descriptions: intervention 1: 5 weeks 10mg dapagliflozin once daily intervention 2: 9 weeks 10mg dapagliflozin once daily

Protocol document:
Prot_SAP_000.pdf: 
Clinical Study Protocol DICE study, © M. Nieuwdorp, AMC-Vumc Diabetes Center, Amsterdam, the Netherlands 
Drug Substance Dapagliflozin 10mg once dailys on top of Crestor 5mg once daily;  a pilot study 
Study Code  01 
Edition Number 05 
Date 25-06-2015 
 
1
Clinical Study Protocol  
Drug Substance 
Dapagliflozin 
Study Code 
DICE study 
Edition Number 
Version 5 
Date 
27-06-2015 
Dapagliflozin on cholesterol metabolism in DM2: dissecting its effect on 
dyslipidemia by using stable isotope based cholesterol and glucose fluxes; a 
pilot study 
Sponsor:  
The following Amendment(s) and Administrative Changes have been made to this protocol since the date of 
preparation: 
Amendment No. 
Date of Amendment 
Local Amendment No: 
Date of Local Amendment 
Administrative 
Change No. 
Date of Administrative 
Change 
Local Administrative 
Change No. 
Date of Local 
Administrative Change 
 
 


Clinical Study Protocol DICE study, © M. Nieuwdorp, AMC-Vumc Diabetes Center, Amsterdam, the Netherlands 
Drug Substance Dapagliflozin 10mg once dailys on top of Crestor 5mg once daily;  a pilot study 
Study Code  01 
Edition Number 05 
Date 25-06-2015 
 
2
PROTOCOL SYNOPSIS 
 
Dapagliflozin on cholesterol metabolism in DM2: dissecting its effect on dyslipidemia (DICE study) 
Principal Investigator 
Prof dr M. Nieuwdorp, internist-endocrinologist AMC-VUmC, Amsterdam, the 
Netherlands 
Study site(s) and number of subjects planned 
AMC-VUmc Diabetes Center, 12 DM2 subjects to be included 
Study period 
 
Phase of development 
Estimated date of first subject enrolled 
Q4 2015 
Phase III/IV 
 
Estimated date of last subject enrolled 
 
Dataanalyses and modelling 
Q4 2017 
 
Q1 2018 
Phase III/IV 
 
Study design 
Single arm intervention trial 
Objectives 
Primary Objective: 
Outcome Measure: 
effect of 5 weeks dapagliflozin 10mg on LDL 
cholesterol in patients with DM2 
Change in LDL synthesis   
 
 
 
 
Secondary Objective: 
Outcome Measure : 


Clinical Study Protocol DICE study, © M. Nieuwdorp, AMC-Vumc Diabetes Center, Amsterdam, the Netherlands 
Drug Substance Dapagliflozin 10mg once dailys on top of Crestor 5mg once daily;  a pilot study 
Study Code  01 
Edition Number 05 
Date 25-06-2015 
 
3
 
Effect of 5 weeks dapagliflozin 10mg on 
and Triglyceride cholesterol fluxes   
 
 
 
 
 
Effect of 5 weeks dapagliflozin 10mg on 
insulin sensitivity and energy expenditure 
 
 
Effect of 5 weeks dapagliflozin 10mg on 
liver fat content (MRI liver) 
  
Change in VLDL  secretion and clearance (as 
determined by 2H3 Leucine enrichment) and 
relation to plasma CETP, PLTP and HDL 
subfractions 
  
Change in De novo lipogenesis (as determined 
by 2H2O deuterated ‘heavy’ water) 
  
oral 1,2,3,4-13C16 – palmitate to measure FFA 
remnant uptake. 
 
Change in hepatic and peripheral insulin 
sensitivity (2 step Hyperinsulinemic 
normoglycemic clamp with 2H2 enriched 
glucose:)  and energy expenditure including 
carbohydrate oxidation and fatty acid oxidation 
rates in breathing air 
Change in liverfat MRI spectrum 
 
  
Target subject population 
Male or postmenopauzal female patients with type 2 diabetes BMI > 25 kg/m2 and more than 
12 weeks a stable dose of metformin treatment > 1500mg (HbA1C ≥6.5% - <8.5%) FPG<132 
mmol/l, LDL cholesterol >2.5 mmol/l, willing to switch to rosuvastatin 10mg once daily for 4 
weeks, and then receive  10 mg dapagliflozin once daily orally, for 5 weeks. Measurements 
(lipid and glucose fluxes) will be done after 4 weeks of rosuvastatin treatment (baseline) and 
after 5 weeks of dapagliflozin  
Duration of treatment 
4 weeks of crestor 10mg once daily treatment in all subjects (baseline) and after 5 weeks of 
dapagliflozin (n=12 DM2 subjects)  
Investigational product, dosage and mode of administration 
Crestor 10 mg once daily for in total 9 weeks orally 
Dapagliflozin 10mg once daily  for 5 weeks orally 


Clinical Study Protocol DICE study, © M. Nieuwdorp, AMC-Vumc Diabetes Center, Amsterdam, the Netherlands 
Drug Substance Dapagliflozin 10mg once dailys on top of Crestor 5mg once daily;  a pilot study 
Study Code  01 
Edition Number 05 
Date 25-06-2015 
 
4
Statistical methods 
All data will be analysed using SPSS for Windows, version 20.0 (SPSS Inc. Chicago, Illinois, 
USA). Multivariate analysis and ANOVA for repeated measures will be used. Wilcoxon’s 
signed-rank test will be used to compare results between the study groups.  Data will be 
expressed as median and range.  
 


Clinical Study Protocol DICE study, © M. Nieuwdorp, AMC-Vumc Diabetes Center, Amsterdam, the Netherlands 
Drug Substance Dapagliflozin 10mg once dailys on top of Crestor 5mg once daily;  a pilot study 
Study Code  01 
Edition Number 05 
Date 25-06-2015 
 
