Metabolic Effects of the SGLT-2 Inhibitor Empagliflozin in Patients With Diabetic Nephropathy (MEDiaN)
Phase: PHASE3
Conditions: Diabetic Nephropathies
Sponsor: Singapore General Hospital

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CLINICAL TRIAL PROTOCOL 
 
PROTOCOL TITLE: Metabolic Effects of the SGLT-2 Inhibitor Empagliflozin in Patients 
with Diabetic Nephropathy (MEDiaN) 
 
 
PROTOCOL NUMBER: MEDiaN2018 
 
 
PROTOCOL VERSION:  
V1.3 
PROTOCOL DATE:  
9 December 2018 
 
PRINCIPAL INVESTIGATOR:  
Lam Yun Rui Amanda, Associate Consultant, Singapore General Hospital 
 
 
CO-INVESTIGATORS: 
Tan Hong Chang, Consultant, Singapore General Hospital  
Jean-Paul Kovalik, Assistant Professor, Duke-NUS Medical School  
Lim Paik Shia, Principal Clinical Pharmacist, Singapore General Hospital  
Choo Chon Jun Jason, Senior Consultant, Singapore General Hospital 
Kwek Jia Liang, Consultant, Singapore General Hospital 
 
STUDY TEAM MEMBERS 
Valerie Lai Jia Hui, Clinical Research Coordinator, Singapore General Hospital 
Vieon Wu Aini, Clinical Research Coordinator, Singapore General Hospital 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Table of Contents 
1 
BACKGROUND AND RATIONALE................................................................................................. 5 
1.1 
GENERAL INTRODUCTION ................................................................................................................... 7 
1.2 
RATIONALE AND JUSTIFICATION FOR THE STUDY ...................................................................................... 7 
1.2.1 
RATIONALE FOR THE STUDY PURPOSE ................................................................................................... 7 
1.2.2 
RATIONALE FOR DOSES SELECTED ........................................................................................................ 7 
1.2.3 
RATIONALE FOR STUDY POPULATION .................................................................................................... 7 
1.2.4 
RATIONALE FOR STUDY DESIGN ........................................................................................................... 8 
2 
HYPOTHESIS AND OBJECTIVES ................................................................................................. 8 
2.1 
HYPOTHESIS ................................................................................................................................... 8 
2.2 
PRIMARY OBJECTIVES ....................................................................................................................... 8 
2.3 
SECONDARY OBJECTIVES ................................................................................................................... 8 
2.4 
POTENTIAL RISKS AND BENEFITS: ........................................................................................................ 8 
2.4.1 
POTENTIAL RISKS ............................................................................................................................ 8 
2.4.2 
POTENTIAL BENEFITS ...................................................................................................................... 11 
3 
STUDY POPULATION ................................................................................................................ 11 
3.1 
LIST THE NUMBER AND NATURE OF SUBJECTS TO BE ENROLLED. ................................................................ 11 
3.2 
CRITERIA FOR RECRUITMENT AND RECRUITMENT PROCESS ....................................................................... 11 
3.3 
INCLUSION CRITERIA ...................................................................................................................... 12 
3.4 
EXCLUSION CRITERIA ...................................................................................................................... 12 
3.5 
SUBJECT REPLACEMENT ................................................................................................................... 13 
4 
STUDY DESIGN ......................................................................................................................... 14 
4.1 
RANDOMISATION AND BLINDING ........................................................................................................ 14 
4.2 
CONTRACEPTION AND PREGNANCY TESTING .......................................................................................... 14 
4.3 
STUDY VISITS AND PROCEDURES ....................................................................................................... 15 
4.3.1 
SCREENING VISITS AND PROCEDURES .................................................... ERROR! BOOKMARK NOT DEFINED. 
4.3.2 
STUDY VISITS AND PROCEDURES .......................................................... ERROR! BOOKMARK NOT DEFINED. 
4.3.3 
FINAL STUDY VISIT: .......................................................................... ERROR! BOOKMARK NOT DEFINED. 
4.3.4 
POST STUDY FOLLOW UP AND PROCEDURES .......................................................................................... 18 
4.4 
DISCONTINUATION/WITHDRAWAL ...................................................................................................... 19 
4.4.1 
DISCONTINUATION CRITERIA ............................................................................................................ 19 
4.4.2 
DISCONTINUATION VISIT AND PROCEDURES ......................................................................................... 19 
5 
TRIAL MATERIALS .................................................................................................................... 19 
5.1 
TRIAL PRODUCT (S) ....................................................................................................................... 19 
5.2 
STORAGE AND DRUG ACCOUNTABILITY ................................................................................................ 20 
6 
TREATMENT .............................................................................................................................. 20 
6.1 
RATIONALE FOR SELECTION OF DOSE .................................................................................................. 20 
6.2 
STUDY DRUG FORMULATIONS ........................................................................................................... 20 
6.3 
STUDY DRUG ADMINISTRATION ......................................................................................................... 20 
6.4 
SPECIFIC RESTRICTIONS / REQUIREMENTS ........................................................................................... 21 
6.5 
BLINDING .................................................................................................................................... 21 
6.6 
CONCOMITANT THERAPY .................................................................................................................. 21 
7 
SAFETY MEASUREMENTS.......................................................................................................... 21 

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7.1 
DEFINITIONS ................................................................................................................................ 21 
7.2 
COLLECTING, RECORDING AND REPORTING OF ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS TO CIRB .......... 22 
7.3 
COLLECTING, RECORDING AND REPORTING OF SERIOUS ADVERSE EVENTS (SAES) TO THE HEALTH SCIENCE 
AUTHORITY (HSA) .................................................................................................................................... 22 
7.4 
SAFETY MONITORING PLAN .............................................................................................................. 23 
7.5 
COMPLAINT HANDLING .................................................................................................................... 23 
8 
DATA ANALYSIS........................................................................................................................ 23 
8.1 
DATA QUALITY ASSURANCE .............................................................................................................. 23 
8.2 
DATA ENTRY AND STORAGE .............................................................................................................. 23 
9 
SAMPLE SIZE AND STATISTICAL METHODS ............................................................................. 24 
9.1 
DETERMINATION OF SAMPLE SIZE ...................................................................................................... 24 
9.2 
STATISTICAL AND ANALYTICAL PLANS ................................................................................................. 24 
10 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS .................................................................... 24 
11 
QUALITY CONTROL AND QUALITY ASSURANCE ...................................................................... 24 
12 
ETHICAL CONSIDERATIONS ..................................................................................................... 24 
12.1 
INFORMED CONSENT....................................................................................................................... 25 
12.2 
CONFIDENTIALITY OF DATA AND PATIENT RECORDS ............................................................................... 25 
13 
PUBLICATIONS ......................................................................................................................... 25 
14 
RETENTION OF TRIAL DOCUMENTS ......................................................................................... 25 
15 
FUNDING AND INSURANCE ...................................................................................................... 25 

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PROTOCOL SIGNATURE PAGE 
 
 
 
Protocol Title: Metabolic Effects of the SGLT-2 Inhibitor Empagliflozin in Patients with Diabetic 
Nephropathy 
 
 
 
Protocol Number: 
 
 
Protocol Version/ Date: V1.3 / 9th December2018 
 
 
Funding body: Tanoto Initiative For Diabetes Research Award (Duke-NUS-TIDR/2018/0010) 
 
 
 
 
Declaration of Investigator 
 
I confirm that I have read the above-mentioned protocol and its attachments. I agree to 
conduct the described trial in compliance with all stipulations of the protocol, regulations and 
ICH E6 Guideline for Good Clinical Practice (GCP) 
 
  
 
 
Principal Investigator Name:  LAM YUN RUI AMANDA  
                                                            
 
Principal Investigator Signature:  _______________________________________ 
                                                            
 
Date:  __________________________________________ 
                                                            
 
 
 
 
 
 
 

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1 
BACKGROUND AND RATIONALE 
 
Diabetic nephropathy (DN) is a common cause of end-stage renal disease (ESRD) and accounts for 
nearly half of all new patients starting dialysis in Singapore, the country with the highest rates of DN in the 
Asia-Pacific region. Despite the scale of the problem, little progress has been made in our understanding of 
the pathogenesis of the disorder and no new therapies have been offered.  
 
