Conditions: Type 2 Diabetes Impaired Renal Function
Keywords: Diabetes Mellitus, Type 2 Endothelial cells Cellular biomarker endothelial dysfunction CD34+ Impaired renal function
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 31
Healthy volunteers: no

Brief summary:
Type 2 diabetes is a national epidemic. Diabetes has undesirable effects on blood vessels which may contribute to heart disease. Endothelial Progenitor Cells (EPCs) are found in the blood. Research has shown that improving the survival of these special blood cells may decrease the harmful effects of diabetes on blood vessels and reduce or reverse heart disease. Linagliptin is an Food and Drug Administration (FDA) approved prescription medicine used along with insulin or with oral medications to lower blood sugar in people with Type 2 diabetes. It is in a class of diabetes medication called Dipeptidyl peptidase-4 (DPP-4) inhibitors. DPP-4 inhibitors have been shown to increase EPCs in patients with Type 2 diabetes.

Hypothesis: Both type 2 diabetes and Chronic Kidney Disease (CKD) are associated with poor stem cell number and function. Poor viability and function of EPCs in CKD and diabetes The investigators hypothesize that use of Linagliptin (along with Insulin) may help reduce cardiovascular risk by improving EPC survival and function above and beyond adequate glucose metabolism control

Detailed description:
Type 2 diabetes is a national epidemic with significant macro and microvascular complications. Insulin resistance in pre-diabetes and overt diabetes are associated with endothelial dysfunction.

A few studies indicate that stem cells particularly EPCs can act as a suitable bio-marker for monitoring cardiovascular morbidity. In this proposal the investigators suggest that EPCs or CD34 positive cells (defined as CD34/vascular endothelial growth factor receptor 2 (VEGFR2+) cells) can act as a suitable cellular biomarker for estimating and following endothelial dysfunction in early type 2 diabetes patients with CKD. EPCs have been shown to be dysfunctional in both CKD patients and type 2 Diabetes Mellitus (DM) patients.

Linagliptin (TRADJENTA) tablets are indicated as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus. No dose adjustment is recommended for patients with renal impairment.

EPCs have been used as a regenerative tool in ischemic myocardium and diabetic wound healing. Endothelial dysfunction with associated inflammation may be a consequence of excess intra-cellular super-oxide presence in a setting of diabetes which is a pro-oxidative stress condition ultimately leading to poor EPC function and senescence.

Though lifestyle modification has been proposed as a main stay for prevention and treatment of early type 2 diabetes, several new therapies for diabetes have been developed in recent years. Incretins and incretin mimetics appear to hold promise. Mechanism of positive effect of exercise and oral hypoglycemic agents can be very different.

DPP-4 inhibitors have been shown to increase EPCs in patients with type 2 diabetes reportedly via stromal cell-derived factor 1 (SDF-1) alpha up-regulation. Interestingly, up-regulation of SDF-1 alpha and vascular endothelial growth factor (VEGF), both chemotactic factors increase mobilization and recruitment of EPCs in the face of acute ischemic injury for repair and regeneration.

Several studies have shown positive effect of incretins (Glucagon like peptide, GLP-1) and incretin receptor agonists (GLP-1 receptor agonists) on cardiovascular risk factors in type 2 diabetes patients and even in patients with chronic heart failure and left ventricular dysfunction who do not have diabetes.

DPP-4 Inhibitors may have cardio-protective effects of their own, as they increase bio-availability of endogenous GLP-1. They improve blood flow and nitric oxide production in endothelium. These are unique properties not demonstrated by other oral diabetes medications. The mechanism underlying these effects may be mediated by increased nitric oxide bioavailability but is not completely known. However these beneficial effects appear to be independent of glycemia reduction.

It is however unknown whether Linagliptin will have any positive effect on human EPC function where two prominent cardiovascular risk factors co-exist such as CKD and type 2 diabetes.

Therefore the investigators plan to investigate if Linagliptin can alter function and gene expression of CD34+ cells in a setting of CKD and type 2 diabetes. The investigators choose to look at non geriatric adult population with early type 2 diabetes (less than 10 years of duration) at an early phase of renal impairment (stages 1-3).

Arms:
arm 1: Matching placebo 1 pill daily for 12 weeks arm 2: Linagliptin 5mg once daily for 12 weeks

Interventions: intervention 1: Linagliptin intervention 2: Placebo
Intervention descriptions: intervention 1: 5 mg tablet once daily for 12 weeks intervention 2: 1 tablet daily for 12 weeks

Protocol document:
Prot_SAP_000.pdf: 
 
Linagliptin-Sen IIS  
Protocol Version: 6.9 
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TITLE PAGE 
 
Role of Linagliptin in improving renal failure by improving 
CD34+ stem cell number, function and gene expression in renal 
function impaired type 2 diabetes patients 
 
Sabyasachi Sen, MD, MRCP (UK), PhD, FACP, FACE 
 
George Washington University Medical Faculty Associates 
2150 Pennsylvania Ave NW, Suite 4-403 
Washington, D.C.  20037 
Phone - (202) 994-8560 
 
 
Protocol Version/Date: Version 6.9 / June 2017 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
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PROTOCOL SYNOPSIS 
Protocol Title: 
Role of Linagliptin in improving renal failure by improving CD34+ stem 
cell number, function and gene expression in renal function impaired 
type 2 diabetes patients 
Site Numbers & 
Names: 
GWU Medical Faculty Associates 
Research 
Hypothesis: 
Both type 2 diabetes and CKD are associated with poor stem cell 
number and function. Poor viability and function of EPCs in CKD and 
diabetes affects the repair and regeneration of the endothelium and 
renal tubules. We hypothesize that use of Linagliptin (along with 
Insulin, metformin, or both) may help reduce cardiovascular risk by 
improving EPC survival and function above and beyond adequate 
glucose metabolism control.   
Study Schema: 
Drugs / Doses / 
Length of Treatment) 
We propose a 2-arm randomized, parallel group, longitudinal study of 
12-week intervention duration. The 12-week time interval has been 
previously shown adequate to observe changes in biochemistry, EPCs 
and, importantly, pulse wave velocity (PWV) changes. Patients will be 
randomized to 2 groups:  
Control, (n=20), Insulin/Metformin + Lifestyle + Placebo.  
Treatment, (n=20) Insulin/Metformin + Lifestyle + Linagliptin 5mg.  
 


 
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Study Objectives: 
• 
Primary: 
• 
Secondary: 
The primary objective is to ascertain if addition of Linagliptin improves CD34+ 
cell function and gene expression in early type 2 diabetes patients with CKD 
stage 1-3, which will be correlated to improvement in 24hr urinary protein 
estimation and creatinine clearance (obtained via micro-albumin/creatinine ratio 
from a spot urine sample). 
The secondary objective is to correlate the cellular outcome measures with other 
measures of endothelial function such as: 
1. Arterial stiffness measures with pulse wave analysis and pulse wave velocity 
measurements  
2. Serum biochemistry looking at surrogates of endothelial health, endothelial 
inflammation, appetite controlling hormone levels and fasting glucose, insulin 
and lipid profile.  
3. Resting Metabolic Rate measurement 
 
Study Outcome Measures 
Primary:  
To investigate the effect of Linagliptin on Endothelial function in patients 
inadequately controlled (HbA1C ≥6.5 % to ≤10%) while being treated 
with stable Insulin, Metformin, or both.  
• 
Cellular markers. We will study pre and post Linagliptin treatment 
changes in number, function and gene expression of patients’ peripheral 
blood-derived CD34+ cells.  
• 
24hr urinary protein estimation and creatinine clearance. 
 
Secondary:  
• 
To investigate the effect of Linagliptin treatment on serum endothelial 
inflammatory markers including high sensitivity C-reactive protein (hs-
CRP), IL-6, TNF-alpha, and fasting lipid profile including ApoA1 and 
ApoB.  
 Glycemic control will be evaluated by measuring fasting blood glucose, 
insulin, and HbA1c levels and assessing insulin resistance using HOMA-
IR 
• 
Adiposity, measured using the Tanita Body Composition Analyzer scale, 
measured as percentage body fat. 
• 
Estimation of Creatinine clearance and Protenuria estimation. 
• 
Vessel health will be assessed by systolic and diastolic blood pressure 
and Arterial stiffness assessed using vascular flow and wave 
measurement equipment, SphygmoCor CP system from ATCOR.  
• 
The sphygmocor system also allows us to estimate central and aortic 
blood pressure. 
• 
Resting Metabolic Rate (RMR, similar to Resting Energy expenditure 
measurement) at baseline, midway point (week 6) and post therapy. 
 
 
 


 
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Study Design: 
Prospective, double-masked, randomized placebo-controlled trial  
Accrual Goal: 
(Total number of 
subjects) 
N=40 
Accrual Rate: 
(Number of subjects 
expected per month) 
Recruit 40 patients in approximately 36 months. 
 
Estimated: 
FPFV: 
LPFV:  
Follow Up: 
(dd-mm-yy) 
FPFV: 09-15-2015.    
LPFV: 09-15-2018 
Co-relative Studies:  
(PK/PD, etc.) 
N/A 
Inclusion Criteria: 
1. Adults aged 40-70 years.  
2. Diagnosis of type 2 diabetes within the previous 15 years using 
criteria of the American Diabetes Association 
3. Currently treated with a stable dose of Insulin, Metformin (1-2 
grams/day), or a stable combination of the two as therapy. 
4. HbA1C between 6.5 to 10% (both inclusive)  
5. BMI 25 to 39.9 kg/m2 (both inclusive)  
6. CKD stages 1-3  


 
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Exclusion Criteria: 
Patients with: 
1. Implanted devices (e.g., pacemakers) that may interact with 
Tanita scale 
2. Previous coronary or cerebrovascular event within 6 months of 
screening or active or clinically significant coronary and/or 
peripheral vascular disease 
3. Low hematocrit (<28 UNITS).   
4. Pre-existing  liver disease and/or ALT and AST >2.5X’s UNL 
5. CKD stage 4 and  
6. History of pancreatitis, or cancer (except basal cell carcinoma 
and cancer that is cured or not active or being treated in the 
past five years) 
7. Statin use started or dose change in the last 3 months  
8. Use of oral anti-diabetic medication other than Metformin, or 
Insulin.  
9. Use of consistent long-term steroid medication in the last 3 
months (oral, inhaled, injected). 
10. Systolic BP> 140 mmHg and Diastolic BP> 90 mmHg  
11. Active wounds or recent surgery within 3 months.  
12. Inflammatory disease, or chronic current use of anti-
inflammatory drugs within the last 3 months 
13. triglycerides >450 mg/dL 
14. untreated hyper/hypothyroidism 
15. Auto antibody confirmed type 1 diabetes 
Additionally, patients who are active smokers, patients who are 
pregnant, nursing women, and post-menopausal women who are on 
hormone replacement therapy will be excluded. 
Patients on low dose oral contraceptives will be allowed to participate 
as these formulations contain lesser amount of estrogens. 
  