5
TABLE OF CONTENTS 
PAGE 
 
TITLE PAGE ........................................................................................................... 1 
PROTOCOL SYNOPSIS ......................................................................................... 2 
TABLE OF CONTENTS ......................................................................................... 5 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ............................ 9 
1. 
INTRODUCTION ................................................................................................. 10 
1.1 
Background and rationale for conducting this study ............................................. 10 
1.2 
Rationale for study design, doses and control groups ............................................ 10 
1.3 
Study Design .......................................................................................................... 12 
2. 
STUDY OBJECTIVES .......................................................................................... 16 
2.1 
Primary objective ................................................................................................... 16 
2.2 
Secondary objectives ..................................... Fout! Bladwijzer niet gedefinieerd. 
2.3 
Safety objectives .................................................................................................... 16 
2.4 
Exploratory objectives ........................................................................................... 17 
3. 
SUBJECT SELECTION, ENROLMENT, RANDOMISATION, 
RESTRICTIONS, DISCONTINUATION AND WITHDRAWAL ...................... 17 
3.1 
Inclusion criteria .................................................................................................... 17 
3.2 
Exclusion criteria ................................................................................................... 17 
3.3 
Subject enrolment and randomization ................................................................... 17 
3.4 
Procedures for handling incorrectly enrolled or randomized subjects ................... 17 
3.5 
Methods for assigning treatment groups ................................................................ 18 
3.6 
Methods for ensuring blinding ............................................................................... 18 
3.7 
Methods for unblinding .......................................................................................... 18 
3.8 
Restrictions ............................................................................................................ 18 
3.9 
Discontinuation of investigational product ............................................................ 18 
3.9.1 
Procedures for discontinuation of a subject from investigational product ............ 18 
3.10 
Criteria for withdrawal ........................................................................................... 18 
3.10.1 
Screen failures ........................................................................................................ 18 
3.10.2 
Withdrawal of the informed consent ...................................................................... 18 
3.11 
Discontinuation of the study .................................................................................. 18 


Clinical Study Protocol DICE study, © M. Nieuwdorp, AMC-Vumc Diabetes Center, Amsterdam, the Netherlands 
Drug Substance Dapagliflozin 10mg once dailys on top of Crestor 5mg once daily;  a pilot study 
Study Code  01 
Edition Number 05 
Date 25-06-2015 
 
6
4. 
STUDY PLAN AND TIMING OF PROCEDURES ............................................. 18 
4.1 
Enrolment/screening period ................................................................................... 21 
4.2 
Treatment period .................................................................................................... 21 
4.3 
Follow-up period .................................................................................................... 21 
5. 
STUDY ASSESSMENTS ..................................................................................... 21 
5.1 
Efficacy assessments .............................................................................................. 21 
5.2 
Safety assessments ................................................................................................. 21 
5.2.1 
Laboratory safety assessments ............................................................................... 21 
5.2.2 
Physical examination ............................................................................................. 22 
5.2.3 
ECG ........................................................................................................................ 22 
5.2.3.1 
Resting 12-lead ECG ............................................................................................. 22 
5.2.4 
Vital signs .............................................................................................................. 22 
5.2.4.1 
Pulse and blood pressure ........................................................................................ 22 
5.2.4.2 
Body temperature ................................................................................................... 22 
5.2.5 
Other safety assessments ........................................................................................ 22 
5.3 
Other assessments .................................................................................................. 22 
5.3.1 
Patient reported outcomes ...................................................................................... 22 
5.3.1.1 
<<Name of PRO method or questionnaire>> Fout! Bladwijzer niet gedefinieerd. 
5.4 
Pharmacokinetics ................................................................................................... 23 
5.4.1 
Collection of samples ............................................................................................. 23 
5.4.2 
Determination of drug concentration ..................................................................... 23 
5.4.3 
Storage and destruction of pharmacokinetic samples ............................................ 23 
5.5 
Pharmacodynamics ................................................................................................ 23 
5.5.1 
Collection of samples ............................................................................................. 23 
5.5.2 
Storage, re-use and destruction of pharmacodynamic samples ............................. 23 
5.6 
Pharmacogenetics .................................................................................................. 23 
5.6.1 
Collection of pharmacogenetic samples ................................................................ 23 
5.6.2 
Storage, re-use and destruction of pharmacogenetic samples ............................... 23 
5.7 
Biomarker analysis ................................................................................................. 23 
5.7.1 
Storage, re-use and destruction of biological samples ........................................... 23 
5.7.2 
Labelling and shipment of biological samples ....................................................... 23 
5.7.3 
Chain of custody of biological samples ................................................................. 23 
5.7.4 
Withdrawal of Informed Consent for donated biological samples ........................ 24 
6. 
SAFETY REPORTING AND MEDICAL MANAGEMENT .............................. 24 
6.1 
Definition of adverse events .................................................................................. 24 
6.2 
Recording of adverse events .................................................................................. 24 
6.2.1 
Time period for collection of adverse events ......................................................... 24 
6.2.2 
Follow-up of unresolved adverse events ................................................................ 24 
6.2.3 
Variables ................................................................................................................ 24 


Clinical Study Protocol DICE study, © M. Nieuwdorp, AMC-Vumc Diabetes Center, Amsterdam, the Netherlands 
Drug Substance Dapagliflozin 10mg once dailys on top of Crestor 5mg once daily;  a pilot study 
Study Code  01 
Edition Number 05 
Date 25-06-2015 
 
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6.2.4 
Causality assessment .............................................................................................. 25 
6.2.5 
Disease progression ............................................................................................... 25 
6.3 
Reporting of serious adverse events....................................................................... 25 
6.4 
Overdose ................................................................................................................ 25 
6.5 
Pregnancy ............................................................................................................... 27 
6.5.1 
Maternal exposure .................................................................................................. 27 
6.5.2 
Paternal exposure ................................................................................................... 27 
6.6 
Management of toxicities <<Dose Reductions>> ................................................. 27 
6.7 
Study governance and oversight ............................................................................ 27 
6.7.1 
Steering Committee ............................................................................................... 27 
6.7.2 
Data Monitoring Committee .................................................................................. 27 
6.7.3 
Scientific Advisory Committee .............................................................................. 27 
7. 
INVESTIGATIONAL PRODUCT AND OTHER TREATMENTS .................... 27 
7.1 
Identity of investigational product(s) ..................................................................... 27 
7.2 
Dose and treatment regimens ................................................................................. 28 
7.3 
Labelling ................................................................................................................ 28 
7.4 
Storage ................................................................................................................... 28 
7.5 
Compliance ............................................................................................................ 28 
7.6 
Accountability ........................................................................................................ 28 
7.7 
Concomitant and other treatments ......................................................................... 28 
7.7.1 
Other concomitant treatment .................................................................................. 29 
7.8 
Post Study Access to Study Treatment .................................................................. 29 
8. 
STATISTICAL ANALYSES ................................................................................ 29 
8.1 
Statistical considerations ........................................................................................ 29 
8.2 
Sample size estimate .............................................................................................. 29 
8.3 
Definitions of analysis sets .................................................................................... 29 
8.3.1 
Efficacy analysis set ............................................................................................... 29 
8.3.2 
Safety analysis set .................................................................................................. 30 
8.3.3 
PK analysis set ....................................................................................................... 30 
8.3.4 
PRO analysis set .................................................................................................... 30 
8.4 
Outcome measures for analyses ............................................................................. 30 
8.5 
Methods for statistical analyses ............................................................................. 30 
8.5.1 
Analysis of the primary variable(s) ........................................................................ 30 
8.5.2 
Analysis of the secondary variable(s) .................................................................... 30 
8.5.3 
Subgroup analysis (if applicable) ........................................................................... 30 
8.5.4 
Interim analysis ...................................................................................................... 30 
8.5.5 
Sensitivity analysis (if applicable) ......................................................................... 30 