Dysregulated fuel metabolism as a major driver of diabetic nephropathy 
 
We have conducted a metabolomics study of human diabetic nephropathy that revealed evidence for 
alterations in mitochondrial fuel metabolism in patients with the disease, a finding also reported in other 
recent studies of human DN1,2. This study, which was conducted in collaboration with clinician-scientists at 
Khoo Teck Puat Hospital (KTPH) in Singapore, was a comprehensive metabolic profiling study of over 300 
human diabetic nephropathy patients using our tandem MS-based metabolomics platforms. The study used 
serum from patients with diabetic nephropathy and matched controls with diabetes but without nephropathy. 
The patients with diabetic nephropathy showed a metabolite pattern suggesting alterations in fatty acid 
oxidation (Figure 1).  
 
 
The metabolomics profiling study showed that patients with DN have elevated levels of several short chain 
acyl-carnitines (Figure 1, top right) including C3 (propionyl-), C4 (butyryl-) and C5 (isovaleryl-carnitine). This 
pattern of elevated short-chain acyl-carnitines resembles what is observed in several inherited metabolic 
disorders including short-chain acyl-coA dehydrogenase (SCAD).  
 
A similar analysis of urine from patients with DN demonstrate alterations in the abundance of several TCA-
cycle related organic acids (JJ Liu et al, manuscript under review). 
 
Based on this we hypothesize that dysregulated mitochondrial fuel oxidation is a major driver of 
Figure 1: Acyl-carnitine species in human subjects with diabetic nephropathy are elevated. The serum acyl-carnitine 
profile of subjects with type 2 diabetes and urine albumin-creatinine ratio >300 (n=149) was compared to the profile from 
matched controls (n=150) with diabetes of equal duration but no evidence of proteinuria. *p<0.05. 

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diabetic nephropathy.  
 
Renal glucose handling and the SGLT-2 inhibitors 
 
The SGLT-2 inhibitor (SGLT-2i) class of drugs is a recently approved treatment for diabetes, which lower 
blood glucose levels by inhibiting renal reabsorption of glucose by SGLT-2 transporters in the proximal 
renal tubule, resulting in glucosuria3.  
 
SGLT-2i and Diabetes Complications 
 
Clinical trials of SGLT-2i in patients with type 2 diabetes (T2DM) have shown unexpectedly strong effects of 
the drug on diabetes complications4,5. In the EMPA-REG OUTCOME trial, the SGLT-2i empagliflozin was 
able to i) reduce the risk of cardiovascular death and ii) slow the progression of DN in patients with 
diabetes. The effect of the drug on cardiac and kidney complications seems to be out of proportion to the 
modest glucose lowering ability of the drug. The reason for the protective effect on cardiac and kidney 
complications remains unexplained. 
 
SGLT-2i and Fuel Metabolism 
Attempts to explain how SGLT-2i protect against diabetes complications have focused on several aspects 
of the drug’s action including the effect on whole body fuel metabolism6–8. The forced excretion of glucose 
in the urine leads to unique physiologic changes. Patients on the drug have lower glucose and insulin levels 
and raised glucagon. There is reduced glucose use, increased lipolysis with a resultant rise in fatty acid 
oxidation and ketogenesis9. Interestingly, recent studies have also reported a rise in activity of the urea 
cycle10. The net effect is a major shift in fuel metabolism away from glucose towards the burning of fat and 
ketone bodies. The shift in fuel metabolism is of particular interest given our recent findings of altered fuel 
metabolism in patients with diabetic nephropathy.  
 
Study aims 
 
The aim of our proposed study is to examine the state of fuel metabolism in patients with DN both 
before and after the use of SGLT-2i. We anticipate that 30 days of treatment with the SGLT-2i 
empagliflozin will induce a shift in fuel metabolism in subjects with DN.  
 
Our hypothesis is that dysregulated fuel metabolism is a major driver of diabetic nephropathy. We propose 
that the beneficial effect of SGLT-2i in DN is related to changes in fuel metabolism. Our anticipated 
outcomes include reversal of many of the metabolomics findings reported in DN. We expect decreased 
fasting glucose and elevated serum ketones, as previously reported. We also predict reductions in the 
levels of short-chain acyl-carnitines, increased C4-OH and long-chain acyl-carnitines as well as reductions 
in urinary organic acid excretion. Unfortunately, the scope of this proposal is too short to expect significant 
changes in albuminuria, although this will be assessed. If completed successfully, this project will help to 
link changes in mitochondrial fuel metabolism with the beneficial effects of the SGLT-2i class of drugs.  
 
Our goals are to better understand the contribution of fuel metabolism to the development of DN, and to 
determine if changes to fuel metabolism can have a positive impact on this disease. 
 
 
 

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1.1 General Introduction 
 
Empagliflozin is a SGLT-2 inhibitor which is indicated as an adjunct to diet and exercise to improve 
glycemic control in adults with type 2 diabetes mellitus, and to reduce the risk of cardiovascular death in 
adult patients with type 2 diabetes mellitus and established cardiovascular disease. By inhibiting sodium-
glucose co-transporter 2 (SGLT2), the predominant transporter responsible for reabsorption of glucose from 
the glomerular filtrate back into the circulation, empagliflozin reduces renal reabsorption of filtered glucose, 
thereby increasing urinary glucose excretion. Presently empagliflozin is not indicated for the treatment of 
diabetic nephropathy (DN), although it has been shown in previous studies to have a beneficial effect on 
DN. 
 
 
1.2 Rationale and Justification for the Study 
 
We hypothesize that treatment with empagliflozin taken orally at 10mg per day for 30 days will lead to 
significant changes in the circulating metabolomics pattern in patients with DN. 
 
Since we are interested in understanding the metabolic effects of the drug, we propose to administer the 
drug for a period of 30 days. We will perform a comprehensive analysis of the state of fuel metabolism in 
these patients before, during and after the administration of empagliflozin using targeted metabolomics and 
other approaches. Our goal is to discover key changes in fuel metabolism in DN patients receiving 
empagliflozin.  
 