 
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Criteria for 
Evaluation:  
(Efficacy, safety,  
stopping rules, etc.) 
 
Subjects will be screened at week -4, then evaluated for end points at 
weeks 0, 6 and 12. Follow up phone call will be done 30 days after last 
dose of study drug to assess for adverse effects.  
Study participation will be stopped for adverse effects at the discretion 
of the study investigator. Subjects may stop study participation at any 
time and for any reason. 
Statistics: 
The total sample size is requested, after accounting for attrition over 
the 12-week period, is 20 subjects per group or 40 subjects total. 
Sample size estimates were based on the effects of exercise on 
CD34+/KDR+ cells as described in the literature.  The effect of a 
single session, as well as extended training, on healthy subjects or 
those with existing cardiovascular conditions appears to increase the 
CD34+/KDR+ cells.    
This is a pilot study as a result appropriate power calculation is not 
feasible. W however did conduct power analysis, which provided us 
with 73% power (see statistics section).  A p-value of less than 0.05 
will be considered statistically significant. 
 


 
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1 
INTRODUCTION 
Type 2 diabetes is a national epidemic (1, 2) with significant macro and microvascular 
complications (3). Insulin resistance in pre-diabetes and overt diabetes are associated 
with endothelial dysfunction (4).  
A few studies indicate that stem cells particularly EPCs can act as a suitable bio-marker 
(5-7, 9) for monitoring cardiovascular morbidity. In this proposal we suggest that EPCs 
or CD34 positive cells (defined as CD34/VEGFR2+ cells) can act as a suitable cellular 
biomarker for estimating and following endothelial dysfunction in early type 2 diabetes 
patients with CKD. EPCs have been shown to be dysfunctional in both CKD patients and 
type 2 DM patients. (5, 7, 31, 35)  
Linagliptin (TRADJENTA) tablets are indicated as an adjunct to diet and exercise to 
improve glycemic control in adults with type 2 diabetes mellitus. No dose adjustment is 
recommended for patients with renal impairment. (29) 
EPCs have been used as a regenerative tool in ischemic myocardium and diabetic 
wound healing (8, 10-13). Endothelial dysfunction with associated inflammation may be 
a consequence of excess intra-cellular super-oxide presence in a setting of diabetes 
which is a pro-oxidative stress condition ultimately leading to poor EPC function and 
senescence (14).  
Though lifestyle modification has been proposed as a main stay for prevention and 
treatment of early type 2 diabetes, (2, 15-19) several new therapies for diabetes have 
been developed in recent years (2). Incretins and incretin mimetics appear to hold 
promise. Mechanism of positive effect of exercise and oral hypoglycemic agents can be 
very different. (44-50) 
DPP-4 inhibitors have been shown to increase EPCs in patients with type 2 diabetes 
(20) reportedly via SDF-1 alpha up-regulation. Interestingly, up-regulation of SDF-1 
alpha and vascular endothelial growth factor (VEGF), both chemotactic factors increase 
mobilization and recruitment of EPCs in the face of acute ischemic injury for repair and 
regeneration. (21-24).  
Several studies have shown positive effect of incretins (Glucagon like peptide, GLP-1) 
and incretin receptor agonists (GLP-1 receptor agonists) on cardiovascular risk factors in 
type 2 diabetes patients (20, 25) and even in patients with chronic heart failure and left 
ventricular dysfunction who do not have diabetes (26, 27).  
DPP-4 Inhibitors may have cardio-protective effects of their own, as they increase bio-
availability of endogenous GLP-1. They improve blood flow and nitric oxide production in 


 
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endothelium (28, 29). These are unique properties not demonstrated by other oral 
diabetes medications (29). The mechanism underlying these effects may be mediated by 
increased nitric oxide bioavailability but is not completely known. However these 
beneficial effects appear to be independent of glycemia reduction.  
It is however unknown whether Linagliptin will have any positive effect on human EPC 
function where two prominent cardiovascular risk factors co-exist such as CKD and type 
2 diabetes.  
Therefore we plan to investigate if Linagliptin can alter function and gene expression of 
CD34+ cells in a setting of CKD and type 2 diabetes. We choose to look at non geriatric 
adult population with early type 2 diabetes (≤15 years of duration) at an early phase of 
renal impairment (stages 1-3). 
We specifically choose to exclude known CVD disease patients and early type 2 DM 
patients with early CKD as this cohort may be still retain capability to reverse or halt 
persistent endothelial damage seen in type 2 diabetes and CKD (3,30,35).   
Our preliminary study results , which just finished, looked at effect of 150min/week of 
aerobic physical activity on EPCs in pre-diabetes population show that interventions 
even though non pharmaceutical can lead to quick (within 4 weeks) improvement in 
gene expression and function of CD34+ cells. These cellular changes are reproducible 
and consistent with other outcome measures such as serum biochemistry and vessel 
wall dilation measures such as flow mediated dilatation. (34)    
Research Hypothesis: 
We hypothesize that Linagliptin, a member of DPP-4 inhibitor group of drugs may be 
able to improve number and function of CD34+  endothelial progenitor cells by up-
regulating chemotactic agent SDF1 alpha (DPP-4 degrades SDF-1) and its receptor 
CXCR4 (7, 20, 21) and thereby improving EPC migration and overall endothelial 
function. We also propose that this expected cardiovascular benefit is independent of 
HbA1C reduction. (30, 31)  
In this proposed study we plan to recruit patients with type 2 diabetes of ≤15 years 
duration (in early phase of diagnosis), who are on a stable dose of insulin, Metformin, or 
both for at least 3 months either short or long acting but still remain inadequately 
controlled with HbA1C between 6.5% and 10% (both values inclusive), but not overtly 
out of control (> 10% of HbA1C).  
The patient will adjust insulin levels so as to keep their HbA1C levels under 10.0%, for 
health measures. If the HbA1c rises above 10.0% then the PI will speak with the patient 
and provide a documented form indicating the insulin dose adjustment that should be 


 
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made. Initially life-style intervention with diet and regular aerobic activity (as per 
Standards of Diabetes Care: Diabetes Care Supplement: Jan 2014) along with insulin 
titration will be undertaken for initial 4 weeks (time between screening visit and 1st visit). 
Subjects will begin taking 5.0 mg of Linagliptin or placebo after initial 4 weeks. Subjects 
will be withdrawn from the study if the medication or placebo is not tolerated.  
2 
STUDY OBJECTIVES 
2.1 
Primary Objective 
To investigate the effect of Linagliptin on Endothelial progenitor cells (CD34 
positive cells) in patients whose HbA1C is between 6.5 to 10% (both inclusive) 
while being on treatment with a stable dose of insulin, Metformin, or both for the 
preceding 3 months before enrollment.  
 
Cellular markers: We will use patient’s peripheral blood derived CD34+ cells looking at 
number, function and gene expression changes pre and post Linagliptin over 12 weeks 
of therapy.  
Urinary Function Marker in CKD: We will estimate 24hr urinary protein estimation and 
creatinine clearance (This is measured via the micro-albumin/Creatinine ratio provided 
from a random spot urine sample). 
 
 
2.2 
Secondary Objectives 
To investigate the effect of Linagliptin treatment on serum endothelium inflammatory 
markers including: C-reactive protein (hs-CRP), IL-6, TNF-alpha, and fasting lipid profile 
including ApoA1 and ApoB (5, 6, 30, 36).  
 
Glycemic control will be evaluated by measuring fasting blood glucose, insulin levels and 
HbA1c. Fasting blood glucose, insulin and lipid profile will be used to assess insulin 
resistance (32, 37)  
 
Adiposity will be measured using the Tanita Body Composition Analyzer scale, 
measured as percentage body fat (33). Serum will be used to estimate levels of SDF1-
Alpha, VEGF, and GLP1 levels by ELISA (25, 28, 29, and 30). 
We also intend to follow resting Metabolic Rate at baseline, midway, and post DPP4 
inhibitor therapy to ascertain if Linagliptin has any effect on RMR (56).  
 
Kidney health will be monitored by creatinine clearance and microalbumin/ creatinine 
levels, which will be obtained from the spot urine sample. 
a. Subjects will have the option of opting into a sub-study that is being 
where 10-20 mL of the excess fresh spot urine that has been 
collected will be used for exosome analysis. This is optional, and is 
not required of subjects. For details of urine-exosome study please 
see the appendix.  


 
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The biochemical or bio-inflammatory markers are an important marker of endothelial 
inflammation and dysfunction along with glycemic markers and markers of arterial 
stiffness (36-43, 55, 57). Literature suggests that DPP4 inhibitors and GLP1modifies and 
modulates hemopoetic stem cells and vaso dilatation (37, 44, 45).  
Vessel health will be assessed by degree of arterial stiffness, using arterial tonometry. 
The central and the aortic pressure is assessed by pulse wave analysis (PWA) and 
pulse wave velocity (PWV) . Arterial stiffness will be assessed using vascular flow and 
wave form analysis equipment, SphygmoCor CP system from ATCOR. (56) 
    
3 
INVESTIGATIONAL PLAN 
3.1 
Study Design and Duration 
Study Schema, 40 Type 2 diabetic Subjects Aged 40-70 on stable dose of Insulin, 
Metformin, or both 
 
 
+/- 3 day window for visits 
 
*Assessed at week 0, 6 and 12: Biochemical and cellular markers of endothelial function 
and Pulse wave analysis and velocity (PWA and PWV), REE and Adiposity. 
 
Week 16 - A telephone call to subjects will be made 30 days after last dose of study 
medication to determine if there have been any adverse events.  
                         
Available related studies and Expected Results: Studies measuring changes 
to EPCs in type 2 diabetes patients are very limited. To the best of our knowledge there 
are no studies examining the effect of Linagliptin in type 2 diabetes patients with CKD. 
Group A: Insulin/Metformin/Both + Placebo, 
n=20 
Wk 6* 
Wk 16 
Follow up 
          Screening 
Group B: Insulin/Metformin/Both + 
Linagliptin 5mg, n=20 
Wk-0* 
Randomization 
Wk 12* 
Wk -4 
 


 
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Use of CD34 positive cells as a cardiovascular risk surrogate has been reported in the 
past. Various studies (11, 34), have shown that various disease states, including those 
seen in diabetes, can deplete and damage EPC, thereby diminishing their regenerative 
potential.  The inability to maintain or repair damaged endothelial tissue leads to 
cumulative vascular dysfunction and cardiovascular disease.  
 
We hypothesize that monitoring number, function and gene expression of 
endothelial progenitors will allow us to quantify cardiovascular disease risk at the 
onset and regenerative potential post intervention at a cellular level. It will also 
help us to identify and correlate the best endothelial function bio-chemical 
inflammatory marker as an early indicator of cardiovascular disease progression 
in early type 2 diabetes with CKD and identify patients that are responders. 
   
Linagliptin along with other gliptins can increase SDF1 alpha levels and possibly CXCR4 
receptor expression which will help EPC mobilization and gene expression of 
hemopoetic CD34 positive stem cells.  
  