Clinical Study Protocol DICE study, © M. Nieuwdorp, AMC-Vumc Diabetes Center, Amsterdam, the Netherlands 
Drug Substance Dapagliflozin 10mg once dailys on top of Crestor 5mg once daily;  a pilot study 
Study Code  01 
Edition Number 05 
Date 25-06-2015 
 
8
8.5.6 
Exploratory analysis (if applicable)) ...................................................................... 31 
9. 
STUDY AND DATA MANAGEMENT ............................................................... 31 
9.1 
Training of study site personnel ............................................................................. 31 
9.2 
Monitoring of the study ......................................................................................... 31 
9.2.1 
Source data ............................................................................................................. 31 
9.2.2 
Study agreements ................................................................................................... 31 
9.2.3 
Archiving of study documents ............................................................................... 31 
9.3 
Study timetable and end of study ........................................................................... 31 
9.4 
Data management ................................................................................................... 31 
10. 
ETHICAL AND REGULATORY REQUIREMENTS ......................................... 32 
10.1 
Ethical conduct of the study ................................................................................... 32 
10.2 
Subject data protection ........................................................................................... 32 
10.3 
Ethics and regulatory review.................................................................................. 32 
10.4 
Informed consent ................................................................................................... 32 
10.5 
Changes to the protocol and informed consent form ............................................. 32 
10.6 
Budget ............................................................ Fout! Bladwijzer niet gedefinieerd. 
11. 
LIST OF REFERENCES ....................................................................................... 33 
 
LIST OF TABLES 
<<Table x 
Laboratory Safety Variables ............. Fout! Bladwijzer niet gedefinieerd. 
 
LIST OF FIGURES 
Figure 1 
Study flow chart ....................................................................................... 12 
list of APPENDICES 
Appendix A 
Signatures 
Appendix B 
Additional Safety Information  
<<Appendix C>> <<IATA 6.2 Guidance document>> 
<<Appendix D>> <<Pharmacogenetics Research>> 
<<Appendix X>> <<Other>> 


Clinical Study Protocol DICE study, © M. Nieuwdorp, AMC-Vumc Diabetes Center, Amsterdam, the Netherlands 
Drug Substance Dapagliflozin 10mg once dailys on top of Crestor 5mg once daily;  a pilot study 
Study Code  01 
Edition Number 05 
Date 25-06-2015 
 
9
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
<<>> 
The following abbreviations and special terms are used in this study Clinical Study Protocol. 
Abbreviation or 
special term 
Explanation 
AE 
Adverse event 
CRF 
Case Report Form (electronic/paper) 
CSA 
Clinical Study Agreement 
CSR 
Clinical Study Report 
CTCAE 
Common Terminology Criteria for Adverse Event 
DAE 
Discontinuation of Investigational Product due to Adverse Event 
DNA 
Deoxyribonucleic acid  
EC 
Ethics Committee, synonymous to Institutional Review Board (IRB) and 
Independent Ethics Committee (IEC) 
GCP 
Good Clinical Practice 
ICH 
International Conference on Harmonisation 
IP 
Investigational Product  
IVRS 
Interactive Voice Response System 
IWRS 
Interactive Web Response System 
LSLV 
Last Subject Last Visit 
OAE 
Other Significant Adverse Event 
PGx 
Pharmacogenetic research 
PI 
Principal Investigator  
SAE 
Serious adverse event  
WBDC 
Web Based Data Capture 
 


Clinical Study Protocol DICE study, © M. Nieuwdorp, AMC-Vumc Diabetes Center, Amsterdam, the Netherlands 
Drug Substance Dapagliflozin 10mg once dailys on top of Crestor 5mg once daily;  a pilot study 
Study Code  01 
Edition Number 05 
Date 25-06-2015 
 
10
1. 
INTRODUCTION 
1.1 
Background and rationale for conducting this study 
There is a worldwide epidemic of obesity, which is a major risk factor for the development of 
common medical conditions such as type 2 diabetes (T2D), dyslipidemia and cardiovascular 
disease (CVD). Obesity and T2D are leading causes of morbidity and mortality in developed 
nations with an estimated cost exceeding 400 billion euro in the last 5 years and an estimated 
250,000 deaths per year (1) The prevalence of obesity and T2D continues to rise: one out of 
three adults is expected to be diagnosed with T2D in 2050 (2) Current therapeutic regimens 
only minimally reduce T2D-related complications and lead to a shift from myocardial 
infarction to peripheral artery disease and kidney failure. Additionally, all-cause morbidity 
and mortality is still 10-fold higher in T2D patients compared to healthy subjects (3) This 
collectively represents a growing unmet clinical need.  
 
In this regard, SGLT2 inhibitors improve glycemic control and are currently investigated in 
large cardiovascular outcome trials  in patients with type 2 diabetes mellitus (4). It has been 
suggested that the beneficial cardiovascular effects are driven by  both improved peripheral 
insulin sensivity (5,6) in conjunction with reduced bloodpressure and bodyweight in subjects 
with type 2 diabetes mellitus, the latter most likely driven  by an osmotic (plasmacontraction) 
effect due to the  increased glycosuria (7,8). However a potential disadvantage of all SGLT2 
inhibitors might be the adverse effect seen on fasting lipid profiles including decreased HDL 
cholesterol and increased LDL-cholesterol  and triglyceride plasma levels (9,10,11). In this 
regard ,there is a striking similarity with the observation of increased APOB synthesis due to 
plasma contraction in patients with nephrotic syndrome (12). Other mechanisms that might 
drive the observed dyslipidemic plasma cholesterol profile might include decreased  hepatic 
LDL cholesterol clearance as well as deleterious effects of Cholesteryl ester transfer protein 
(CETP) resulting altered HDL cholesterol plasma levels. Epidemiological studies have 
unequivocally shown that high TG and low HDL-C represent strong independent risk factors 
of CVD. (13) Emerging evidence suggests that TG and HDL lipoproteins are strong 
interrelated biological parameters and linked to glucose homeostasis.  Circulating triglycerides 
in part derive from VLDL-secretion from the liver as well as from Free Fatty Acid (FFA) 
uptake in the small intestine, a metabolic flux that is highly regulated by insulin (14). In obese 
insulin resistant subjects, insulin-mediated suppression of VLDL-synthesis and secretion is 
reduced resulting in high plasma TG and TG accumulation in the liver (15). Finally, SGLT2 is 
widely expressed throught the human body including in the small intestine (near GLP1 
producing L cells) and in the liver (16). Thus suggesting that SGLT2 inhibitors can also affect 
glucose and lipid uptake in the gut. 
We thus propose to further dissect the underlying mechanisms regarding dyslipidemic effects 
of SGLT2 inhibitors in relation to insulin sensivitity and energy metabolism in uncomplicated 
DM2 patients. This will be done using previously published stable isotope based techniques to 
study VLDL, LDL and HDL synthesis (15)  as well as for determinination of hepatic and 


Clinical Study Protocol DICE study, © M. Nieuwdorp, AMC-Vumc Diabetes Center, Amsterdam, the Netherlands 
Drug Substance Dapagliflozin 10mg once dailys on top of Crestor 5mg once daily;  a pilot study 
Study Code  01 
Edition Number 05 
Date 25-06-2015 
 
11
peripheral insulin sensivitiy (5). All of these stable isotopes are GMP produced and analyses 
techniques have been previously used and published by our group (17, 18 and 19). 
 