 
1.2.1 Rationale for the Study Purpose 
 
 
Clinical trials of SGLT-2i in patients with type 2 diabetes (T2DM) have shown unexpectedly strong effects of 
the drug on diabetes complications, including slowing the progression of DN. The mechanisms underlying 
the protective effect on renal complications of diabetes have not been fully elucidated. 
 
Existing literature suggest that the forced excretion of glucose in the urine resulting from SGLT-2i use leads 
to unique physiologic changes, leading to a major shift in fuel metabolism away from glucose towards the 
burning of fat and ketone bodies. The shift in fuel metabolism is of particular interest given our recent 
findings of altered fuel metabolism in patients with diabetic nephropathy.  
 
The aim of our study is to examine the state of fuel metabolism in patients with DN both before and after the 
use of SGLT-2i. If completed successfully, this study will help to link changes in mitochondrial fuel 
metabolism with the beneficial effects of the SGLT-2i class of drugs. A better understanding of the 
relationship between mitochondrial fuel metabolism, DN, and the metabolomic effects of SGLT-2i may point 
a way to more therapies for DN which target mitochondrial fuel metabolism.  
 
1.2.2 Rationale for Doses Selected 
 
The dose of empagliflozin 10mg per day is selected as it is the recommended initiation dose according to 
the prescribing information provided by the manufacturers. The dose selection is similar to dosages used in 
patients in standard clinical practice and in large clinical trials.  
 
1.2.3 Rationale for Study Population 
 
DN is the most common cause of end-stage renal disease (ESRD) in the US and also accounts for nearly 

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half of all new patients starting dialysis in Singapore, the highest in the Asia-Pacific region. ESRD is also a 
major cause of death, accounting for over 65% of deaths in T1DM patients with nephropathy and the 5-year 
survival rate being typically less than 10% in elderly patients with T2DM. Despite these sobering statistics, 
little progress has been made in early diagnostic markers or the availability of increased treatment options 
for the clinical management of DN. Current mainstay of therapy relies on control of hyperglycemia and 
blood pressure and pharmacologic blockade of the renin-angiotensin system (RAS) with ACE inhibitors or 
angiotensin receptor blockers (ARBs).  Despite these treatments, DN typically progresses over time. Finally, 
no new therapies for DN have been offered the past few years and to our knowledge, there are only a 
couple of ongoing clinical trials in the US for potential new treatments for DN (clinicaltrials.gov). 
 
 
1.2.4 Rationale for Study Design 
 
We will conduct a single-arm open-label intervention study to examine the effects of a standard dose of 
empagliflozin on fuel oxidation patterns in patients with type 2 diabetes and DN. No randomization or 
blinding will be performed as the response to empagliflozin is stereotypical and the measured parameters 
are objective and thus not subject to observer bias. 
 
2 
HYPOTHESIS AND OBJECTIVES 
 
2.1 Hypothesis 
 
Our study hypothesis is that dysregulated mitochondrial fuel oxidation, as measured by changes in the 
circulating metabolomics pattern of DN patients, is a major driver of diabetic nephropathy. Furthermore, we 
hypothesize that treatment with empagliflozin over a period of 30 days will reverse the metabolomics 
changes seen in patients with DN.   
 
2.2 Primary Objectives 
 
The primary objective is to study the effects of empagliflozin on fuel metabolism patterns in patients with 
DN. We will conduct a single arm intervention study to examine the effects of a standard dose of 
empagliflozin on fuel oxidation patterns in patients with type 2 diabetes and DN. Since we are interested in 
understanding the metabolic effects of the drug, we propose to administer the drug for a period of 30 days. 
We will perform a comprehensive analysis of the state of fuel metabolism in these patients before and after 
the administration of empagliflozin using targeted metabolomics and other approaches. Our goal is to 
discover key changes in fuel metabolism in DN patients receiving empagliflozin. 
 
 
2.3 Secondary Objectives 
 
N.A. 
 
2.4 Potential Risks and Benefits:  
 
2.4.1 Potential Risks 
Venepuncture 
This study requires blood to be drawn from a vein in the arm. For most people, blood draws using needle 
punctures do not cause any serious problems. However, drawing blood may result in pain at the point of 

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puncture, a feeling of faintness, irritation of the vein and bruising or bleeding at the site of needle puncture. 
There is also a very slight possibility of an infection at needle puncture site. An experienced staff will 
perform these procedures and every precaution will be taken to prevent infection. 
Indirect calorimetry 
Indirect calorimetry involves the placement of a transparent plastic hood with ventilation over the head for 
30-minute intervals during the 2nd and 3rd study visit. It is well tolerated but some people may feel 
claustrophobic. To avoid this, a brief trial run will be performed for subjects to familiarize with the equipment 
and measurement. 
 
Adverse Effects Related to empagliflozin 
A) Allergic Reactions 
All drugs have a potential risk of an allergic reaction. Allergic reactions range from mild reactions such as 
skin itchiness, rash, eye swelling to more severe allergic reactions that affects the blood pressure (allergic 
anaphylaxis). Severe allergic reactions could become life threating if not treated quickly.   
 
B) Hypotension 
Empagliflozin causes intravascular volume contraction. The incidence of symptomatic hypotension is 
greater in patients with renal impairment, elderly individuals, in patients with low systolic blood pressure, 
and patients on diuretics. Subjects will be advised to avoid prolonged periods of fasting or water 
deprivation. They will be advised to maintain an adequate intake of water especially during periods of 
greater insensible fluid loss (eg: exercise or hot weather). All subjects will be educated about signs and 
symptoms of hypotension. Individuals above age 65 years, or with more severe renal impairment will be 
excluded from this study. 
 
C) Ketoacidosis 
Reports of ketoacidosis, a serious condition requiring urgent hospitalization have been reported in patients 
taking empagliflozin. Some of these cases were identified in patients with type 1 diabetes, in whom 
empagliflozin is not indicated. We have excluded patients with type 1 diabetes from our study. In patients 
with T2DM receiving empagliflozin, the FDA identified potential triggering factors for ketoacidosis including 
intercurrent illness, reduced food and fluid intake due to illness or surgery, reduced insulin doses, history of 
alcohol intake, and pancreatic disorders suggesting insulin deficiency (eg: type 1 diabetes, history of 
pancreatitis, pancreatic surgery). All subjects will be assessed by a physician for factors that may 
predispose to ketoacidosis, including pancreatic insulin deficiency from any cause, caloric restriction, and 
alcohol abuse. Subjects in whom these factors are identified will be excluded from the study. All subjects 
will be educated to temporarily discontinue empagliflozin in situations known to predispose to ketoacidosis 
(eg: intercurrent illness, prolonged fasting due to acute illness or surgery), and how to identify symptoms 
suggestive of ketoacidosis and to seek medical attention. Subjects may contact the study team should they 
fall ill. The study team will advise patients regarding period of discontinuation of study drug, if applicable.   
 