Linagliptin 5 mg or placebo will be added to patients who have type 2 diabetes 
diagnosed ≤15 years with HbA1C of 6.5 to 10 % (both inclusive) while being on Insulin, 
metformin, or both. 
Insulin will be titrated upwards if HbA1C is above 10% with minimal dose adjustments so 
as to keep the adjusted HbA1C below overtly uncontrolled levels (above 10%).  
 
Our study population will include equal numbers of adult male and female Type 2 
diabetes (T2DM) patients aged 40-70 years, enrolled in both arms of the study. 
Patients will be treated with 5 mg of Linagliptin or placebo for 12 weeks and with 
adequate lifestyle modification for 16 weeks (as per American Diabetes Association 
guidelines).  This time interval has been previously shown to be adequate to observe 
changes to Endothelial Progenitor Cells (EPCs), (5, 6). 
 
3.2 
Study Population 
For entry into the study, the following criteria MUST be met. 
3.2.1 
Inclusion Criteria 
1) Signed Written Informed Consent 
• 
Before any study procedures are performed, subjects will have the details of 
the study described to them, and they will be given a written informed 
consent document to read. Then, if subjects consent to participate in the 
study, they will indicate that consent by signing and dating the informed 
consent document in the presence of study personnel. 
2) Target Population 
• 
Subjects with a diagnosis of Type 2 diabetes mellitus within the previous 15 
years using criteria of the American Diabetes Association. 


 
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• 
Currently treated with Insulin (long or short acting or combination), Metformin 
(stable dose of 1-2 grams/day), or a stable dose of both. 
• 
HbA1C between 6.5% and 10% (both inclusive)   
• 
BMI 25-39.9 kg/m2 both inclusive. 
• 
History of Stage 1-3 CKD shown by a laboratory test taken within the past 2 
years 
CKD Staging: 
Estimated Glomerular Filtration Rate Serum Cr should be used to estimate 
glomerular filtration rate (GFR). Estimated GFR (eGFR) is commonly reported by 
laboratories or can be estimated using formulae such as the Modification of Diet in 
Renal Disease (MDRD) study equation or the Chronic Kidney Disease Epidemiology 
Collaboration (CKD-EPI) equation. The latter is the preferred GFR estimating 
equation. GFR calculators are available at http:// www.nkdep.nih.gov.  
Abnormal urinary albumin excretion and eGFR may be used to stage chronic kidney 
disease (CKD). The National Kidney Foundation classification (Table below) is based 
on both kidney damage (UACR at or above 30 mg/g Cr) and eGFR. 
Normal UACR is defined as less than 30 mg/g Cr, and increased urinary albumin 
excretion is defined as at or above 30 mg/g Cr. Because of variability in urinary 
albumin excretion, ideally two (at least one) specimens of UACR should have been 
collected within a 3- to 6-month period before considering a patient to have 
albuminuria. 
 
Stage -- Description  -- GFR (mL/min/1.73 m2 )  
Stage 1 Kidney damage* with normal or increased GFR >=90  
          2 Kidney damage* with mildly decreased eGFR 60–89  
          3 Moderately decreased eGFR 30–59  
          4 Severely decreased eGFR 15–29  
          5 Kidney failure ,15 or dialysis  
*Kidney damage is defined as abnormalities on pathological, urine, blood, or imaging 
tests.1 
 
 
3) Age and Reproductive Status 
Men and women, 40 to 70 years of age.  
• 
Enrolled women must not be pregnant, should not be breast-feeding and 
should not be taking hormone replacement therapy (HRT) during 
randomization. 
The following women are WOCBP: 
• 
Women using the following methods to prevent pregnancy: Oral contraceptives, 
other hormonal contraceptives (vaginal products, skin patches, or implanted or 
injectable products), or mechanical products such as intrauterine devices or 
barrier methods (diaphragm, condoms, spermicides). 
• 
Women who are practicing abstinence. 


 
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• 
Women who have a partner who is sterile (e.g., due to vasectomy).WOCBP must 
be using an acceptable method of contraception to avoid pregnancy throughout 
the study and for up to 4 weeks after the last dose of study drug in such a 
manner that the risk of pregnancy is minimized.  
• 
WOCBP must have a negative serum or urine pregnancy test result (minimum 
sensitivity 25 IU/L or equivalent units of HCG) within 0 to 72 hours before the first 
dose of study drug.  
• 
Women must not be breast-feeding. 
Post-menopause is defined as:  
• 
Women who have had amenorrhea for  12 consecutive months (without 
another cause) and/or who have a documented serum follicle-stimulating 
hormone (FSH) level > 35 mIU/mL. 
• 
Women who have irregular menstrual periods and a documented serum FSH 
level > 35 mIU/mL. 
• 
Women who are taking hormone replacement therapy (HRT). 
 
 
3.2.2 
Exclusion Criteria 
• 
Type 1 diabetes mellitus 
• 
History of diabetic ketoacidosis or hyperosmolar non-ketotic coma 
• 
Low hematocrit (< 28 Units) 
• 
History of pancreatitis, or cancer (except basal cell carcinoma and cancer that is 
cured or not active or being treated in the past five years) 
• 
Previous coronary event or history of cerebrovascular event within 6 months of 
screening or active or clinically significant coronary and/or peripheral vascular 
disease 
• 
CKD Stages 4 and 5 (estimated CrCl <30 mL/min) 
• 
Beginning Statin medications or of change of Statin dose in the last 3 months,  
• 
Use of oral or injectable anti-diabetic medication other than Insulin and 
Metformin. We did not want to bring in too many variables in the control arm and 
wanted to avoid a combination of insulin and other OHAs as the results might be 
difficult to interpret. 
• 
Patients will be off other OHA (other than Metformin) before randomization and 
inclusion in the study. 
• 
Use of consistent long-term steroid medication (oral, inhaled, injected) within the 
last 3 months 
• 
Untreated Systolic BP> 140 mmHg and Diastolic BP> 90mmHg  
• 
Active wounds or recent surgery within 3 months  
• 
Inflammatory disease, or current chronic use of anti-inflammatory drugs in the 
last 3 months.  


 
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• 
Untreated hyper/hypothyroidism 
• 
Contraindications for moderate exercise 
• 
Implanted devices (e.g., pacemakers) that may interact with Tanita scale 
 
Physical and Laboratory Test Findings 
• 
Pre-existing  liver disease and/or ALT and AST >2.5X’s UNL, 
• 
serum creatinine levels ≥2.0 with estimated CrCl < 30 mL/min) 
• 
Triglycerides >450 mg/dL  
Allergies and Adverse Drug Reactions 
• 
Subjects with a history of any serious hypersensitivity reaction to Linagliptin 
or another DPP-4 inhibitor.  
Sex and Reproductive Status 
• 
Women in reproductive age group will be included in the study but 
encouraged to use contraceptive method to avoid pregnancy within 16 weeks 
of study duration. 
• 
Women who are pregnant or breast-feeding will be excluded. 
Prohibited Treatments and/or Therapies 
Treatment with a strong cytochrome P450 3A4 (CYP3A4) or P-gp inducer (i.e. rifampin) 
Other Exclusion Criteria 
• 
Prisoners or subjects who are involuntarily incarcerated. 
• 
Subjects who are compulsorily detained for treatment of either a psychiatric 
or physical (e.g., infectious disease) illness.  
 
Additionally, patients who are active smokers, patients who are pregnant, nursing 
women, and women who are on hormone replacement therapy will be excluded.  
Patients on low dose oral contraceptives will be allowed to participate as these 
formulations contain very low amounts of estrogens. 
Eligibility criteria for this study have been carefully considered to ensure the safety of the 
study subjects and to ensure that the results of the study can be used. It is imperative 
that subjects fully meet all eligibility criteria. 
3.2.3 
Discontinuation of Subjects from Treatment 
Subjects MUST discontinue investigational product (and non-investigational product at 
the discretion of the investigator) for any of the following reasons: 
• 
Withdrawal of informed consent (subject’s decision to withdraw for any reason). 
• 
Any clinical adverse event, laboratory abnormality, or inter-current illness which, 
in the opinion of the investigator, indicates that continued participation in the study 
is not in the best interest of the subject. 


 
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• 
Patients in CKD stages 1-3 who progress to stages 4 and beyond, post 
screening, will be excluded also. 
• 
Concomitant treatment with a strong systemic cytochrome P450 3A4 (CYP 3A4) 
or P-gp inducer such as rifampicin. Use of alternative treatments to Linagliptin is 
recommended. 
• 
Pregnancy 
− 
Pregnant patients will be advised to contact the investigator or study staff 
immediately if they suspect they might be pregnant (e.g., missed or late 
menstrual period) at any time during study participation.  
• 
Loss of ability to freely provide consent through imprisonment or involuntary 
incarceration for treatment of either a psychiatric or physical (e.g., infectious 
disease) illness. 
• 
If pancreatitis is suspected, promptly discontinue treatment with Linagliptin 
All subjects who discontinue should comply with protocol-specified follow-up 
procedures outlined before. The only exception to this requirement is when a subject 
withdraws consent for all study procedures or loses the ability to consent freely (i.e., is 
imprisoned or involuntarily incarcerated for the treatment of either a psychiatric or 
physical illness). If a subject withdraws before completing the study, the reason for 
withdrawal must be documented appropriately. 
4 
TREATMENTS 
4.1 
Study Treatment: Linagliptin 
Definition of Investigational Product: A pharmaceutical form of an active substance or 
placebo being tested or used as a reference in a clinical study, including products 
already with a marketing authorization but used or assembled (formulated or packaged) 
in a way different from the authorized form, or used for an unauthorized indication, or 
when used to gain further information about the authorized form. In this protocol, the 
investigational product is Linagliptin. 
Definition of Non-Investigational Product: Other medications used in the study as 
support or escape medication for preventative, diagnostic, or therapeutic reasons as 
components of a given standard of care. In this protocol, the non-investigational product 
is Insulin, Metformin, or both.  Patients will continue on Insulin, Metformin, or both as per 
their study entry dose added to Linagliptin or placebo. As mentioned before, patients on 
oral hypoglycemic agents other than Metformin will not be invited to join the study as 
OHA plus DPP4 inhibitor could have a different effect on vasculature or hemopoetic 
CD34+ stem cells compared to insulin and DPP4 inhibitor.   