1.2 
Rationale for study design, doses and control groups 
Study Hypothesis and rationale  
Dapagliflozin 10mg once daily for 5 weeks increases hepatic LDL and VLDL secretion driven 
by altered CETP in DM2 patients on stable statin therapy (10mg rosuvastatin once daily)  
Study design:  
Male or postmenopauzal female patients with type 2 diabetes (BMI > 25 kg/m2 and more than 
12 weeks a stable daily dose of metformin treatment > 1500mg) with good glycemic control 
(HbA1C ≥6.5% - <8.5% and fasting plasma glucose <132 mmol/l) and on stable statin 
regiment and willing to switch  to crestor 5 mg once daily during the study. After 4 weeks of 
rosuvastatin treatment (20), baseline measurements for glucose and lipid fluxes will be 
performed and therafter DM2 patients will be randomized to receive 10 mg dapagliflozin 
(n=12) orally with rosuvastatin 10mg once daily. Measurements (lipid and glucosefluxes) will 
be repeated after 5 weeks of dapagliflozin treatment. 
 
Sample size  
Previous studies have reported a  10 % increase in fasting plasma LDL plasma levels upon 
dapagliflozin 10mg once daily treatment in DM2 patients on statin therapy (20)   As it can 
expected that metabolic effects of SGLT2 inhibition stabilize after 4 weeks (5,6) and lipid 
fluxes upon intervention are usually determined upon 4-5 weeks after start of intervention 
(21), we suggest to treat for 5 weeks. .A reduction was seen in 8 DM2 subjects of  plasma 
LDL (from 3.1 ± 0.8 to   1.5 ± 0.4 mmol/l)  on rosuvastatin treatment, that corresponded with 
a similar decrease in  LDL- ApoB poolsize /synthesis (2.8 ± 0.4 to 1.5  ± 0.4  gram/day, see 
ref 22) . We thus expect  10% higher plasma LDL level (from 3.1 ± 0.8 to 1.7 ± 0.4 mmol/l ) 
with concomitant less decrease in LDL- ApoB poolsize /synthesis (1.8 ± 0.4   gram/day) upon 
rosuvastatin  combined with dapagliflozin 10mg treatment in DM2. Using single sided test 
(with alfa of 0.05  and  85% power) and using difference in LDL-apoB synthesis of 0.3 
gram/day with SD of 0.4, the sample size needs to be 11 DM2 subjects, on dapagliflozin 10mg 
treatment. Taking a 10% patient dropout rate, we aim to include 12 DM2 subjects in total.  
 
Benefit/risk and ethical assessment 
Both rosuvastatin and dapagliflozin have been approved by FDA/EMEA and are widely 
prescribed. The effect of rosuvastatin alone on lipidfluxes in this group of DM2 patients has 
already been established (22) which thus allows us to study the mere effect of SGLT2 on 


Clinical Study Protocol DICE study, © M. Nieuwdorp, AMC-Vumc Diabetes Center, Amsterdam, the Netherlands 
Drug Substance Dapagliflozin 10mg once dailys on top of Crestor 5mg once daily;  a pilot study 
Study Code  01 
Edition Number 05 
Date 25-06-2015 
 
12
lipidfluxes in DM2 patients on stable statin therapy in a single arm trial. The use of stable 
isotope infusion is not associated with adverse events. Thus, the risk for patients to participate 
in this study is minimal. 
1.3 
 
Study Design  
 
 
  
 
Figure 1 
 
 
Study day 1: Assessment of cholesterol fluxes 
VLDL, LDL, HDL kinetics:  Subjects will drink 2H2O (2g per kg body weight), in the 
evening before the study day. First a blood sample will be drawn for determination of 
background enrichments. The subjects will sip the deuterated water between 18.00 and 
22.00h. Thereafter, they are allowed to drink deuterated water (0.5% enriched) only to prevent 
dilution of the isotopic label. 


Clinical Study Protocol DICE study, © M. Nieuwdorp, AMC-Vumc Diabetes Center, Amsterdam, the Netherlands 
Drug Substance Dapagliflozin 10mg once dailys on top of Crestor 5mg once daily;  a pilot study 
Study Code  01 
Edition Number 05 
Date 25-06-2015 
 
13
The next day, subjects will be admitted to the metabolic unit at 7.30 AM after a 10 hour 
overnight fast. A catheter will be inserted into an antecubital vein for infusion of the stable 
isotope tracers. Another catheter will be inserted into a contralateral hand vein and kept in a 
thermo-regulated (60 C) plexiglas box for sampling of arterialized venous blood. Saline will 
be infused as NaCl 0.9% at a rate of 50 mL/h to keep the catheter patent. At T=0 (8.00 AM) a 
bolus of 7mg/kg of L[ 5.5.5-2H3]-leucine(99% enriched; Cambridge isotopes, Andover, USA) 
and simultaneously a [1,1,2,3,3-2H5] glycerol (99% enriched; Cambridge isotopes, Andover, 
USA) bolus of 500mg will be given. (23,24)  
At T=0:02,0:04,0:06,0:08,0:10,0:12,0:15,0:20,0:30,0:45, 1,2,3, 4, 6, 8, 10 and 12h blood 
samples will be drawn for the determination of [2H3] leucine concentration in plasma. For the 
measurement of [2H3]-leucine and [2H5] glycerol in VLDL, LDL and HDL, blood samples 
will be drawn at T=-0.30, 0:30, 0:45, 1, 1:15, 1:30, 2, 2:30, 3, 4, 5, 6, 7, 8, 10, and 12h. In 
addition, at the above time-points blood samples will be drawn for measurement of plasma 
cholesterol, cholesteryl esters, FFA, apoB, apoAI and glycerol. At T=0, 4, 8 and 12h the 
particle composition and Apo-B mass of the VLDL and LDL fractions will be determined and 
the apoAI composition of HDL. See the study outline shown in figure 2. 
At T=2.00 subjects will be served an oral fat load containing the dietary triglyceride tracer 
1,2,3,4-13C16 – palmitate (1-2 grams). The meal will consist of bread, butter, cheese, low fat 
milk and tea or coffee (63 grams carbohydrates, 56 grams fat (P/S ratio 0.11) and 35.9 grams 
protein). Blood samples are drawn up to 8 hours after the meal (see figure 2). 
During the test only deuterated water will be served ad libitum and the subjects will remain 
physically inactive. After the last blood sample at T=12h patients will be offered a 
meal.  Time series data from enrichments of plasma leucine, leucine in apoB100 from VLDL 
and LDL and glycerol in VLDL  and LDL are used as input to the kinetic model together with 
pool size measurements to simultaneously determine the kinetics of VLDL-TG and apo-B100 
and LDL apoB and cholesterol. (23,24).    
 