 
D) Acute kidney injury and impairment in renal function 
Treatment with empagliflozin is known to cause an initial mild fall in eGFR which is related to one of its 
underlying mechanism of actions in altering tubuloglomerular feedback. This initial mild fall in eGFR is 
followed by a progressive recovery of GFR during the following weeks. Empagliflozin may also result in 
acute kidney injury or significantly increased serum creatinine as a result of intravascular volume 
contraction. Factors that may predispose to acute kidney injury include more severe renal impairment, 

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congestive heart failure, and some concomitant medications (including diuretics, ACE inhibitors, 
angiotensin receptor blockers, and NSAIDs). Subjects with more severe renal impairment will be excluded 
from this study. Subjects will be educated on situations that predispose to acute kidney injury, such as 
reduced oral intake or increased fluid losses, and to temporarily discontinue empagliflozin if these occur. All 
subjects will have their kidney function tested before and monitored during this study. 
 
E) Genital mycotic infections, urinary tract infections, urosepsis and pyelonephritis 
Treatment with SGLT-2i increases the risk for genital mycotic infections and urinary tract infections, which 
are usually mild. Serious urinary tract infections including urosepsis and pyelonephritis requiring 
hospitalization in patients receiving SGLT-2i have been described. Patients with a history of recurrent 
urinary tract or genital mycotic infections, and patients with a history of serious urinary tract infections will 
be excluded from this study.  
 
F) Fournier’s gangrene 
On 29th August 2018, the U.S. Food and Drug Administration (FDA) warned that cases of a rare but serious 
infection of the genitals and area around the genitals have been reported with SGLT2 inhibitors. This 
serious rare infection, called necrotizing fasciitis of the perineum, is also referred to as Fournier’s gangrene. 
In the five years from March 2013 to May 2018, 12 cases of Fournier’s gangrene were identified in patients 
taking an SGLT2 inhibitor (7 men and 5 women), all of whom were hospitalized and required surgery. In 
2017, an estimated 1.7 million patients received a dispensed prescription for an SGLT2 inhibitor from U.S. 
outpatient retail pharmacies. Fournier’s gangrene developed within several months of the patients starting 
an SGLT2 inhibitor. In accordance with recommendations made by the U.S. FDA, patients will be 
counselled regarding the risk of Fournier’s gangrene before commencing treatment with empagliflozin. 
Patients will be counselled to seek medical attention immediately if they experience any symptoms of 
tenderness, redness, or swelling of the genitals or the area from the genitals back to the rectum, and have a 
fever above 38.0 degrees Celsius, or a general feeling of being unwell.  
 
G) Hypoglycemia 
Treatment with empagliflozin is expected to result in a fall in blood glucose. Hypoglycemia is a potential 
side effect of all glucose-lowering medications, including empagliflozin. Patients who are at high risk of 
significant hypoglycaemia will be excluded from the study. Patients at high risk of significant hypoglycaemia 
include those with 1 or more episodes of severe hypoglycaemia in the last 1 year, and frequent (at least 
once a week) hypoglycaemia episodes of any severity. 
 
Issues Related to Pregnancy and Breast-Feeding 
There is inadequate data about the use of empagliflozin in pregnant or breast-feeding women. Therefore, 
pregnant or breast-feeding women cannot participate in this study. Sexually active women of childbearing 
potential must use a reliable method of birth control while participating in this study. Reliable methods of 
birth control are considered to be abstinence (not having sex), oral contraceptives (the pill), intrauterine 
device (IUD), DepoProvera, Norplant, tubal ligation (tubes tied) or vasectomy of the partner. An acceptable 
although less reliable method, involved the careful use of condoms and/or a spermicidal foam or gel along 
with a diaphragm, cervical cap, or sponge. Study subjects would be encouraged to discuss this issue 
further with their doctor if they any questions.  
 
Issues Related to Pediatric Use 
The safety and effectiveness of empagliflozin in pediatric patients under 18 years of age have not been 
established. Patients under 21 years of age will be excluded from this study. 
 

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2.4.2 Potential Benefits 
There is unlikely any direct benefit from participating in this study as treatment with empagliflozin for one 
month is unlikely to provide any long-term benefit. However, completion of the study will provide us with a 
better understanding of the relationship between empagliflozin use and mitochondrial fuel oxidation, and the 
biochemical changes surrounding the development of DN. The results of the study may point a way to more 
therapies for DN which target mitochondrial fuel metabolism. 
 
 
3 
STUDY POPULATION 
 
3.1       List The Number and Nature of Subjects to be Enrolled.  
Assuming that treatment with empagliflozin would result in a significant shift in metabolic fuel selection, 40 
subjects with DN will be required. Assuming a screen failure rate of 1 in 3, a total of 60 subjects will need to 
be recruited from the Singapore General Hospital (SGH) Diabetes and Metabolism Centre (DMC) clinics. 
There are no subject restrictions based on race of the subject in this study.   
 
 
3.2      Criteria for Recruitment and Recruitment Process 
 
This will be a single-centre study conducted in SGH. Subjects from this trial will be patients attending the 
Diabetes and Metabolism Centre (DMC) clinics. Physicians at the DMC can refer any interested and 
suitable patients to the study team.   
 
The study team will provide additional details regarding this study to all interested patients and they will be 
scheduled for consent taking and screening visit at the SGH Clinical Trials Research Centre (CTRC).  
 
 

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3.3      Inclusion Criteria 
 
Subject must meet all of the inclusion criteria listed below to participate in this study:   
 
1. Man or woman between 21 and 65 years of age 
 
2. Type 2 diabetes mellitus as defined by: 
- 
Fasting plasma glucose ≥7.0mmol/l, or 
- 
Symptoms of hyperglycemia with casual plasma glucose ≥11.1 mmol/L, or 
- 
2-hour plasma glucose ≥11.1 mmol/l after a 75-gram oral glucose load, or  
- 
Known type 2 diabetes mellitus diagnosed by a medical practitioner 
 
3. Two or more measurements indicating increased urine protein excretion within 1-year 
 
Increased urine protein excretion is defined as: 
- 
Urine microalbumin/creatinine ratio (ACR) > 3.3 mg/mmol creatinine or 
- 
Urine total protein/creatinine ratio (PCR) > 0.2 g/urine creatinine 
 
4. Known diabetes duration > 3 months 
 
5. HbA1c 7.5 – 9% (within 3 months prior to enrolment)  
 
6. Not currently treated with an SGLT-2 inhibitor, and have not received SGLT-2 inhibitor therapy 
within the last 10 weeks. 
 
7. Stable diabetes therapy for at least 3months as defined as: 
- 
No change in dose of diabetes medications by more than two-fold or  
- 
No new agents added within the previous 3 months 
 
8. Stable doses of angiotensin converting enzyme (ACE) inhibitors or angiotensin AT(1)-receptor 
blockers (ARBs) for at least 3 months. 
 
9. Capable of providing informed consent 
 
Subjects with HbA1C between 7.5 – 9% will be recruited because treatment with SGLT-2i is indicated in 
these patients according to local clinical practice guidelines. Subjects with HbA1C > 9% will be excluded 
due to safety considerations from uncontrolled hyperglycemia. We will exclude subjects on insulin treatment 
as insulin treatment may interfere with measured parameters. 
 
We will not be studying children, or adults above the age of 65. The risk of drug related adverse effects 
increases in the elderly. All methods of contraception are allowed in women of childbearing age. 
 
 
3.4     Exclusion Criteria 
 
Exclusion criteria were selected to enhance safety and adherence. All subjects meeting any of the 
exclusion criteria at baseline will be excluded from participation.  
 