 
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This study is being conducted under FDA mandated guideline for use in type 2 diabetes. 
Though in this particular study Linagliptin is referred to as the investigational drug, it is 
not a novel drug and we should not need a separate IND for execution of this study. 
4.2 
Method of Assigning Subjects to a Treatment 
Subjects, as per selection criteria below will be randomized to treatments using a 
permuted block design, developed by the Epidemiology & Biostatistics Research Core. 
This approach ensures groups will be approximately balanced at any time during the 
study and at study completion.  
4.3 
Selection and Timing of Dose for Each Subject 
The recommended dose of Linagliptin is 5 mg PO will be given once daily. Linagliptin 
can be taken with or without food. 
4.3.1 
Dose Modifications 
No dose modification is necessary in CKD stage 1-3 
4.4 
Blinding/ Unblinding 
Blinding is critical to the integrity of this clinical study. However, in the event of a medical 
emergency or pregnancy in a subject, in which knowledge of the investigational 
product is critical to the subject's management, the blind for that subject may be 
broken.  
Before breaking the blind of an individual subject’s treatment, the investigator 
should have determined that the information is necessary, i.e., that it will alter the 
subject’s immediate management. In many cases, particularly when the emergency is 
not investigational product-related, the problem may be properly managed by assuming 
that the subject is receiving active product without the need for un-blinding.  
End of Study Unblinding Protocol 
Upon completion of the research study, we will undergo the following protocol in order to 
unblind the research data. 
Unblinding is the process by which the allocation code is broken so that the investigator, 
clinical staff and the trial statistician becomes aware of which intervention each subject 
enrolled in the research study was taking.  
Unblinding at the end of the study is required in order to make unmasked analysis in 
accordance with the study analysis plan. It is also conducted in order to inform the 
participants of which investigational product they were assigned to.  


 
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Time to unblind: 
Unblinding shall be conducted when all subjects enrolled in the research study have 
finished treatment, and all follow up visits. There must be no plan to recruit any more 
subjects in the research study. Additionally, all data points and outcome measures for 
each research subject must have been collected, and ideally compiled. Prior to 
unblinding there will be a data lock on clinical outcome measures and basic side 
outcome measures and associated  research data collected for the study.  
 
Procedure to unblind 
Once data (from both the clinical and the basic science side) and has been compiled 
and is data locked, the investigator can choose to unblind. The Principal Investigator 
must contact the study sponsor, and receive permission to un-blind. If, for unforeseen 
reasons, at the pre-determined date for full study unblinding the data analysis on the 
cellular or basic aspects of the study is lagging behind the clinical data outcome 
measures (though the data has been acquired) the Principal Investigator in consultation 
with the study sponsor may choose to un-blind the clinical outcome measures before the 
basic side data has been analyzed but compiled. Upon confirmation from the study 
sponsor, the principle investigator must make a written request to the designated party 
to unblind, hereto referred at the “unblinder”. The unblinder is the bio-statistician of the 
MFA, Dr. Richard Amdur. Upon receipt of an instruction to unblind, the unblinder will 
sign the request form, indicating their agreement to unblind. This form will then be taken 
to the pharmacy, MFA’s IDS, where a member of the IDS staff will take the form, and 
will give the unblinding study binder to the unblinder. The pharmacy will sign to indicate 
their release of the binder, and the unblinder will sign to indicate receipt of the binder. At 
this point in time the chain of custody of the pharmacy unblended binder has been 
transferred to the designated party to unblind. The form with all of the signatures will be 
provided to the study coordinator to be kept in the regulatory binder.  
 
Possible Adverse Events, Definitions of Adverse Events and Management 
Adverse event 
An adverse event (AE) is defined as any untoward medical occurrence, including an 
exacerbation of a pre-existing condition, in a patient in a clinical investigation who 
received a pharmaceutical product. The event does not necessarily have to have a 
causal relationship with this treatment. 


 
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Serious adverse event 
A serious adverse event (SAE) is defined as any AE which results in death, is 
immediately life-threatening, results in persistent or significant disability / incapacity, 
requires or prolongs patient hospitalisation, is a congenital anomaly / birth defect, or is to 
be deemed serious for any other reason if it is an important medical event when based 
upon appropriate medical judgement which may jeopardise the patient and may require 
medical or surgical intervention to prevent one of the other outcomes listed in the above 
definitions. Patients may be hospitalised for administrative or social reasons during the 
study (e.g. days on which infusion takes place, long distance from home to site,). These 
and other hospitalisations planned at the beginning of the study do not need to be 
reported as a SAE in case they have been reported at screening visit in the source data 
and have been performed as planned. 
 “Adverse Events of Special Interests” or “AEs of Special Interest” shall include: 
 
1. Severe Allergic Reactions 
2. Skin changes 
3. Angio-edema 
4. Heart attack or cerebral attack 
5. Neurological manifestations 
6. Acute Pancreatitis or acute hepatic failure 
7. Acute worsening of renal function 
8. Development of malignancy, occult or overt 
9. Severe Documented Hypoglycemia (lab blood sugar less 
than 40mg%) 
AEs of special interest are a list of medical topics that are under close surveillance. AE 
should be reported in an expedited manner similar to Serious Adverse Events even if 
they do not meet the seriousness criteria.  
Please refer to the site master file for the complete list of AESIs (27-30).  
 
Intensity of adverse event 
The intensity of the AE should be judged based on the following: 
• Mild: Awareness of sign(s) or symptom(s) which is/are easily tolerated 
• Moderate: Enough discomfort to cause interference with usual activity 
• Severe: Incapacitating or causing inability to work or to perform usual activities 
Causal relationship of adverse event 
Medical judgment should be used to determine the relationship, considering all relevant 
factors, including pattern of reaction, temporal relationship, de-challenge or re-challenge, 
confounding factors such as concomitant medication, concomitant diseases and relevant 
history. Assessment of causal relationship should be recorded in the case report forms.  
Yes: 
There is a reasonable causal relationship between the investigational product 
 
administered and the AE.  


 
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No: 
There is no reasonable causal relationship between the investigational product 
 
administered and the AE.  
Worsening of the underlying disease or other pre-existing conditions 
Worsening of the underlying disease or of other pre-existing conditions will be recorded 
as an (S)AE in the (e)CRF. 
Changes in vital signs, ECG, physical examination, and laboratory test results 
Changes in vital signs, ECG, physical examination and laboratory test results will be 
recorded as an (S)AE in the (e)CRF , if they are judged clinically relevant by the 
investigator. 
Responsibilities for SAE reporting: The Sponsor shall report (i.e., from signing the 
informed consent onwards through the trial defined follow-up period) all SAEs and non-
serious AEs which are relevant for a reported SAE and Adverse Events of Special 
Interest (AESI) by fax or other secure method using BI IIS SAE form to the BI Unique 
Entry Print in accordance with timeline specified below. 
 
• within five (5) calendar days upon receipt of initial and follow-up SAEs 
containing at least one fatal or immediately life-threatening event;  
 
• Within ten (10) calendar days upon receipt of any other initial and follow-up 
SAEs.  
 
Address for contact: 
BIPI Unique Entry Point:  
 
Boehringer Ingelheim Pharmaceuticals, Inc.  
 
900 Ridgebury Road Ridgefield, CT  
 
Fax: 1-203-837-4329  
 
E-mail: PV_global_casemanagement@boehringer-ingelheim.com.  
For each adverse event, the investigator will determine the expectedness of the 
investigational drug to the AEs as defined in the Listed Adverse Events section of 
the Boehringer Ingelheim’s (BI’s Summary of Product Characteristics (SmPC) or 
Product Information (PI) for the authorised Study Drug provided by BI.  
 
 
 
 


 
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4.5 
Study Schematic. Time and Events Schedule 
Time and Events Schedule for Protocol  
Procedure 
Screening 
Visit 
(Week -4) 
Baseline/ 
Randomization  
Visit 1 
(Week 0) 
During 
Treatment 
Visit 2 
(Week 6) 
End-of-
Treatment 
Visit 3 
(Week 12) 
Follow up 
phone 
call 
(week 16) 
Eligibility Assessments 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
Inclusion/Exclusion Criteria 
X 
 
 
 
 
Medical History 
X 
 
 
 
 
Safety Assessments 
 
 
 
 
 
Physical Examination 
X 
 
 
X 
 
Targeted Physical Examination 
(as needed) 
 
X 
X 
X 
 
Vital Signs 
X 
X 
X 
X 
 
Assessment of Signs and 
Symptoms 
 
X 
X 
X 
 
Adverse Events Assessment 
X 
X 
X 
X 
X 
Laboratory Tests (Biochemical) 
screening 
X 
X 
X 
 
Urine Pregnancy (If needed) 
X 
X 
X 
X 
 
Spot Urine Sample 
X 
X 
X 
X 
 
Tanita Scale Body Composition 
 
X 
X 
X 
 
Waist / Hip 
 
X 
X 
X 
 
Efficacy Assessments 
 
 
 
 
 
Peripheral blood draw for 
CD34+cell harvest 
 
X 
X 
X 
 
Pulse wave analysis and Pulse 
wave velocity assessment 
RMR                                             
 
X 
X 
X 
X 
X 
X 
 
Clinical Drug Supplies 
 
 
 
 
 
Randomize 
 
X 
 
 
 
Dispense Study Treatment 
 
X 
X 
 
 
 
Screening visit= week-4; Visit 1= week 0, Visit 2= week 6 and Visit 3= week 12.
 
 


 
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Research Study Design and Methods: 
 
Patient Definition and Selection: Adults aged 40-70 years will be recruited from the 
GWU MFA research patient database provided by MFA Information Technology, and 
then contacted via email and phone, from the endocrinology and other provider clinics at 
GWU and through referrals from the Veterans Affairs (VA) clinics. Suitable 
advertisements for the study patient enrollment will be posted. Patients will be included 
in the study if they have been diagnosed with type 2 diabetes within the previous 15 
years using criteria of the American Diabetes Association (2), and are currently treated 
with a stable dose of Insulin, Metformin, or both. We plan to enroll subjects with HbA1C 
between 6.5% and 10% (both inclusive) and with BMI 25-39.9 kg/m2 (both inclusive). 
Post enrollment, patients will be randomized to Linagliptin 5 mg or placebo as detailed in 
Study Design for direct comparison between the two regimens.  
Before any study procedures are performed, subjects will have the details of the study 
described to them, and they will be given a written informed consent document to read. 
Then, if subjects consent to participate in the study, they will indicate that consent by 
signing and dating the informed consent document in the presence of study personnel. 
Recruitment: Subjects will be recruited from the physician clinics at GWU, Medical 
Faculty Associates, where Dr. Sen is a clinician, as well as through referral from the VA. 
All patients will receive counseling regarding the study aims and methods.  
Study Design Overview: We propose a 2-arm randomized, double-masked, placebo-
controlled, parallel group, longitudinal study of 12-weeks duration. (See study design 
below), recruited over 3 years.  The 12 week time interval has been previously shown 
adequate to observe changes in endothelial progenitor stem cells, biochemistry and 
importantly, PWV changes [20, 36]. Patients will be randomized to 2 groups: 
Control, (n=20), Insulin/Metformin/Both + Lifestyle + Placebo 
Treatment, (n=20) Insulin/Metformin/Both + Lifestyle + Linagliptin 5mg 
This proposed study is based on patients with type 2 diabetes of ≤15 years duration, 
who are on stable dose of insulin, Metformin, or both for at least 3 months but still 
remain poorly controlled with HbA1C between 6.5% and 10% (both inclusive),  but not 
overtly out of control (> 10% of HbA1C). Our study design is similar to that of Fadini et al 
[20] where Sitagliptin was compared to placebo with endothelial progenitor cells as an 
outcome measure in patients with type 2 diabetes over 4 weeks.  
Though our study is over 12 weeks we do not anticipate significant drop in HbA1C in 
between the two groups in this relatively short time period to act as a confounding factor 
on CD34+ cell number, function and gene expression.  