 
 


Clinical Study Protocol DICE study, © M. Nieuwdorp, AMC-Vumc Diabetes Center, Amsterdam, the Netherlands 
Drug Substance Dapagliflozin 10mg once dailys on top of Crestor 5mg once daily;  a pilot study 
Study Code  01 
Edition Number 05 
Date 25-06-2015 
 
14
 
Figure 2: study design VLDL-HDL kinetics and visit 2 and 5. 
 
 
 
Study day 2: 
Assessment of glucose metabolism and lipolysis during a 2-step hyperinsulinemic 
euglycemic clamp 
The clamp will be performed after an overnight fast. A catheter will be inserted into an 
antecubital vein for infusion of the stable isotope tracers (19). Another catheter will be 
inserted retrogradely into a contra lateral hand vein and kept in a thermo-regulated (60oC) 
plexiglas box for sampling of arterialized venous blood. Saline will be infused as NaCl 0.9% 
at a rate of 50 mL/h to keep the catheter patent. [6,6-2H2] glucose will be used as a glucose 
tracer (>99% enriched; Cambridge Isotopes, Andover, USA) to study total glucose production 
and insulin sensitivity. [1,1,2,3,3-2H5]-glycerol (>99% enriched; Cambridge Isotopes, 
Andover, USA) will be used to study glycerol turnover (as a measure of total triglyceride 
hydrolysis/lipolysis). 
At T = -2, blood samples will be drawn for determination of the background enrichments and 
primed continuous infusions will be started: [6,6-2H2] glucose (prime =100 minutes of 
continuous infusion; continuous, 0.11 μmol/kg·min); [1,1,2,3,3-2H5]- glycerol (prime, 1.6 
μmol/kg; continuous, 0.11 μmol/kg·min) and continued until the end of the study. At T=0, 
blood samples will be drawn for the determination of enrichments of glucose and glycerol 3 
times with a 5 min interval. In addition, blood samples will be drawn for measurements of 
glucoregulatory hormones, free fatty acids (FFA), inflammatory markers and adipokines. 


Clinical Study Protocol DICE study, © M. Nieuwdorp, AMC-Vumc Diabetes Center, Amsterdam, the Netherlands 
Drug Substance Dapagliflozin 10mg once dailys on top of Crestor 5mg once daily;  a pilot study 
Study Code  01 
Edition Number 05 
Date 25-06-2015 
 
15
Resting energy expenditure (REE) will be measured during the final 20 minutes of these 2 
hours and during the final 20 minutes of the study day. This will be done by indirect 
calorimetry. Oxygen consumption and CO2 production will be measured using a ventilated 
hood system. These measurements are then used to calculate REE and the respiratory 
quotient.At T=0:15, infusion of insulin (20mU/m2·min; Actrapid 100 IU/ml; Novo Nordisk 
Farma B.V., Alphen aan den Rijn, the Netherlands) and glucose 20% (to maintain a plasma 
glucose level of 5 mmol/L) will be started. [6,6-2H2] glucose will be added to the 20% 
glucose solution to achieve glucose enrichments of 1% to approximate the values for 
enrichment reached in plasma and thereby minimizing changes in isotopic enrichment due to 
changes in the infusion rate of exogenous glucose. Plasma glucose levels will be measured 
every 10 minutes at the bedside. At T=2:15, 5 blood samples with an interval of 5 minutes will 
be drawn to determine glucose enrichments, glucoregulatory hormones, free fatty acids (FFA). 
At T=2:35 the insulin infusion will be increased (60mU/m2·min; Actrapid 100 IU/ml; Novo 
Nordisk Farma B.V., Alphen aan den Rijn, the Netherlands). Plasma glucose levels will be 
measured every 10 minutes at the bedside. At T=4:35, 5 blood samples with an interval of 5 
minutes will be drawn for measurements of above mentioned parameters. At T= 4:55 insulin 
and the isotope tracers will be stopped and patients will be offered a bread meal. The glucose 
infusion will be tapered down to avoid hypoglycaemia.The stable glucose isotope ([6,6-
2H2]glucose) will be obtained at the hospital pharmacy (AMC) and prepared for infusion by 
the coordinating investigator (19). 
 
MRI based Quantification of intrahepatic triglycerides (IHTG) 
IHTG will be quantified by 1H-MRS on study day 2 before the clamp procedure. 1H- MRS 
spectra will be acquired using a 1.0T Tesla open MRI (25). During the measurements, 
participants remain in the supine position within the MRI scanner. IHTG content will be 
obtained using single-voxel 1H-MRS, using a body array coil as the transmitter and phased 
surface coils as receivers. MRS measurements will be acquired during breath-hold, using 
single-voxel stimulated acquisition mode. Volumes of interest in the liver will be located 
away from major vascular structures and bile ducts. The water and fat resonance peaks, 
located at 4.65 and 1.3 ppm, will be integrated using jMRUI software and relative fat content 
will be expressed as the ratio of the fat peak area over the cumulative water and fat peak areas. 
Calculated peak areas of water and fat will be corrected for T2 relaxation (T2water, 34 ms, 
T2fat, 68 ms) and the percentage hepatic fat content will be calculated. The measurements 
will take approximately 30 minutes. 
 
 
 


Clinical Study Protocol 
Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
16
2. 
STUDY OBJECTIVES 
Primary objective Objectives 
Primary Objective: 
Outcome Measure: 
effect of 5 weeks dapagliflozin 10mg on LDL 
cholesterol in patients with DM2 
Change in LDL synthesis   
 
 
 
 
Secondary Objective: 
Outcome Measure : 
 
Effect of 5 weeks dapagliflozin 10mg on 
and Triglyceride cholesterol fluxes   
 
 
 
 
 
Effect of 5 weeks dapagliflozin 10mg on 
insulin sensitivity and energy expenditure 
 
 
Effect of 5 weeks dapagliflozin 10mg on 
liver fat content (MRI liver) 
  
Change in VLDL  secretion and clearance (as 
determined by 2H3 Leucine enrichment) and 
relation to plasma CETP, PLTP and HDL 
subfractions 
  
Change in De novo lipogenesis (as determined 
by 2H2O deuterated ‘heavy’ water) 
  
oral 1,2,3,4-13C16 – palmitate to measure FFA 
remnant uptake. 
 