1. Type 1 diabetes mellitus 
2. Insulin treatment 
3. Ketosis-prone diabetes 
4. Previous diabetic ketoacidosis 

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5. Known peripheral vascular disease or history of lower limb amputations 
6. History of Fournier’s gangrene or skin and soft tissue infections of the perineum 
7. Recurrent or severe urinary tract or genital mycotic infections, or history of genitourinary infection 
within 2 weeks prior to informed consent 
8. Known allergic reaction to empagliflozin or other SGLT2 inhibitor 
9. Significant renal impairment (estimated Glomerular Filtration Rate < 45 ml/min/1.73m2**) 
10. Dialysis or kidney transplant 
11. Renal artery stenosis 
12. Alanine aminotransferase or aspartate aminotransferase above 3x upper limit of normal 
13. Significant change in weight (≥10% in the preceding 6 months) 
14. Treatment with anti-obesity drugs 
15. Previous bariatric surgery or other gastrointestinal surgeries that induce chronic malabsorption 
16. Treatment with systemic glucocorticoids 
17. Blood dyscrasias or clinically significant anaemia (Haemoglobin < 10 g/L) 
18. Cancer within the last 5 years (except basal cell carcinoma) 
19. Medical condition likely to limit survival to less than 3 years 
20. Uncontrolled thyrotoxicosis, untreated hypothyroidism  
21. Any ongoing acute medical illnesses 
22. Hospitalization within 1 month prior to enrolment 
23. Nursing mothers  
24. Pregnancy, currently trying to become pregnant, or of child-bearing potential and not practicing an 
acceptable method of birth control or do not plan to continue using this method throughout the study 
25. Excessive alcohol intake (> 1 unit per day for women and > 2 units per day for men) 
26. History of drug abuse 
27. Pancreatic insulin deficiency from any cause (history of pancreatitis, pancreatic surgery) 
28. Known intolerance or allergic reactions to empagliflozin or other SGLT-2 inhibitors 
29. Current participation in another clinical trial, or ingestion of investigational drug in another trial within 
30 days prior to enrolment. 
30. Chronic liver disease: 
- 
Hepatitis B  
- 
Hepatitis C 
- 
Autoimmune hepatitis 
- 
Hemochromatosis 
31. Presence of any non-DN renal glomerular disease  
(e.g. IgA nephropathy, lupus nephritis, membranous glomerulonephritis, focal segmental glomerular 
sclerosis) 
32. Any previous organ transplantation 
33. Any factors likely to limit adherence to interventions (e.g. dementia; alcohol or substance abuse; 
history of unreliability in medication taking or appointment keeping; significant concerns about 
participation in the study from spouse, significant other or family members) 
34. Failure to obtain informed consent from participant 
 
**MDRD formula: eGFR (ml/min) = 175 x [Screatinine (umol/L)/88.4]-1.154 x [age]-0.203 x 0.742 [if patient is female] 
Ann Intern Med. 1999 Mar 16;130(6):461-70. Am J Kidney Dis. 2002 Feb;39(2 Suppl 1):S1-266. 
 
 
3.5     Subject Replacement 
There will be no replacement of subjects who drop out of the study. 
 
 

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4      STUDY DESIGN 
 
In this single-centre, open-label study, 40 adults with DN will be recruited over 2 years. Following screening 
and baseline metabolic evaluations, eligible subjects will be treated with empagliflozin for 30-days and re-
assessed.  
 
 
 
4.1 
Randomisation and Blinding 
 
No randomization or blinding will be performed as the response to empagliflozin is stereotypical and the 
measured parameters are objective and thus not subject to observer bias. 
 
4.2 
Contraception and Pregnancy Testing 
 
There are no adequate data from the use of empagliflozin in pregnant woman. Therefore, pregnant women 
cannot participate in this study. Female subjects of childbearing potential will be tested for pregnancy using 
urine pregnancy test kits during their screening (Visit 1) and dosing visits (Visit 2). Sexually active women 
of childbearing potential must use a reliable method of birth control while participating in this study. Reliable 
methods of birth control are considered to be abstinence (not having sex), oral contraceptives (the pill), 
intrauterine device (IUD), DepoProvera, Norplant, tubal ligation (tubes tied) or vasectomy of the partner. An 
acceptable although less reliable method, involved the careful use of condoms and/or a spermicidal foam or 
gel along with a diaphragm, cervical cap, or sponge. Study subjects would be encouraged to discuss this 
issue further with their doctor if they have any questions. 
 
 

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4.3 
Study Visits and Procedures 
 
4.3.1 Patients who meet inclusion criteria and do not meet any of the exclusion criteria will be invited to 
participate during their visit to the DMC at SGH by the PI, Co-Is or their managing physician. 
Interested patients will be scheduled for their first study visit (Visit 1) within 28 days at SGH CTRC.  
 
 
4.3.2 VISIT 1: Informed Consent and Screening   
 
Written informed consent will be taken by the principal investigator, co-investigators or study team members 
assigned by the PI. After consent has been taken, study procedures in Table 1 will be performed. The PI, 
Co-I or study team member(s) assigned by the PI will review screening results to determine whether 
subjects are eligible to continue with the study. Information regarding all concomitant therapy taken by the 
patients will be collected. 
 
Table 1. Study Procedures  
 
 
Visit 1 
Visit 2  
Visit 3 
  
Consent 
&  
Dosing & 
Metabolic 
Evaluation 
Post-
treatment 
and Final 
Visit 
Screening 
Administrative 
  
  
  
Informed consent 
X 
  
  
Inclusion/Exclusion criteria 
X 
  
  
Dispense Medication 
  
X 
  
Monitoring of drug compliance 
  
  
X 
Clinical Procedures/Assessment  
  
  
  
Fasting (≥8 hours) 
 
X  
X 
Medical History/Assessment 
X 
  
X 
Drug History 
X 
  
X 
Adverse Effects Monitoring 
  
  
X 
General Physical Examination 
X 
  
X 
BP/Pulse 
X 
  
X 
Weight (triplicate) * 
X 
  
X 
Height (triplicate) * 
X 
  
  
Hip/Waist Circumference (triplicate) ** 
X 
  
X 
Indirect Calorimetry *** 
 
X  
X 
Laboratory Procedures 
  
  
  
HbA1C  
X 
  
  
Fasting Glucose    
 
 X 
X 
Full Blood Count   
X 
  
  
Potassium   
X 
  
X 

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Creatinine  
X 
  
X 
eGFR Calculation  
X 
  
X 
Lipid Panel  
 
X  
X 
Urine βHCG   
X 
X 
  
(female of child bearing potential) 
ALT  
X 
  
X 
AST  
X 
  
X 
Metabolomic Profiling  
 
X  
X 
Urine ACR  
X 
  
X 
Urine PCR  
 
X  
X 
 
 
* Weight will be measured in light clothing to the nearest 0.1 kg on a balance scale and height with a stadiometer three 
times and then averaged 
** Waist circumference will be measured from the inferior margin of the last rib and hip circumference measured at the 
crest of the ileum to the nearest 0.1 cm three times and averaged 
*** Indirect calorimetry using a canopy hood will be performed for 30 minutes for measurement of basal energy 
expenditure, CO2 production and O2 consumption 
ALT = Alanine Transaminase 
AST = Aspartate Transaminase 
 
 
SAMPLE COLLECTION 
 
A total of 37ml (~8 teaspoons) of blood and 32ml (~7 teaspoons) of urine will be collected for the entire 
study. During visit 1 (Screening), 11ml (~2 teaspoons) of blood and 6ml (~1 teaspoon) of urine will be 
collected. During visit 2 (Dosing visit), 13ml (~3 teaspoons) of blood and 11ml (~3 teaspoons) of urine will 
be collected. During visit 3 (Post-treatment and final visit), 13ml (~3 teaspoons) of blood and 15ml (~3 
teaspoons) of urine will be collected. If female subjects are actively menstruating at the time of study visit, 
urine tests will not be sent for biochemical or metabolomics analysis. 
 