 
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Most clinical studies looking at HbA1C reduction were of a much longer duration of 6 
months or above and most DPP-4 inhibitor studies are with animal EPCs where 
comparison with human CD34+ population is difficult to make. In one study (58) where 
saxagliptin was used for 12 weeks in drug-naïve type 2 diabetes patients there was 
placebo-subtracted HbA1c reduction noted of 0.45-0.63%. It showed mean placebo-
subtracted reductions in fasting serum glucose of 20 mg/dL. From our laboratory studies 
of in vitro human EPC studies we do not think glucose level change of 20 mg/dL is 
significant enough to account for changes in EPC number, function and gene 
expression. However we will take the possibility of glycemia change/ reduction in 
experimental arm into account during statistical analysis of the results between the 
Linagliptin and placebo groups. 
As per recent clinical guidelines (2), all patients at entry will receive dietary advice by 
study coordinator, diabetes educator, or registered dietician specifically geared towards 
type 2 diabetes patients. They will be advised to adhere to 150 minutes of weekly 
aerobic exercise (50-70% of maximal heart rate). Patient’s activity will be monitored 
using accelerometers and will be downloaded at visit 2 and visit 3 by the study staff. 
Subjects will be taking 5.0 mg of Linagliptin or placebo. Dose titration of insulin will be 
allowed so that the HbA1C is maintained below 10%. 
Visits with outcome measurements will be scheduled for the morning while overnight 
fasting (except water) to reduce diurnal variability, and clinical parameters will be 
obtained at scheduled office visits as depicted in the study schema and will be measured 
at each outcome visit as follows: 
Week -4: Subjects will be screened and eligibility will be verified based on physical and 
laboratory tests. Equal numbers of male and female patients will be recruited. All 
patients will be educated on the recommended lifestyle changes of maintaining a level of 
aerobic activity of 150 minutes/ week (non-supervised) and adherence to a healthy diet 
as advised by the study coordinator, a diabetes educator, or a dietitian as part of the 
type 2 diabetes patient management plan [2].  We will allow 4 weeks for the patients to 
reach a steady state while implementing diet and exercise changes. 
Week 0: At this stage patients will be randomized into the study. Subjects will have the 
first set of measurements. We will make sure that patients still meet the inclusion criteria. 
They will be started on medication or placebo and will be encouraged to continue 150 
minutes/week of moderate physical exercise and dietary management for the remainder 
of the study. 


 
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Weeks 0, 6 and 12: Subjects will all have the same measurements and will continue on 
study medication or placebo. All primary and secondary measures as outlined before will 
be carried out. 
The cellular outcome measures are as follows: 
 
From MNC population (pre magnetic column sorting): 
A. FACS Analysis, 1 million cells in each tube/assay column. 12 tubes-- total 12 million. 
(All labelling antibodies are obtained from Miltenyi Biotec except Sytox blue-Invitrogen 
and anti-VEGEFR2/KDR-R&D systems)  
1. Unlabelled 
2. CD34 
3. CD309 (VEGFR2-KDR)  
4. CD184 (CXCR4), receptor substrate for SDF1 alpha 
5. CD31 (PECAM-1, a mature circulating endothelial cell marker) 
6. CD133 (a progenitor marker) 
7. CD144 (Vascular Endothelial cadherin) 
8. Sytox Blue OR Propidium Iodide staining (to detect apoptotic cells)  
9. Combinations of 2+3+4+8, to assay percentage of double or triple positive cells 
10. (3+4+5+8), to assay percentage of double or triple positive cells 
11. 500,000 CD34+ cells, post column will be stained with – PE labelled + Sytox Blue (as a 
quality control measure, post column) 
  
B. Plate 5 million cells in each well of a 6 well or 12 well plates (2 million) BD 
Biosciences Human Fibronectin coated plate, total 2 wells, total 10 million mononuclear 
cells (prior to CD34 magnetic bead sorting) using CFU Hill Colony forming unit (Stem 
Cell Technologies- Cat#05900) media for Colony Forming unit (CFU) Assay.  
2 days post plating, harvest non-adherent cells from each well, counted and will be re-
plated at 1.0 x 106 cells/well in 12-well fibronectin coated plate. It is important than cell 
number of 1.0 x10e6 is maintained in each well.  
CFU needs to be counted on Day 7 post plating (from initial plating).  
 
From CD 34+ cell population (Post Sorting): 
A. Migration Assay using SDF1 Alpha concentrations of 0, 10, 100ng/ml using 100,000 
cells in 300ul serum free cell suspension media per insert. If sufficient cells are available 
we will note migration in response to VEGF-A (0, 20, 50 ng/mL).  
SDF1 Assay alone will need 100,000*9 = 0.9 million cells. Use triplicates. 
We will use 24 well plate using 3 micron pore migration membrane inserts from BD or 
Corning (354575). 
 
Genes to be assessed on CD34 positive, CD34 negative cells: 
 
Endothelial lineage cell surface markers: CD34, VEGFR2 (KDR), CD31, CD144 
For Anti-oxidant gene expression: Superoxide dismutase (SOD) 1, 2 and 3, catalase, 
glutathione- peroxidase. 
Apoptosis pathway: p53, p21, Bcl2, caspase-3 


 
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Endothelial Function Assay Gene: eNOS 
Genes associated with progenitor cell chemotaxis: VEGF-A, SDF1 alpha, CXCR4 
  
Secondary Measures:  
• 
Highly selective C-reactive protein (hs-CRP), IL-6, IL-10, TNF-alpha, and fasting 
lipid profile including ApoA1 and ApoB (3, 6) 
• 
Serum SOD activity and SDF1 alpha (ELISA), VEGF-A (ELISA) and GLP-1 
(ELISA) levels will also be assessed (14, 20, 24, 39). 
• 
Assessment of insulin sensitivity using the HOMA-IR (32), calculated from 
individual serum measures (fasting glucose (mg/dl)* insulin (µU/mL)/405) [37].  
• 
Adiposity will be measured using the Tanita Body Composition Analyzer scale, 
measured as percentage body fat, BMI, waist-hip measurements (5, 55) 
• 
Fasting lipid profile will be checked as a marker of insulin resistance and lipo-
toxicity at weeks 0, 6 and 12.  
• 
We also intend to acquire Pulse wave analysis and Vascular Flow using 
SphygmoCor CP system from ATCOR to measure central arterial pressure and 
arterial stiffness (38-42). The secondary measures are indirect measures of 
endothelial inflammation in early type 2 diabetes patients. (30, 31, 55, 56, 57).  
• 
Obtain indirect calometric measurements of basal metabolic rate using machine 
obtained from Korrs Medical. This is a non invasive procedure. We plan to 
measure the oxygen that the body consumes.  It will help us calculate a patient’s 
Resting Energy Expenditure (REE), commonly referred to as a Resting Metabolic 
Rate (RMR).   As our protocol is directed to overweight and obese individuals 
using incretin therapy, it will be beneficial to note if the treatment modifies RMR. 
Description: The machine ReeVue by KORR directly measures the concentration 
of oxygen breathed out by each patient.  The patient breathes through a simple 
mouthpiece as all the exhaled air is collected and analyzed.  Because there is a 
direct correlation between oxygen consumed and calories burned (4.813 calories 
for every milliliter of oxygen consumed), an accurate measurement of oxygen 
consumption is an effective measurement of calorie consumption. The machine 
uses a one-way valve in the disposable (MetaBreather) mouthpiece.  This draws 
in fresh room air with each inspiratory breath and eliminates concerns about 
cross contamination.  The disposable mouthpiece is thrown away. It is un-
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equipment is FDA approved and has been used in physician offices as an 
oxygen uptake test with associated CPT code #94690. (58) 
• 
Urine pregnancy testing for women of child bearing potential 
• 
Creatinine Clearance and proteinuria estimation via random spot urine. 
We will obtain a total of 85 mL of peripheral blood per visit. Of these 85 mL, 60 mL 
will be used to obtain CD34+ cells from MNC population and 25 mL for 
biochemistry and serum ELISA assays.  
We may obtain 95mL of peripheral blood at visits 2 and 3 if necessary, and the 
patient agrees. Of this 95mL, 75mL will be used to obtain CD34+ cells from MNC 
population and 20mL for biochemistry and serum ELISA assays.  
 
 
4.7 
Study Materials 
BI, manufacturers of Linagliptin are expected to provide Linagliptin and matching 
placebo at no cost for this study.  
4.8 
Safety Assessments 
Study drug toxicities will be assessed continuously. Adverse events will be evaluated on 
a continuous basis while the patient is on study and until 30 days after the last dose of 
study drug. Patients should be followed until all treatment-related adverse events have 
recovered to baseline or are deemed irreversible by the principal investigator.  
5 
STATISTICAL CONSIDERATIONS 
Statistical Analysis:  
Sample Size Estimation:  
The total sample size requested, accounting for attrition over the 12-week period, is 20 
subjects per group or 40 subjects total. We plan to allow for 25% attrition (drop out), 
therefore plan to screen 55 patients over three years. 
 
Sample size estimates were based on the effects of exercise on CD34+/KDR+ cells and 
VEGF, as described in the literature (47-50).   
 
This is a pilot study and accurate power calculation is not feasible. 
 
The effect of a single session, as well as extended training, on healthy subjects or those 
with existing cardiovascular conditions appears to increase the CD34+/KDR+ cells and 
VEGF.   


 
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To compute sample size we used the approach suggested by Diggle, Liang, & Zeger 
(1994)(51) which compares the rates of change in the two study groups over time.  This 
approach incorporates the number and interval of time points and the correlation among 
repeated measures.  For this study, we will employ one baseline and two follow-up 
measures at 6 and 12 weeks.  Further, we will assume a correlation 0.60 among 
repeated measures of the outcome. We consider this a conservative estimate since 
Frison and Pocock (1992) (52) suggest a correlation of 0.65 as reasonable in the 
absence of an existing estimate. We also note that as this correlation increases, 
statistical power also increases.  
 
The results in the table below show the expected mean difference in study groups at the 
end of follow-up, as well as the average rate of change in the two groups at 80% power 
and 90% power.  To estimate the effect of Linagliptin on the CD34+/KDR+ cells, we 
expect that the effect would be at least 25% greater than the effect seen for exercise 
alone.  Using the results from Sandri et al. (2005)49 for the rate of change and the 
variability, the CD34+/KDR+ cells increased an average rate of about 4/wk with a 
standard deviation of about 15.  Thus, for a 25% increase in the rate of change for the 
CD34+/KDR+ cells due to Linagliptin, a sample size of 18 subjects per group would 
provide about 84% power, assuming measures taken at baseline and 2 equally-spaced 
time points over 12 weeks. At the conclusion of follow-up, we would expect study groups 
to differ by an average of 12 cells.  If the effect of Linagliptin is only 20%, a sample of 18 
would provide about 70% power, whereas a sample of 20 would provide about 73% 
power. 
 