Change in hepatic and peripheral insulin 
sensitivity (2 step Hyperinsulinemic 
normoglycemic clamp with 2H2 enriched 
glucose:)  and energy expenditure including 
carbohydrate oxidation and fatty acid oxidation 
rates in breathing air 
Change in liverfat MRI spectrum 
 
  
2.1 
Safety objectives 
Safety Objective: not applicable 
Outcome Measure : 


Clinical Study Protocol 
Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
17
n/a 
n/a 
 
 
 
2.2 
Exploratory objectives   
Exploratory Objective: 
Outcome Measure : 
n/a 
n/a 
 
 
3. 
SUBJECT SELECTION, ENROLMENT, RANDOMISATION, 
RESTRICTIONS, DISCONTINUATION AND WITHDRAWAL  
<<>> 
3.1 
Inclusion criteria 
Male or postmenopauzal female patients ; 
  
Type 2 diabetes mellitus (HbA1C ≥6.5% - <8.5%) 
At least 12 weeks a stable dose of metformin treatment > 1500mg FPG<132 mmol/l  
LDL cholesterol >2.5 mmol/l,  
Willing to switch used statin to crestor 10mg once daily for 9 weeks  
3.2 
Exclusion criteria 
History of cardiovascular event 
Smoking 
Creatinin clearance < 60ml/min 
Alcohol abuse (>4 units/day) 
AST or ALT elevation (>2.5x upper limit) 
3.3 
Subject enrolment and randomization 
  
 All patients will receive dapagliflozin 10mg once daily. 
3.4 
Procedures for handling incorrectly enrolled or randomized 
subjects 
Patients will be replaced by new patients until all 12 DM2 patients have completed the trial 


Clinical Study Protocol 
Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
18
3.5 
Methods for assigning treatment groups 
n/a 
3.6 
Methods for ensuring blinding 
n/a 
3.7 
Methods for unblinding  
n/a 
3.8 
Restrictions 
None 
3.9 
Discontinuation of investigational product 
  
3.9.1 
Procedures for discontinuation of a subject from investigational product  
Patients will be replaced by new patients until all 12 DM2 patients have completed the 
trial.Dropout patients will be followed up until 3 months after cessation of the trial. 
3.10 
Criteria for withdrawal  
none 
3.10.1 
Screen failures  
When patients do not match inclusion criteria, they will not be enrolled in the trial.  
3.10.2 
Withdrawal of the informed consent 
Patients will be replaced by new patients until all 12 DM2 patients have completed the 
trial.Dropout patients will be followed up until 3 months after cessation of the trial. 
3.11 
Discontinuation of the study 
Dropout patients will be followed up until 3 months after cessation of the trial. 
4. 
STUDY PLAN AND TIMING OF PROCEDURES  
 


Clinical Study Protocol 
Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
19
Table 1 Study Plan DICE study detailing the procedures 
Visit 
Visit window 
  Days –28 to 0 for Visit 
1 
 7 ±1 day for Visit 2& 3 
 7 ±1 day for Visits 4   
 7 ±1 day for Visit 5&6 
 
1  
Screening 
and start 
rosuvastatin 
2 
3 
4 
5 
6 
  
  
  
For details see 
Protocol 
Section 
Week 
 
0 
0 
2.5 
5 
5 
   
    
 
  
Day 
- 
  
  
  
  
   
  
  
  
 
Written informed consent 
(including tissue samples) 
X 
 
 
 
 
 
 
 
 
 
Demographics  
X 
 
 
 
 
 
 
 
 
 
Physical examination, 
height and weight 
X 
 
 
X 
 
X 
 
 
 
 
Medical/surgical 
history/ECG 
X 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 
X 
  
 
 
 
 
 
 
 
 
Fluxes and MRI 
 
X 
X 
 
X 
X 
 
 
 
 
Vital signs 
X 
X 
X 
X 
X 
X 
  
 
 
 
Start study treatment 
 
 
X 
 
 
 
 
 
 
 
Treatment 
dispensed/returned 
X 
X 
 
 
 
 
X 
  
  
 
Concomitant medication  
X 
X 
X 
X 
X 
X 
X 
  
  
 


Clinical Study Protocol 
Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
20
Visit 
Visit window 
  Days –28 to 0 for Visit 
1 
 7 ±1 day for Visit 2& 3 
 7 ±1 day for Visits 4   
 7 ±1 day for Visit 5&6 
 
1  
Screening 
and start 
rosuvastatin 
2 
3 
4 
5 
6 
  
  
  
For details see 
Protocol 
Section 
Week 
 
0 
0 
2.5 
5 
5 
   
    
 
  
Day 
- 
  
  
  
  
   
  
  
  
 
Adverse event review (AEs 
and SAEs) 
 
X 
X 
X 
X 
X 
  
  
  
 
Blood samples for 
haematology and clinical 
chemistry   
X 
X 
X 
X 
X 
X 
  
  
 
 
24h Urinalysis 
  
X 
X 
  
X 
X 
  
  
 
 
  
 
  
 
 
 
 
 
 
 
 
 
 


Clinical Study Protocol 
Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
21
4.1 
Enrolment/screening period 
Q3 2015  
4.2 
Treatment period 
5 weeks 
 
4.3 
Follow-up period 
none 
5. 
STUDY ASSESSMENTS   
  
5.1 
Efficacy assessments 
Change in lipolysis  using plasma VLDL-TG clearance  (as determined by 2H5 glycerol 
enrichment) 
Change in VLDL-TG secretion and clearance (as determined by 2H3 Leucine enrichment) in 
relation to plasma CETP, PLTP and HDL subfractions 
Change in cholesterol mobility (13C cholesterol enrichment in LDL and HDL) 
  
Change in De novo lipogenesis (as determined by 2H2O deuterated ‘heavy’ water)   
oral 1,2,3,4-13C16 – palmitate to measure FFA remnant uptake. 
5.2 
Safety assessments 
5.2.1 
Laboratory safety assessments 
  
  
 


Clinical Study Protocol 
Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
22
Haematology/Haemostasis (whole blood) 
Clinical Chemistry (serum or plasma) 
B-Haemoglobin (Hb) 
S/P-Creatinine 
B-Leukocyte count 
S/P-Bilirubin, total 
B-Leukocyte differential count (absolute count) 
S/P-Alkaline phosphatise (ALP) 
B-Platelet count 
S/P-Aspartate transaminase (AST) 
 
S/P-Alanine transaminase (ALT) 
Urinalysis (dipstick) 
S/P-Albumin 
U-Hb/Erythrocytes/Blood  
S/P-Potassium 
U-Protein/Albumin  
S/P-Calcium, total 
U-Glucose  
S/P-Sodium 
 