Blood and urine samples for laboratory testing at the clinical laboratory (Table 1) will be despatched for 
immediate analysis. Blood and urine samples for metabolomic profiling will be stored in the SGH CTRC at -
80°C, and then sent to Duke-NUS laboratory for batch analysis.  
 
Informed consent will be taken from participants regarding storage of biological materials. The blood and 
urine specimens will be coded and stored for future research if participants consent to this. The blood 
specimens will not be transferred out of Singapore. 
 
Subjects who developed drug-related adverse reactions may require additional blood and urine taken for 
clinical assessment. The estimated amount required for these additional assessments are 10 ml (~2 
teaspoons) of blood and 10 ml (~2 teaspoons) of urine. 
 
 

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LABORATORY MEASUREMENTS 
 
 
A) Blood Chemistry  
 
Screening and standard laboratory tests including full blood count, Potassium, Creatinine, ALT, AST, β-
HCG (for female subjects of childbearing age), lipid panel, glucose and HbA1c will be measured using 
standard methods.  
 
B) Metabolomic Profiling 
 
Metabolomic profiling of lipids, acylcarnitines (ACs), organic acids and ketones will be measured using 
liquid and gas chromatography and tandem mass spectrometry (LC-MS/MS and GC-MS/MS) at the Duke-
NUS metabolomics laboratory. Metabolomic analysis will be performed on serum (acyl-carnitines, amino 
acids and organic acids) as well as urine (organic acids). 
ACs represent intermediaries of metabolic fuel oxidation and provides a “snap-shot” of in vivo metabolism 
at the cellular level. The measurement of the various long-, intermediate-chain ACs provide an indicator of 
fatty acid oxidation efficiency. Ceramides are toxic lipid intermediaries that are implicated in the 
pathogenesis of insulin resistance.  Lactate, pyruvate, succinate, fumarate, malate, alpha-ketoglutarate and 
citrate are organic acids involved in TCA cycle activity. Beta hydroxybutyrate is a ketone which will also be 
measured with the organic acids. 
 
 
C) Urine Protein Excretion 
 
Urine ACR and PCR will be performed to quantify urine protein excretion. 
 
 
4.3.3 VISIT 2: Initiation of Study Drug 
 
Subjects who satisfy the inclusion and exclusion criteria will return to the CTRC within the next 14 days for 
visit 2 to receive the study drug, empagliflozin. Empagliflozin 10mg daily is to be taken orally for 30 ± 7 
days. Subjects will take the first dose of empagliflozin under supervision of the clinical research coordinator 
at SGH CTRC.  
 
Patients will be instructed to take empagliflozin once daily with water. To ensure a dose interval of about 24 
hours, the medication should be taken at the same time every day, with or without food. If a dose is missed 
by more than 12 hours, that dose should be skipped and the next dose should be taken as scheduled. No 
double doses should be taken, and dose reductions are not permitted.  
 
 
4.3.4 VISIT 3: 4-Week Post-Treatment and Final Visit 
 
Visit 3 will take place 30 ± 7 days from Visit 2. Subjects will be assessed to quantify changes in metabolic 
fuel utilization after treatment with empagliflozin. These changes are determined based on metabolomic 
profiling of acyl-carnitines, organic acids and amino acids and indirect calorimetry measurements. The list 
of procedures performed is listed in Table 1. 
 
Subjects will take their final dose of study medication on the day of visit 3. Subjects will be asked to bring all 
trial medication containers (with or without any remaining tablets) with them to visit 3. Subjects who forget 
to return trial medication containers will be asked to bring them at a separate visit. The tablets will be 
counted by the PI, Co-I or study team members and compliance will be calculated according to the formula: 
 

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Compliance (%) =                  Number of tablets actually taken since visit 2                            x 100% 
                               Number of tablets which should have been taken in the same period  
 
In addition, study subjects will be asked to fill up a Subject Treatment Diary to record the number of doses 
of empagliflozin 10mg taken. The Subject Treatment Diary will be reviewed by the PI, Co-I or study team 
member at visit 3.  
The study drug will be discontinued upon completion of visit 3, and any remaining study drug tablets will be 
returned to the study team. 
 
Should study subjects misplace any empagliflozin 10mg tablets between visit 2 and visit 3, the exact 
number of misplaced tablets will be resupplied to them by the study team. 
 
Study drug will be discontinued for subjects upon occurrence of any adverse drug reaction to empagliflozin. 
 
 
 
4.3.5 Post Study Follow up and Procedures 
 
Subjects will continue their routine follow-up with their regular physicians upon completion of the final study 
visit. There will be no post study follow-up procedures. 
 
 

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4.4 
Discontinuation/Withdrawal 
 
4.4.1 Discontinuation Criteria 
 
Study drug will be discontinued following completion of visit 3 (final study visit).  
 
Study drug will be discontinued upon the development of any of the following adverse reactions: 
1. Any allergic reaction to study drug 
2. An increase in creatinine by > 50% compared to baseline 
3. Ketoacidosis 
4. Urosepsis and pyelonephritis 
5. Fournier’s gangrene  
 
Individual patients will also be withdrawn from the trial if: 
1. The patient withdraws consent, without the need to justify the decision. 
2. If a patient becomes pregnant during the trial, the investigational drug will be stopped, the patient 
will be discontinued from treatment, and the patient will be followed up until birth or otherwise 
termination of the pregnancy. 
 
All patients who drop out of the study after visit 1 (Consent & Screening), but before visit 2 (Dosing) will be 
considered a screening failure, and no further follow-up is required. 
 
 
4.4.2 Discontinuation Visit and Procedures 
 
Should any suspected study drug-related adverse effects occur, study subjects may contact the study 
coordinator. If required, they would be scheduled for a visit at SGH CTRC within the next 72 hours. They 
will be assessed by a member of the study team who is a registered medical practitioner for their suitability 
to continue with the study drug. Subjects may be referred back to their primary physician or other 
physicians as necessary upon the discovery of any drug-related adverse reaction to be given appropriate 
care under medical supervision until the symptoms of any adverse event resolve, or the subject’s condition 
becomes stable. In the event of any medical emergency or urgency, subject will be referred to the 
Emergency Department.   
 
 
Subjects who have taken at least one dose of the study drug and withdrew from this study (voluntary 
withdrawal or discontinuation due to adverse reaction), are encouraged to return for their third visit (30 ± 7 
days from Visit 2) any and complete all planned study procedures as listed in Table 1. Subjects who had 
not taken any study drug will not be required to return for any subsequent study visits.  
 