For VEGF, the results from Sandri et al. (2005)(49) were mixed with one study group 
showing a .6 pg/mL per week increase and another showing a 0.5 pg/mL per week 
decrease after 4 weeks of training.  If we assume that Linagliptin will increase VEGF 
consistently at 0.3 pg/mL per week difference in slopes compared with exercise alone, 
then assuming a standard deviation of 5 and correlation or 0.60 among repeated 
measures, a sample size of 18 per group would provide about 77% power to detect a 
difference in slopes of 0.3 pg/mL per week or a mean difference in the groups at the end 
of the study of 3.6 pg/mL.  A sample size of 27 would provide about 90% power to detect 
the same effects. 
 
Thus, we feel that a sample size of at least 18 subjects per group with complete data 
would provide sufficient power for the study outcomes.  In order to insure that we will 
have 18 per group who complete the study, we will enroll 20 subjects per group in order 
to account for attrition over the 12-week intervention period. 
 
 
Biochemical 
Measure 
Mean difference  
at end of 12 weeks 
Sample 
Size per 
group 
Power 
CD34+/KDR+ 
12 cells 
18 
0.84 


 
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cells 
(25% increase;  
4/wk vs. 5/wk) 
22 
0.90 
VEGF, pg/mL 
3.6 pg/mL 
(50% increase;  
(0.6/wk vs. 0.9/wk) 
18 
0.77 
27 
0.90 
 
Sub-Group Subject Number Breakdown: 
To estimate the effect of linagliptin on CD34+/KDR+ cells, we originally proposed 
a sample size of 20 per group in 2 groups, giving us power = .73, assuming 
alpha=.05, between time points of .60 and equal allocation between groups. 
Thus, the group x time interaction effect size we were modeling was an 
interaction that explained 3% of total variance, or a partial eta-square = .0291.  
Using 3 groups (Insulin only, metformin only, metformin + insulin), with the same 
effect size, we would achieve this same level of power using a total sample size 
of 51 (17 per group). However, given the relatively small numbers of potential 
participants with insulin-only (n=21) and insulin+metformin (n=54), and the 
relative large number with Metformin only (260), we will need to change the 
allocation ratio between groups. Maintaining the same total sample size as 
originally proposed (40), we will allocate patients from each group in the following 
numbers: Metformin only (22), Metformin+insulin (12), Insulin only (6). We 
recognize that we will be underpowered for comparisons between the Metformin 
+ insulin versus Insulin groups. However, comparisons with the Metformin-only 
group will still have reasonable power.  Given the small number of potential 
patients with Insulin-only, this is a feasible strategy for including this group in the 
analysis.  
 
 
Data Analysis.   
 
The distributional assumptions of all measures will be examined. Means and standard 
deviations will be computed for continuous measures and proportions for categorical 
variables. Graphical representations of the mean group slopes and individual slopes will 
be generated and inspected.  Study groups will be compared to determine whether any 
imbalance between the groups on patient characteristics remain after randomization.  If 
imbalance is evident, by inspection, all models will be adjusted for the unbalanced 
covariates.  Change in outcome over time will be examined using a multilevel approach 
with linear mixed models (LMM) (53, 54).  The longitudinal multilevel modeling approach 
will enable us to examine characteristics of within-person change, as well as between-
group differences that may influence change.  LMMs handle missing data more 
efficiently than traditional ANOVA designs. In the linear mixed models, the patient is 
considered a random effect and the outcome measured at specific time points is nested 
within patient.   In an initial model containing only the variable TIME, we will use a 
likelihood ratio test to determine whether a random effect for time should be modeled.  


 
Linagliptin-Sen IIS  
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ij
ij
i
i
i
ij
i
ij
ij
Time
u
u
Group
Time
Group
Time
Y





+
+
+

+
+
+
=
1
0
3
2
1
0
)
(
ij
ij
i
i
ij
ij
Time
u
u
Time
Y



+
+
+
+
=
1
0
1
0
The model below represents a random intercept for patient and a random coefficient for 
TIME. 
 
 
We will examine whether TIME may be modeled linearly by testing the added 
contribution of TIME (represented with two indicator variables) to a model with TIME as 
a single continuous variable.  Assuming time may be modeled as a continuous variable, 
subsequent analyses will add study group and other patient characteristics as 
covariates. We are particularly interested in whether there is a significant interaction 
between study group and time, adjusting for other patient level covariates.  A significant 
interaction would indicate that the slopes in outcome over time differ by study group.  If 
the global test of interaction is significant, subsequent significance testing of the pair 
wise comparisons of group slopes group will be adjusted for multiple comparisons.  The 
model also includes coefficients for the group-by-time interactions and random effects 
for patient and time. 
 
 
In the event that TIME is modeled using indicator variables, two indicator variables will 
be used to represent the three time points, with baseline serving as the reference 
category.  The interaction between GROUP and TIME will be modeled in a similar 
fashion using two additional indicator variables.   The above multivariable model enables 
us to adjust for time-varying covariates, such as glucose.  Since glucose will be 
measured contemporaneously with the outcomes at each visit, however, causal 
interpretations of glucose and any outcome are suspect.  As such, we will consider 
glucose as a nuisance covariate, and consider any relationship between glucose and an 
outcome as merely associative.   
 
APPENDIX A: OPTIONAL URINE EXOSOME STUDY 
A urine sample is already required as a part of Dr. Sen’s Linagliptin protocol. This urine 
will be used for urine pregnancy tests (if applicable), and labcorp tests for 
microalbumin/creatinine ratio. There will likely be excess urine.  
 
 
Subjects will have the option to opt in or out of a research study in which the urine will 
undergo a urine exosome analysis.  
 
Purpose of this Study: Exosomes are cell-derived vesicles that are present in many and 
perhaps all biological fluids, including blood, urine, and cultured medium of cell cultures. 
The reported diameter of exosomes is between 30 and 100 nm, which is larger than LDL, 
but much smaller than for example, red blood cells. Exosomes are either released from 
the cell when multivesicular bodies fuse with the plasma membrane or they are released 
directly from the plasma membrane.[3] Evidence is accumulating that exosomes have 
specialized functions and play a key role in, for example, coagulation, intercellular 
signaling, and waste management.[1] Consequently, there is a growing interest in the 
clinical applications of exosomes. Exosomes can potentially be used for prognosis, 
therapy, and biomarkers for health and disease. In this study we are particularly interested 


 
Linagliptin-Sen IIS  
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in exosomes released from podocytes as a marker of podocyte function and podocyte 
inflammation. Podocytes are often considered a modified endothelium and one of its 
more commonly used parameter is microalbuminuria. 
In this study we are using urine exosomes as an added parameter of kidney function other 
than microalbuminuria and GFR calculation. 
 
The procedure for this study is as follows: 
From this sample, 10-20 mL of the fresh spot urine (or as much as possible) is placed into 
a urine cup containing 1 tab of cOmplete Ultra - protease inhibitor cocktail (Roche) for 
exosome analysis. The specimen can be refrigerated at 4ºC for ~ 1 week or frozen at -
80ºC until assay, or will be processed for exosomes as follows;  
 
Spot urine for exosomes isolation has been successfully employed in our laboratories 
from ~15 ml urine using a differential centrifugation method2,3. The supernatant will be 
subjected to ultracentrifugation, with final centrifugation at 200,000g for 2 hr at 4ºC to 
obtain the urinary exosome pellet. The pellet will be tested for exosomes markers, 
namely CD63, CD9, CD81, Hsp70, and WT-1, nephrin and podocalyxin, using one of 
two methods. Preferably mRNA will be extracted and then RT-PCR will be run. 
Alternative analysis will be done using gel electrophoresis followed by Western blot 
techniques.   
 
Method 1: Urine exosome mRNA extraction 
mRNA extraction from exosomes will be accomplished by using commercial kits 
(Norgen Biotek Corp., Catalog # 47200). Thereafter, a pre-amplification step will take 
place, this is a necessary step to measure mRNA in exosomes and will be followed by 
quantification of specific endothelium and podocyte proteins such as CD9, WT-1, 
nephrin and podocalyxin by RT-PCR. 
 
Total urinary protein content, albumin and creatinine will be measured via Lapcorp for 
the same urine samples that have been collected.  
 
Method 2: Gel electrophoresis and Western blot  
 Exosome pellet will be dissolved in Laemli buffer and proteins will be resolved by gel 
electrophoresis (4-12% polyacrylamide gel) under denaturing conditions. Subsequently, 
proteins will be transferred to a PVDF membrane by Western blotting using a dry 
blotting method (iBlot). The blot will be incubated with blocking solution for 1h, and 
subsequently exposed to a solution with the first antibody overnight, at 4ºC. After 
washing (5 x 5 min), the membrane will be exposed to a secondary antibody (horseradish 
peroxidase [HRP]-conjugated) for 1h at room temperature. Finally, the membrane will be 
incubated with an HRP substrate reagent to reveal the proteins bound to the antibodies 
tested. Quantitative analysis will be performed by densitometry readings of the positive 
signals corrected to creatinine concentration of the urine aliquot equivalent to each 
exosome sample.  


 
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End-point: Exploratory but in conjunction with urine albumin/creatinine ratio will help 
to establish impact of Linagliptin on renal function in diabetes related CKD population. 
 