  
>> 
5.2.2 
Physical examination 
Regular  
5.2.3 
ECG 
  
  
5.2.3.1 
Resting 12-lead ECG 
Done on screeningsvisit 
5.2.4 
Vital signs 
Will be taken according to AMC Vascular Medicine SOP  
5.2.4.1 
Pulse and blood pressure 
Will be taken according to AMC Vascular Medicine SOP  
5.2.4.2 
Body temperature 
Will not be taken routinely 
5.2.5 
Other safety assessments 
  
 
n/a 
5.3 
Other assessments 
  
5.3.1 
Patient reported outcomes 
Not used 


Clinical Study Protocol 
Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
23
5.4 
Pharmacokinetics 
5.4.1 
Collection of samples 
n/a 
5.4.2 
Determination of drug concentration 
n/a 
5.4.3 
Storage and destruction of pharmacokinetic samples 
n/a 
5.5 
Pharmacodynamics 
 
5.5.1 
Collection of samples 
n/a 
5.5.2 
Storage, re-use and destruction of pharmacodynamic samples 
n/a 
5.6 
Pharmacogenetics 
 
5.6.1 
Collection of pharmacogenetic samples 
n/a 
5.6.2 
Storage, re-use and destruction of pharmacogenetic samples 
n/a 
5.7 
Biomarker analysis  
 
5.7.1 
Storage, re-use and destruction of biological samples 
Samples will be stored at -80C at AMC and used for analyses. Upon 5 years after 
completion of the study, samples will be destroyed. 
5.7.2 
Labelling and shipment of biological samples 
Samples will be  labelled according to entry number; upon shipment, they will be only 
be shipped by Worldcourier 
5.7.3 
Chain of custody of biological samples  
Samples will be owned by AMC hospital 


Clinical Study Protocol 
Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
24
5.7.4 
Withdrawal of Informed Consent for donated biological samples  
Samples will be destroyed upon retraction of informed consent. 
6. 
SAFETY REPORTING AND MEDICAL MANAGEMENT 
  
6.1 
Definition of adverse events 
  
Adverse events are defined as any undesirable experience occurring to a subject during 
a clinical trial, whether or not considered related to the investigational drug or deterioraton of 
a pre-existing medical condition. All adverse events reported spontaneously by the subject or 
observed by the investigator or his staff will be recorded. 
  
6.2 
Recording of adverse events 
Will be recorded in CRF 
6.2.1 
Time period for collection of adverse events 
During 9 weeks of being in the study 
6.2.2 
Follow-up of unresolved adverse events 
Patients will be followup until 3 months after end of trial visit 
6.2.3 
Variables  
Adverse events are defined as any undesirable experience occurring to a subject 
during a clinical trial, whether or not considered related to the investigational drug. 
All adverse events reported spontaneously by the subject or observed by the 
investigator or his staff will be recorded.  The following variables should be 
collected for each AE; 
 
AE (verbatim) 
 
The date and time when the AE started and stopped 
 
Whether the AE is serious or not 
 
Investigator causality rating against the Investigational Product (yes or no) 
 
Action taken with regard to investigational product 
 
AE caused subject’s withdrawal from study (yes or no) 


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Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
25
 
Outcome. 
 
In addition, the following variables should be collected for SAEs: 
 
Date AE met criteria for serious AE 
 
Date Investigator became aware of serious AE 
 
AE is serious due to... 
 
Date of hospitalisation 
 
Date of discharge 
 
Probable cause of death 
 
Date of death 
 
Autopsy performed 
 
Causality assessment in relation to Study procedure(s) 
 
Description of AE 
 
Causality to assessment in relation to study procedure(s) 
 
Causality assessment in relation to other medication 
 
(Causality assessment in relation to additional study drug) 
 
  
 
6.2.4 
Causality assessment 
6.2.5 
Causality will be assessed by study physician and Principle Investigator. 
6.2.6 
Disease progression 
Not applicable in such a short timeframe 
6.3 
Reporting of serious adverse events 
A serious adverse event is  an important medical event that may jeopardize the subject or may 
require medical intervention to prevent one of the outcomes listed above any untoward 
medical occurrence, effect or deterioration of a pre-existing medical condition that at any 
dose:  


Clinical Study Protocol 
Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
26
 results in death; 
 is life-threatening (at the time of the event); 
 requires hospitalization or prolongation of existing inpatients’ hospitalization; 
 results in persistent or significant disability or incapacity; 
 is a congenital anomaly or birth defect; 
  The Principal Investigator is responsible for ensuring that all staff involved in the 
study is familiar with the content of this section. In accordance to section 10, 
subsection 1, of the WMO, the investigator will inform immediately the subjects, the 
reviewing accredited MEC and concurrently AstraZeneca if anything occurs, on the 
basis of which it appears that the disadvantages of participation may be significantly 
greater than was foreseen in the research proposal. The study will be suspended 
pending further review by the accredited MEC/IRB, except insofar as suspension 
would jeopardise the subjects’ health. The investigator will take care that all subjects 
are kept informed. 
 
 
The investigator will report the SAE’s through the web portal ToetsingOnline to the 
accredited METC that approved the protocol, within 15 days after the investigator has first 
knowledge of the serious adverse reaction and concurrently to AZ. 
SAE’s that result in death or are life threatening should be reported expedited. The expedited 
reporting will occur not later than 7 days after the investigator has first knowledge of the 
adverse reaction. This is for a preliminary report with another 8 days for completion of the 
report Also, a concurrently SAE report and accompanying cover page will be send by e-mail  
(AEMailboxClinicalTrialTCS@AstraZeneca.com ) to AstraZeneca TCS Data Entry Site. 
On behalf of the sponsor, the PI will submit once a year an Annual Safety Report to the 
accredited MEC and competent authority. 
 
6.4 
Overdose 
Patients will be admitted to AMC hospital (known symptoms:  


Clinical Study Protocol 
Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
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6.5 
Pregnancy 
Pregnancy test will be performed at inclusion, since all females will be 
postmenopausal no checkup will be done thereafter 
6.5.1 
Maternal exposure 
Pregnancy test will be performed at inclusion, since all females will be 
postmenopausal no checkup will be done thereafter 
 
6.5.2 
Paternal exposure  
Will not be studied 
6.6 
Management of toxicities <<Dose Reductions>> 
Will not be studied 
6.7 
Study governance and oversight 
  
6.7.1 
Steering Committee 
Will not be installed 
 
6.7.2 
Data Monitoring Committee 
Study will be monitored by Clinical Research Unit of the AMC (head: JJ van Dalen) 
6.7.3 
Scientific Advisory Committee 
Will not be installed 
 