 
 
5 
TRIAL MATERIALS 
 
 
5.1 
Trial Product (s) 
 
Information on trial product (empagliflozin 10mg) is detailed in the prescribing information attached in 
Appendix 2. 
 
 

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5.2 
Storage and Drug Accountability 
 
Empagliflozin (Jardiance) will be stored in the original package in order to protect from light and moisture, at 
a room temperature not exceeding 30 °C. It will be stored securely in the investigator product cabinet at the 
SGH CTRC IP Room with restricted access. Study drug will be stored securely and the study coordinator 
will maintain a dispensing log. IP will be monitored by a 24-hour Temperature Monitoring System (TMS) 
and reports will be generated on a fortnightly basis. Alarms to any excursion will be triggered and the CTRC 
staff and FRS will be informed.  
 
 
 
6 
TREATMENT 
6.1 
Rationale for Selection of Dose 
 
Dosage of empagliflozin is similar to that used for the treatment of type 2 diabetes mellitus in clinical 
practice. Eligible subjects will be given empagliflozin 10mg once daily to be taken orally for 30 ± 7 days. 
 
6.2 
Study Drug Formulations 
 
Study drug will be dispensed as empagliflozin (Jardiance) 10mg film-coated tablets. 
 
 
6.3 
Study Drug Administration 
 
Study drug is to be taken orally. Subjects will be warned to look out for the following symptoms and contact 
the study coordinator if they occur: 
 
a) Development of allergic reactions (eg: new rash) 
b) Symptoms suggestive of hypotension (eg: light-headedness) 
c) Symptoms suggestive of ketoacidosis (eg: nausea, vomiting, abdominal pain, generalized malaise, 
shortness of breath) 
d) Factors predisposing to ketoacidosis which may warrant discontinuation of empagliflozin (eg: acute 
febrile illness, reduced caloric intake due to illness or surgery) 
e) Factors predisposing to acute kidney injury which may warrant discontinuation of empagliflozin (eg: 
reduced oral intake, fluid losses) 
f) Symptoms suggestive of urinary tract infection (eg: fever, dysuria) and genital mycotic infection (eg: 
genital discharge or pruritus) 
g) Symptoms suggestive of Fournier’s gangrene (eg: fever, malaise, or tenderness, redness, or 
swelling of the genitals or the area from the genitals back to the rectum) 
 
 
 

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6.4 
Specific Restrictions / Requirements  
 
Individuals who are treated with insulin or in whom insulin treatment is clinically indicated will be excluded 
from this study. 
6.5 
Blinding 
 
This is an open-label study. 
 
6.6 
Concomitant therapy 
 
All medications (prescription and over the counter), vitamin and mineral supplements, and / or herbs taken 
by the participant will be documented. 
 
7 
SAFETY MEASUREMENTS 
7.1 
Definitions 
 
An adverse event (AE) is any untoward medical occurrence in a patient or clinical investigation subject 
administered a pharmaceutical product and which does not necessarily have a causal relationship with this 
treatment. 
 
A serious adverse event (SAE) or reaction is any untoward medical occurrence that at any dose: 
• 
results in or contributes to death 
• 
is life-threatening 
• 
requires inpatient hospitalisation or prolongation of existing hospitalisation 
• 
results in or contributes to persistent or significant disability/incapacity or 
• 
results in or contributes to a congenital anomaly/birth defect 
• 
results in such other events as may be prescribed  
• 
is a medical event that may jeopardize the patient and may require medical or surgical 
intervention to prevent one of the outcomes listed above. 
 

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7.2 
Collecting, Recording and Reporting of Adverse Events and Serious Adverse Events to 
CIRB  
 
Reporting of adverse events involves the PI submitting to the approving CIRB the completed SAE 
Reporting Form within the stipulated timeframe. PI is responsible for informing the institution representative 
(local SAE resulting in death), sponsor or regulatory bodies as required and appropriate. 
 
Reporting timeline to CIRB:  
 
• 
SAE that result in death, regardless of causality, should be reported immediately - within 24 
hours of the PI becoming aware of the event.  
• 
Local life-threatening (unexpected/ expected) SAE should be reported no later than 7 
calendar days after the Investigator is aware of the event, followed by a complete report 
within 8 additional calendar days. 
• 
Local unexpected SAE that are related events, but not life-threatening, should be reported no 
later than 15 calendar days after the investigator is aware of the event. 
• 
An increase in the rate of occurrence of local expected SAE, which is judged to be clinically 
important, should be reported within 15 calendar days after the PI is aware of the event.  
• 
Local expected SAE should be reported annually (together with Study Status Report for 
annual review). 
• 
Local unexpected and unlikely related SAE that are not life-threatening should also be 
reported annually (together with Study Status Report for annual review). 
• 
Local unexpected AE that are related events should be reported at least annually (together 
with Study Status Report for annual review). 
• 
Non-local unexpected SAE that are fatal or life threatening and definitely/probably/possibly 
related should be reported not later than 30 calendar days after the PI is aware of the event. 
 
 
7.3 
Collecting, Recording and Reporting of Serious Adverse Events (SAEs) to the Health 
Science Authority (HSA) 
 
All SAEs that are unexpected and related to the study drug will be reported to HSA. All SAEs will be 
reported to HSA.  Please refer to the HSA website for more information on Safety Reporting Requirements 
for Clinical Trials.  
 
The investigator is responsible for informing HSA no later than 15 calendar days after first knowledge that 
the case qualifies for expedited reporting. Follow-up information will be actively sought and submitted as it 
becomes available. For fatal or life-threatening cases, HSA will be notified as soon as possible but no later 
than 7 calendar days after first knowledge that a case qualifies, followed by a complete report within 8 
additional calendar days. 
 
 
 

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7.4 
Safety Monitoring Plan 
 
Any adverse effects will be reported. The PI and Co-investigators will monitor and report to the CIRB and 
HSA of any SAE. There will be no DSMB. Risks of study drug will be continually reassessed throughout the 
study period. Closed monitoring meetings with the research team will take place every 4 weeks to discuss 
the data collected from monitoring reports.  
 
7.5 
Complaint Handling  
 
Contact information of the study team and SingHealth CIRB will be provided to all study participants and 
available in the participant informed consent sheet. 
 
The PI will be informed immediately about any complaint from study subjects. The PI will contact the 
relevant study subjects to resolve the complaint. 
 
 
7.6 
Incidental findings 
 
Incidental findings are defined as findings which are discovered during the course of conducting the study, 
and which have potential health or reproductive importance to study participants, but are unrelated to the 
purposes, objectives or variables of the study. Participants will be asked to indicate whether they wish to be 
re-identified and notified in the event of an important incidental finding that is related to them.  
 
Should participants agree to be re-identified and notified, the PI or other study team member delegated by 
the PI will explain the incidental finding to the participant, and advise them on the next appropriate step. 
The costs for any care needed to diagnose or treat an incidental finding will be borne by the study 
participant.  
 
 
8 
DATA ANALYSIS 
8.1 
Data Quality Assurance 
 
Discrepancy and inconsistent data will be resolved by looking at the source document.  
 