 
 
Guidance document for Adverse Events of Special Interest 
 
Adverse events of special interest are events that we are keeping under close 
surveillance and we would be very grateful if non-serious adverse events relating 
to the following medical topics can be reported using the SAE form so that the 
Global Pharmacovigilance Team receives it as soon as possible: 
 
Hepatic adverse events 
Renal adverse events 
Hypersensitivity reactions 
Skin lesions 
Pancreatitis 
Pancreatic cancer 
 
Thyroid neoplasia or cancer 
Cardiac failure 
 
 
The following information is key in assisting case evaluation. If possible please 
provide the following when reporting an Adverse Event of Special Interest: 
 
Hepatic adverse events 
-     Alcohol intake (e.g. grams or units per week) present and past 
-     Jaundice (personal or family history) 
-     Hepatobiliary gallstone disease 
-  Known hepatic disease- eg metabolic, autoimmmune, malignancy or -
metastatic disease 
-     Metabolic-induced liver disease, non-alcoholic steatohepatitis (NASH) 
-     Recent administration of other drugs with known hepatic toxicity- please 
state reason if these are not recorded as co-suspect drugs 
-     Environmental exposure to liver toxins (e.g. CCL4, death cap mushroom, 
vinyl chloride) 
-     Substance abuse/Intoxications/Intravenous drug use 
-     History of drug allergy/hypersensitivity reaction 
-     Infectious diseases: 
-     Bacterial, Protozoic/parasitic hepatic infection 
-     Viral hepatic serology 
-     Travel history 
-     Blood transfusions history 
-     Site, radiation and character of pain 
-     Biopsy results &/or hepatology consult reports 
-     Radiological investigations eg sonography 


 
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Renal adverse events 
-     Clarity on whether the patient has pre-renal, renal-renal or post-renal failure 
and the cause of this, eg hypovolaemia due to diarrhoea, glomerulonephritis or 
bladder obstruction  
-     Fluid balance if possible 
-     Baseline renal function and past history of renal insufficiency 
-     Renal and bladder stone disease- if possible with type of stone (eg Ca 
oxalate, etc) 
-     Site, radiation and character of pain 
-     Biopsy results &/or nephrology consult reports 
-     Known renal or urinary tract disease 
-     All BUN / U&Es results 
-     All urine investigation results including microscopy and culture 
-     Creatinine clearance 
-     Radiological investigation reports, eg Kidney-Ureter-Bladder X-ray, 
Intravenous pyelogram 
Hypersensitivity reactions 
-     Clarity on whether the patient has a diagnosed condition- eg angioedema or 
anaphylaxis as opposed to reporting symptom of tongue swelling 
-     For systemic hypersensitivity syndromes, all laboratory and radiological 
investigations and differential diagnoses 
-     Clear description of clinical presentation, time course and treatments 
-     If there are consults from other specialities, please include a copy of the 
report if possible 
Skin lesions 
-     Clarity on whether the patient has a diagnosed condition as opposed to 
reporting symptoms 
-     If a dermatology consult has occurred, please send a copy of the report 
-     Results of any biopsies 
-     Estimate of the percentage of the total body surface area that is involved 
-     Clearly state whether or not there is mouth ulceration 
-     Clearly state whether or not the rash has blisters 
-     Please provide any evidence of systemic reaction, laboratory abnormalities 
 
Pancreatitis 
-     Pancreatic enzymes and liver enzyme values, if possible on presentation and 
during course of the event 
-     Baseline and current lipid investigations 
-     Report of Radiological diagnostic procedures, eg sonography, CT, ERCP 
-     Clinical symptoms with description of site radiation and character of pain 
-     Clarity on the diagnosis in the verbatim reported term- eg whether the patient 
had clinical pancreatitis or assymptomatic enzyme rise 
-     Permission to contact treating physician for follow up information 
-     All relevant abnormalities in the pancreaticobiliary tree, eg gallstones, stenosis, 
CBD thickening 
-     Alcohol intake 


 
Linagliptin-Sen IIS  
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-     Clarity on whether the patient has chronic pancreatitis with a known aetiology 
 
Pancreatic cancer 
-     Please clearly state the start date of the study drug, the start date of the 
presenting symptoms and the date of diagnosis of cancer 
-     Please state method of diagnosis, staging, grading and (if available) histology 
-     Family history of cancer or pancreatitis 
-     BMI of the patient 
-     If previous radiology had not shown abnormalities, please confirm if vascular 
imaging techniques had been requested 
-     Please state all previous diabetic medication 
-     Has the patient had previous chemo/radiotherapy? If so please provide details 
-     Please provide permission to contact pancreatic oncologist 
 
Thyroid neoplasia or cancer 
 
-     Please clearly state the start date of the study drug, the start date of the 
presenting symptoms and the date of diagnosis of cancer 
-     Please state method of diagnosis, staging, grading and (if available) histology 
-     Family history of cancer 
-     Please state all previous diabetic medication 
-     Has the patient had previous chemo/radiotherapy? If so please provide details 
-     Please provide permission to contact oncologist 
-     Please clarify whether the lesion is benign or not 
-     Please list previous medications used to treat thyroid disease 
 
 
Cardiac failure 
-     Please clearly state the start date of the study drug, the start date of the 
presenting symptoms and the date of diagnosis of heart failure 
-     Did the patient have a history of heart failure, coronary artery disease, renal 
failure, arrhythmia, of heart valve abnormality? 
-     If EchoCG was done, please provide the report 
-     Provide NYHA class of CHF 
 
 
 
 
 
 
 
 
References- Linagliptin Study 
1. NIDDK. National diabetes statistics: 2007 and 2011 fact sheet. Bethesda, MD, 
USA. Department of Health and Human Services, NIH, 2008 and National Center 
for Chronic Disease Prevention and Health Promotion, 2011. 


 
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2. Standards of medical care in diabetes-2014; American Diabetes Association. Diabetes 
Care January 2014 37:S14-S80 
3. Sheetz MJ, King GL. Molecular understanding of hyperglycemia’s adverse 
effects for diabetic complications.  JAMA. 2002;288(20):2579–2588. 
4.  Rask-Madsen C, King GL: Mechanisms of disease: Endothelial dysfunction in 
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5. Werner N, Kosiol S, Nickenig G, et al:. Circulating endothelial progenitor cells 
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N 
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6. Jialal I, Devaraj S, Singh U, Huet BA. Decreased number and impaired 
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7. Hill JM, Quyyumi AA, Finkel T et al. Circulating endothelial progenitor cells, 
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8. Losordo DW, Schatz RA, White C et al. Intramyocardial transplantation of 
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12. Marrotte EJ, Chen DD, Hakim JS, Chen AF. Manganese superoxide dismutase 
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13. Elsharawy MA, Naim M, Greish S. Human CD34+ stem cells promote healing of 
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senescence: contribution to oxidative stress. Curr Cardiol Rev 2008; 4:275-286 


 
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15. Knowler WC, Barrett-Connor E, Fowler SE et al. Reduction in the incidence of 
type 2 diabetes with lifestyle intervention or metformin. NEJM 2002 Feb 
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16. Ratner R, Goldberg R, Haffner S et al. Impact of intensive lifestyle and metformin 
therapy on cardiovascular disease risk factors in the diabetes prevention 
program. Diabetes Care; 2005 Apr;28(4):888-94.  
17. Knowler WC, Fowler SE, Hamman RF et al. 10-year follow-up of diabetes 
incidence and weight loss in the Diabetes Prevention Program Outcomes Study. 
Lancet. 2009 Nov 14;374(9702):1677-86.  
18. Padilla J, Simmons GH, Bender SB, Arce-Esquivel AA, White JJ, Laughlin MH. 
Vascular effects of exercise: endothelial adaptations beyond active muscle beds. 
Physiology. 2011 26: 132-145.  
19. Exercise and Type 2 Diabetes: American College of Sports Medicine and the 
American Diabetes Association: Joint Position Statement  Medicine & Science in 
Sports & Exercise. 42(12):2282-2303, December 2010. 
20. Fadini GP, Boscaro E, Albiero M, et al: The oral dipeptidyl peptidase-4 inhibitor 
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21. Herrera C, Morimoto C, Blanco J, et al: Comodulation of CXCR4 and CD26 in 
human lymphocytes. J Biol Chem 2001; 276:19532-19539 
22. Zheng H, Fu G, Dai T, et al: Migration of endothelial progenitor cells mediated by 
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23. Yin Y, Zhao X, Fang Y, et al: SDF-1alpha involved in mobilization and 
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24. Moore MA, Hattori K, Heissig B, et al: Mobilization of endothelial and 
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serum levels of SDF-1, VEGF, and angiopoietin-1. Ann N Y Acad Sci 2001; 
938:36-45;  
25. Verge D, López X., Impact of GLP-1 and GLP-1 Receptor Agonists on 
Cardiovascular Risk Factors in Type 2 Diabetes, Curr Diabetes Rev. 2010 Apr 
12. Review. 
26. Sokos GG, Nikolaidis LA, Mankad S, Elahi D, Shannon RP. Glucagon-like 
peptide-1 infusion improves left ventricular ejection fraction and functional status 
in patients with chronic heart failure. J Card Fail. 2006 Dec;12(9):694-699.  
27. Nikolaidis LA, Mankad S, Sokos GG, Miske G, Shah A, Elahi D, Shannon RP. 
Effects of glucagon-like peptide-1 in patients with acute myocardial infarction and 


 
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left ventricular dysfunction after successful reperfusion. Circ. 2004 Mar 
2;109(8):962-5.  
28. Muniyappa R., Iantorno M., Quon M.J. An integrated view of insulin resistance 
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29. Lovshin JA, Drucker DJ. Incretin based therapies for type 2 diabetes mellitus. Nat 
Rev Endocrinol. 2009 May;5(5):262-9. Review.  
30. Sen S, Strappe P, O'Brien T. Gene transfer in endothelial dysfunction and 
hypertension. Methods in Molecular Medicine. 2005;108:299-314.  
31. Krenning G, Dankers PY, Drouven JW, Waanders F, Franssen CF, van Luyn MJ, 
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32. Matthews DR, Hosker JP, Rudenski AS, et al. Homeostasis model assessment: 
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34. Sen S, Chou C, Islam A: Effect of aerobic exercise on Endothelial Progenitor 
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SessionsL. 
35. Jie KE, Zaikova MA, Bergevoet MW, Westerweel PE, Rastmanesh M, Blankestijn 
PJ, Boer WH, Braam B, Verhaar MC. Progenitor cells and vascular function are 
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36. Hattori Y, Jojima T, Tomizawa A, Satoh H, Hattori S, Kasai K, Hayashi T. A 
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production and exerts anti-inflammatory action in endothelial cells. Diabetologia. 
2010 Oct;53(10):2256-63.  
37. Cabou C, Campistron G, Marsollier N, Leloup C, Cruciani-Guglielmacci C, 
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38. Delp MD. Effects of exercise training on endothelium-dependent peripheral 
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39. Scheen AJ: Dipeptidylpeptidase-4 inhibitors (gliptins): focus on drug-drug 
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40. Tepper OM, Galiano RD, Capla JM, Kalka C, Gagne PJ, Jacobowitz GR, Levine 
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CD31+/annexin V+ apoptotic microparticles correlate with coronary endothelial 
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42. Hambrecht R, Wolf A, Gielen S, Linke A, Hofer J, Erbs S, Schoene N and 
Schuler G. Effect of exercise on coronary endothelial function in patients with 
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77(8):736-742. 