7. 
INVESTIGATIONAL PRODUCT AND OTHER TREATMENTS 
  
7.1 
Identity of investigational product(s) 
 
Investigational product 
Dosage form and strength 
Manufacturer 
Dapagliflozin 
 
10mg 1dd 
 
Astrazeneca 


Clinical Study Protocol 
Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
28
Rosuvastatin 
10mg 1dd 
Astrazeneca 
<<>> 
7.2 
Dose and treatment regimens 
10mg dapagliflozin once daily for 5 weeks ; rosuvastatin 10mg once daily for 9 weeks. Both 
will be supplied by AstraZeneca Netherlands, free of charge. 
7.3 
Labelling 
Will be done by AMC clinical pharmacy 
7.4 
Storage 
Will be done at AMC clinical pharmacy 
 
7.5 
Compliance 
Will be checked by physician and nurse countil returned pills 
7.6 
Accountability 
Will be done by AMC clinical pharmacy 
 
7.7 
Concomitant and other treatments  
  
Restricted Medication/Class of drug: 
Usage: 
insulin 
  
 
 
 
Prohibited Medication/Class of drug: 
 
insulin 
 
 
 
 
Rescue/Supportive Medication/Class of 
drug: 
Usage: 
n/a 
  


Clinical Study Protocol 
Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
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7.7.1 
Other concomitant treatment 
  
7.8 
Post Study Access to Study Treatment 
Both rosuvastatin and dapagliflozin can be prescribed by physicians in the Netherlands 
8. 
STATISTICAL ANALYSES  
  
8.1 
Statistical considerations 
8.2 
Sample size estimate 
Previous studies have reported a  10 % increase in fasting plasma LDL plasma levels 
upon dapagliflozin 10mg once daily treatment in DM2 patients on statin therapy (20)   
As it can expected that metabolic effects of SGLT2 inhibition stabilize after 4 weeks 
(5,6) and lipid fluxes upon intervention are usually determined upon 4-5 weeks after 
start of intervention (21), we suggest to treat for 5 weeks. .A reduction was seen in 8 
DM2 subjects of  plasma LDL (from 3.1 ± 0.8 to   1.5 ± 0.4 mmol/l)  on rosuvastatin 
treatment, that corresponded with a similar decrease in  LDL- ApoB poolsize 
/synthesis (2.8 ± 0.4 to 1.5  ± 0.4  gram/day, see ref 22) . We thus expect  10% higher 
plasma LDL level (from 3.1 ± 0.8 to 1.7 ± 0.4 mmol/l ) with concomitant less decrease 
in LDL- ApoB poolsize /synthesis (1.8 ± 0.4   gram/day) upon rosuvastatin  combined 
with dapagliflozin 10mg treatment in DM2. Using single sided test (with alfa of 0.05  
and  85% power) and using difference in LDL-apoB synthesis of 0.3 gram/day with 
SD of 0.4, the sample size needs to be 11 DM2 subjects, on dapagliflozin 10mg 
treatment. Taking a 10% patient dropout rate, we aim to include 12 DM2 subjects in 
total.  
 
8.3 
Definitions of analysis sets 
Before and after dapagliflozin treatment  
8.3.1 
Efficacy analysis set 
Effect dapagliflozin on HDL catabolic fractional rate and plasma CETP levels 
Effect dapagliflozin on triglyceride/VLDL synthesis rate and liver fat MRI signal 
Effect dapagliflozin on hepatic and peripheral insulin sensitivity 


Clinical Study Protocol 
Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
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8.3.2 
Safety analysis set 
n/a 
8.3.3 
PK analysis set 
n/a 
8.3.4 
PRO analysis set 
n/a 
8.4 
Outcome measures for analyses 
 
8.5 
Methods for statistical analyses 
  
8.5.1 
Analysis of the primary variable(s) 
All data will be analysed using SPSS for Windows, version 20.0 (SPSS Inc. Chicago, 
Illinois, USA). Multivariate analysis and ANOVA for repeated measures will be used. 
Wilcoxon’s signed-rank test will be used to compare results between the study groups. 
Data will be expressed as median and range. Spearman’s rank test will be used to 
calculate correlations. 
 
8.5.2 
Analysis of the secondary variable(s) 
All data will be analysed using SPSS for Windows, version 20.0 (SPSS Inc. Chicago, 
Illinois, USA). Multivariate analysis and ANOVA for repeated measures will be used. 
Wilcoxon’s signed-rank test will be used to compare results between the study groups. 
Data will be expressed as median and range. Spearman’s rank test will be used to 
calculate correlations. 
 
8.5.3 
Subgroup analysis (if applicable) 
n/a 
8.5.4 
Interim analysis 
N/a 
8.5.5 
Sensitivity analysis (if applicable) 
n/a 


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Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
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8.5.6 
Exploratory analysis (if applicable))  
n/a  
9. 
STUDY AND DATA MANAGEMENT  
  
9.1 
Training of study site personnel 
Personall will be trained according to NFU/BROK regulations. 
9.2 
Monitoring of the study 
Will be done by Clinical Research Unit of AMC 
9.2.1 
Source data 
Data will be stored in a SPSS based database, all source data can be verified by 
monitor upon request. 
9.2.2 
Study agreements 
AstraZeneca and AMC will draft a contract for this project. The sponsor will have 
no say in the analyses or publication of the data. 
9.2.3 
Archiving of study documents 
Source documents will be stored at AMC archive 
9.3 
Study timetable and end of study 
Study will run for 2-2.5 years  with another 3-6 months for data analyses/modelling 
9.4 
Data management  
  
Serious Adverse Event (SAE) Reconciliation 
All SAE will be reported to the accredited AMC IRB/MEC that approved the protocol, 
according to the requirements of the AMC IRB/MEC 
Data Management of genotype data 
n/a 
Data associated with human biological samples 
 According to local AMC rules 


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Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
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Management of external data 
10. 
ACCORDING TO LOCAL AMC RULESETHICAL AND 
REGULATORY REQUIREMENTS 
10.1 
Ethical conduct of the study 
The study will be approved by local AMC IRB (MEC) 
10.2 
Subject data protection 
All data will be anonymous (patients will receive entry number upon enrolment in the 
study) 
10.3 
Ethics and regulatory review 
See above 
10.4 
Informed consent 
The Principal Investigator(s) at each centre will: 
Delegate informed consent procedure to study physicians 
10.5 
Changes to the protocol and informed consent form 
IRB/MEC will be informed upon changes to procotol 
 
10.6 
Budget aanpassen 
 
Salary MD PhD student 27 months   
125k 
Patient participation+travel fee  
15k 
Stable isotopes (GMP produced)  
 
70k 
Biochemistry + MassSpec analyses   
10k:   
15% AMC overhead incl AMC pharmacy 
33k 
+ 
total Budget    
 
 
 
253.000 euro 
 


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Study Code DICE study 
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Study Code DICE study 
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Date 28-02-2015 
 
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Drug Substance Dapagliflozin  
Study Code DICE study 
EditionVersion 5 
Date 28-02-2015 
 
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