8.2 
Data Entry and Storage 
 
Data will be stored in paper form and electronically. Paper documents will be stored in a locked cabinet in 
the PI’s office. All electronic data will be de-identified and password protected. Electronically entered data 
will be stored in the network drive of Singapore General Hospital. Source documents will be retained until at 
least 6-years after the end of the study, as per CIRB policy recommendation.  
 
The PI, Co-Is and Research Coordinators will have access to the data for tracking and following up with the 
participants and for academic analysis purposes. For randomisation and statistical analysis purposes, data 
access may also be granted to Duke-NUS Biostatisticians. 
 

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9 
SAMPLE SIZE AND STATISTICAL METHODS 
 
9.1 
Determination of Sample Size  
 
The effect of short-term treatment with empagliflozin to cause a shift in metabolic fuel oxidation in DN is 
unknown. We anticipate that a significant shift in metabolic fuel utilization towards lipid oxidation will occur 
after 23 – 30 days of treatment with SGTL2i. 40 subjects with DN will be required. Assuming a screen 
failure rate of 1 in 3, a total of 60 subjects will need to be screened. 
 
9.2 
Statistical and Analytical Plans 
 
For the measurement of metabolic fuel utilization and related parameters at baseline and post-treatment, 
values will be compared using the paired Student’s t-test. If the data are not normally distributed, the 
appropriate transformation will be used or a non-parametric approach will be considered. Hypotheses 
testing will be non-directional (two sided), with values < 0.05 as significance. Software programs SPSS 
(version 21; SPSS, Chicago, IL) and GraphPad Prism (version 6, GraphPad Software) will be used for all 
statistical analysis. There will be no planned interim analyses. 
 
 
10 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
 
The investigator(s)/institution(s) will permit study-related monitoring, audits and/or IRB review and 
regulatory inspection(s), providing direct access to source data/document. 
 
 
 
11 
QUALITY CONTROL AND QUALITY ASSURANCE 
 
All study team members will undergo protocol training to ensure adherence with the protocol and for 
accuracy in relation to source documents.  
 
 
12 
ETHICAL CONSIDERATIONS 
 
This study will be conducted in accordance with the ethical principles that have their origin in the 
Declaration of Helsinki and that are consistent with the Good Clinical Practice and the applicable regulatory 
requirements. 
 
This final study protocol, including the final version of the Patient Information and Informed Consent Form, 
will be approved in writing by the Centralised Institutional Review Board (CIRB) and regulatory approval 
from Health Sciences Authority (HSA), prior to enrolment of any patient into the study. 
 
The principle investigator is responsible for informing the CIRB and HSA of any amendments to the protocol 
or other study-related documents. 
 

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12.1 Informed Consent 
 
Informed consent will be taken during Visit 1 at SGH CTRC. Consent will be taken by the the PI or 
investigators assigned by the PI. There will be a provision for a translator for non-English speaking 
subjects. Illiterate subjects have the option to have a family member present during the consent taking 
process. Vulnerable subjects will not be recruited for this study. In obtaining and documenting informed 
consent, the investigator will comply with the GCP guidelines and to the ethical principles that have their 
origin in the Declaration of Helsinki. 
 
12.2 Confidentiality of Data and Patient Records 
 
Confidentiality of study participants will be respected and all potential participants will be assigned a study 
ID. Data and samples collected from study participants will be de-identified. 
 
 
13 
PUBLICATIONS 
 
Publication-related decisions will be in discussed among all investigators and collaborators but the final 
decision lies within the PI. 
 
 
14 
RETENTION OF TRIAL DOCUMENTS 
 
Records for all participants, including CRFs, all source documentation (containing evidence to study 
eligibility, history and physical findings, laboratory data, results of consultations, etc.) as well as CIRB 
records and other regulatory documentation will be retained by the P.I. in a secure storage facility. The 
storage will be in accordance with SingHealth CIRB guideline on Retention of Research Data and Records 
and will be stored for a minimum storage period of 7 years after the completion of the study. The records 
will be accessible for inspection and copying by authorized authorities. 
 
 
15      FUNDING and INSURANCE 
 
The study is funded by the Tanoto Initiative for Diabetes Research Award (Duke-NUS 
TIDR/2018/0010) for a 2-year period. 
 
This study will be covered under the National Clinical Trial Insurance Policy 
  

Version 1.3, Dated 9th December 2018  
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List of Attachments 
 
Appendix 1 
Blood sampling schedule 
 
TOTAL VOLUME  
  
Blood 
(ml) 
(Teaspoon) 
Visit 1 
11 
2 
Visit 2 
13 
3 
Visit 3 
13 
3 
TOTAL 
37 
8 
Urine 
(ml) 
(Teaspoon) 
Visit 1 
6 
1 
Visit 2 
11 
3 
Visit 3 
15 
3 
TOTAL 
32 
7 
 
 
Appendix 2       Jardiance (Empagliflozin) prescribing information 

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Appendix 3 
    References 
 
1. Liu, J.-J. et al. Profiling of Plasma Metabolites Suggests Altered Mitochondrial Fuel Usage and 
Remodeling of Sphingolipid Metabolism in Individuals With Type 2 Diabetes and Kidney Disease. 
Kidney Int. Rep. 2, 470–480 (2017). 
2. Sharma, K. et al. Metabolomics reveals signature of mitochondrial dysfunction in diabetic kidney 
disease. J. Am. Soc. Nephrol. JASN 24, 1901–1912 (2013). 
3. Vasilakou, D. et al. Sodium-glucose cotransporter 2 inhibitors for type 2 diabetes: a systematic 
review and meta-analysis. Ann. Intern. Med. 159, 262–274 (2013). 
4. Wanner, C. et al. Empagliflozin and Progression of Kidney Disease in Type 2 Diabetes. N. Engl. J. 
Med. 375, 323–334 (2016). 
5. Zinman, B. et al. Empagliflozin, Cardiovascular Outcomes, and Mortality in Type 2 Diabetes. N. 
Engl. J. Med. 373, 2117–2128 (2015). 
6. Ferrannini, E., Mark, M. & Mayoux, E. CV Protection in the EMPA-REG OUTCOME Trial: A ‘Thrifty 
Substrate’ Hypothesis. Diabetes Care 39, 1108–1114 (2016). 
7. Perrone-Filardi, P. et al. Mechanisms linking empagliflozin to cardiovascular and renal protection. 
Int. J. Cardiol. 241, 450–456 (2017). 
8. Mudaliar, S., Alloju, S. & Henry, R. R. Can a Shift in Fuel Energetics Explain the Beneficial 
Cardiorenal Outcomes in the EMPA-REG OUTCOME Study? A Unifying Hypothesis. Diabetes 
Care 39, 1115–1122 (2016). 
9. Ferrannini, E. et al. Metabolic response to sodium-glucose cotransporter 2 inhibition in type 2 
diabetic patients. J. Clin. Invest. 124, 499–508 (2014). 
10. 
Kappel, B. A. et al. Effect of Empagliflozin on the Metabolic Signature of Patients With Type 2 
Diabetes Mellitus and Cardiovascular Disease. Circulation 136, 969–972 (2017).