ICF_001.pdf: 
 
 
Ver. 03/07/2018 
IRB # 121439 
1 
 
 
Research Subject Information and Consent Form 
 
Title:  
Role of Linagliptin in Improving Renal Failure by Improving CD34+ Cell  
 
 
 
Number, and Gene Expression in Renal Function Impaired Type 2  
 
 
 
 
Diabetes Patients 
 
 
Sponsor: 
  Medical Faculty Associates, George Washington University 
Funding Source:  
Boehringer Ingelheim (BI) 
 
Principal 
Investigator: 
 
 Research 
Coordinator: 
 
 
Dr. Sabyasachi Sen, MD, PhD 
The George Washington University 
School of Medicine and Health Sciences 
2300 I Street, Ross Hall 450 
Washington DC, 20037 
202-994-8560 
 
Fiona Dore, B.S.  
Medical Faculty Associates 
2300 M Street, NW 
Washington, DC 20037 
 
fdore@mfa.gwu,edu 
202-741-2342 
 
 
WHY ARE YOU BEING ASKED TO TAKE PART IN THIS RESEARCH?  
You have been invited to participate in a research study. You are being asked to take part in this 
research study because 1) you have type 2 diabetes 2) mild form of kidney disease 3) taking a stable 
injectable form of Insulin, stable dose of Metformin, or stable dose of both, and are not taking any other 
oral diabetic medications 3) have not yet achieved the level of glucose control that is best for you. We 
will be enrolling male and female subjects who are 30-70 years old. 
Whether or not you take part in this study is up to you. If you choose not to participate in the study it 
will not affect the quality of medical care you will receive. Your academic standing/employment status 
will not be affected in any way should you choose not to take part or to withdraw at any time. 
This form gives you important information. Please read it carefully and ask questions before you make a 
decision. You may want to talk about this research study with your family, your friends, and your other 
health care providers. Please take your time. You should not sign this form until all of your questions 
are answered. 
 
WHY IS THIS RESEARCH STUDY BEING DONE?  
Type 2 diabetes is a national epidemic. Diabetes has undesirable effects on blood vessels which may 
contribute to heart disease. Endothelial Progenitor Cells (EPCs) are found in the blood. Research has 
shown that improving the survival of these special blood cells may decrease the harmful effects of 
diabetes on blood vessels and reduce or reverse heart disease. Linagliptin is an FDA (Food and Drug 
Administration) approved prescription medicine used along with insulin or with oral medications to 
lower blood sugar in people with Type 2 diabetes. It is in a class of diabetes medication called DPP-4 
inhibitors. DPP-4 inhibitors have been shown to increase EPCs in patients with Type 2 diabetes. The 
IRB Approval Date 10APR2018


 
 
Ver. 03/07/2018 
IRB # 121439 
2 
 
purpose of this research study is to explore the effect of Linagliptin in addition to a stable dose of 
Insulin, stable dose of Metformin, or stable dose of both in patients diagnosed with type 2 diabetes with 
creatinine clearance less than 90 and above 29 (or with microalbinuria), on the EPCs. A creatinine 
clearance is a test that compares the amount of creatinine (a breakdown product from muscle) in your 
urine to the amount in your blood. This is used to test how well the kidneys are working. In this study, 
Linagliptin will be compared to placebo used together with Insulin/Metformin/Both (which you are 
already taking). Linagliptin is a medication in pill form taken once a day. The placebo is a pill that looks 
like Linagliptin but will have no active ingredients. If you take the placebo medication it will be like 
taking no medication at all. 
 
HOW IS THIS RESEARCH STUDY BEING FUNDED?  
This research is being funded by an Investigator Initiated Independent Study Grant funded by 
Boehringer Ingelheim (BI), the maker of Linagliptin. 
 
HOW MANY PEOPLE WILL TAKE PART IN THIS STUDY?  
The research is being conducted only at GWU Medical Faculty Associates. We expect to enroll 40 
subjects in this study. 
 
HOW LONG WILL YOU BE IN THIS STUDY?  
Your participation in this research study is expected to last for 18 weeks. You will need to visit the 
research department four (4) times, including the screening visit. Each visit is anticipated to last 
approximately 90 minutes. 
Your participation in this study may be stopped if the study doctor thinks (1) it is in your best interest to 
stop, (2) if you do not follow the study requirements, or (3) or if the study is stopped for any reason. 
The study doctor will tell you about new information that might affect your health or could change your 
decision to be in this study. If this occurs, you may be asked to sign a new consent form. 
 
PARTICIPATION IN THIS STUDY IS VOLUNTARY  
Taking part in this study is voluntary. You may choose not to take part or to leave the study at 
any time. Your decision will not affect your relationship with your doctor or with GWU and will 
not result in any penalty or loss of benefits to which you are otherwise entitled. 
You can stop taking part in this study at any time. Tell the study doctor or research coordinator if 
you are thinking about stopping or have decided to stop. The study doctor may also ask you to have 
some follow up care or tests done. 
 
WHAT WILL YOU DO IN THIS STUDY?  
Screening: 
To make sure that you are eligible for this research study you will need to have the following exams, 
tests, and/or procedures. This process is called “screening”. If you had some of these done recently, they 
may not need to be repeated.  
• A medical history and physical exam will be performed, to make sure you don’t have anything 
that could keep you out of the study, such as a heart disease or medical conditions that make it 
unsafe for you to exercise. 
• You will receive exercise counseling by the study coordinator, a registered dietician, or a 
diabetes educator. 
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• Your heart rate, blood pressure, temperature, respiratory rate, weight and height will be 
measured 
• Lab tests will be done to check different things in your body. They include: 
o Complete Blood Count(CBC) and Sed Rate (Infection, anemia, and others) 
o Hemoglobin A1C(HbA1C) (Blood sugar) 
o ALT/AST (Liver function) 
o Lipid Panel (Cholesterol) 
o Blood Urea Nitrogen (BUN) (Kidney function) 
o Electrolytes (Kidney function) 
o Fasting Glucose (Blood sugar) 
o Thyroid Stimulating Hormone (TSH) (Thyroid Function) 
o Microalbumin/Creatinine Ratio (Kidney function) - URINE 
o Pregnancy test (if applicable) - URINE 
If the results of the screening tests show that you are found to be eligible for this study, you will be 
randomly assigned(randomized)into one of the two study groups by chance (like flipping a coin). You 
will have an equal chance (50%) of being in either group. Half of the people will get a placebo and half 
will get the study drug, Linagliptin. The same tests and procedures will happen to both study groups. 
After the first two weeks of changes to your diet and exercise, you will begin to receive the study 
medication, either placebo or Linagliptin. Neither you nor the study team can choose or know which 
group you are in. 
 
If you are accepted into the study you will have the following exams, tests or procedures: 
• Lab tests will be done at study Visit 1, 2, and 3 for research purposes. This includes a fasting 
blood draw, and a urine sample. We will be looking at many things including a check of your 
blood sugar level called HbA1C, and many other lab tests including cholesterol. The amount of 
blood drawn will be approximately 85 milliliters, but may be up to 95 mL for visit 2 and 
visit 3. 
• Your heart rate, blood pressure, weight and height will be measured 
• Your waist and hip circumference measurements will be done. 
• Your weight and body fat will be measured with bare feet on the Tanita Body Composition 
Analyzer Scale. This scale sends a mild electrical current through your body when you step on 
it. The electrical current is so small that you will not feel it. This is a research related procedure 
only and is not usually done as part of a physical exam. 
• You will need to do mild to moderate exercise (~150 minutes/week), in order to maintain a 
healthy life style. 
• You will be required to wear an Actigraph for 7 continuous days after visit 1 and 7 continuous 
days after visit 2. This device is worn on the hip with a belt or clip either under or above your 
clothing and will measure your activity during the day. You will take it off before you go to bed 
and put it back on when you wake up. It cannot get wet, so if you bathe or swim during the day, 
you will need to remove it. This device will help us monitor your level of physical activity. This 
is a research related procedure. It is not routinely done. 
• Vascular flow and wave measurements (like a mini ultrasound) of the blood vessels (pulse) in 
your wrist, neck, and groin area. This is a research related procedure, not routinely done. 
• Resting Metabolic Rate (RMR) will be measured using a machine called ReeVu. This is a non-
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invasive procedure. You will breathe through a simple mouthpiece and all the exhaled air is 
collected and analyzed. Because there is a direct relationship between oxygen used and calories 
burned an accurate measurement of oxygen use is an effective measurement of calories burned. 
The machine uses a one-way valve in the disposable (MetaBreather) mouthpiece. This draws in 
fresh room air with each inspiratory breath and eliminates concerns about cross contamination. 
The disposable mouthpiece is thrown away. This procedure is not routinely done for the care of 
diabetic patients. 
The following will happen to everyone in the study (in all groups): 
 
When 
What happens 
Screening  
(Week -2) 
Today’s visit (described just before this table) 
•  Consent form, inclusion/exclusion 
checklist, list of current medications 
• Screening lab tests, including pregnancy 
test (if applicable) 
• History and physical exam 
• Vital signs (Blood Pressure, Respiratory 
Rate, Heart Rate, and Temperature)  
• Height, weight, and hip and waist 
measurements 
• Diet and exercise counseling 
Study Visit 1 
(Week 0) 
• Randomization 
• Vital signs (Blood Pressure, Respiratory 
Rate, Heart Rate, and Temperature) 
• Height, weight/body composition (Tanita 
Scale), and hip and waist measurements 
• Resting Metabolic Rate measurement 
using ReeVue device 
• Dietary advice 
• Actigraph device given and exercise 
motivation 
• Fasting Lab work 
• Vascular flow and wave measurements 
• General diabetes education 
• Begin taking study medication or placebo 
• Urine Sample from patient 
Study Visit 2  
(Week 6) 
• Vital signs (Blood Pressure, Respiratory 
Rate, Heart Rate, and Temperature) 
• Height, weight/body composition (Tanita 
Scale), and hip and waist measurements 
• Resting Metabolic Rate measurement 
using ReeVue device 
• Dietary advice 
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• Actigraph device given and exercise 
motivation 
• Fasting Lab work 
• Vascular flow and wave measurements 
• General diabetes education 
• Return unused study drug and packaging 
• Dispense study medication or placebo 
• Urine pregnancy test (if applicable) 
• Urine Sample from patient 
Study Visit 3 
(Week 12) 
• Vital signs (Blood Pressure, Respiratory 
Rate, Heart Rate, and Temperature) 
• Height, weight/body composition (Tanita 
Scale), and hip and waist measurements 
• Resting Metabolic Rate measurement 
using ReeVue device 
• Dietary advice 
• Actigraph device and exercise motivation 
• Fasting Lab work 
• Vascular flow and wave measurements 
• General diabetes education 
• Return unused study drug and packaging 
and Actigraph device 
• Dispense study medication or placebo 
• Urine pregnancy test (if applicable) 
• Urine Sample from patient 
Follow up telephone call 
(Week 16) 
• Determine if you are experiencing any 
side effects after stopping the study 
medication 
 
OPTIONAL: Urine Exosome Study  
As a part of the Linagliptin study, you will be providing a urine sample. This urine will be used for urine 
pregnancy tests (if applicable), and tests for microalbumin/creatinine ratio. There will be extra urine left over.  
If you want, you can be a part of a sub-study in which your extra urine will undergo a urine exosome analysis.  
 
Exosomes are bubbles that form from cells, and travel between parts of the body to communicate information. 
They also are used to get rid of “garbage” in the cell. The information in these bubbles can tell us more about 
someone’s kidney function, and also about how some diseases (such as chronic kidney disease) may be 
changing.  
 
You have the option at the end of this consent form to either opt in or opt out of this sub-study.  
If you opt in, then the excess urine will be used for exosome analysis. There will be no additional 
compensation provided, and you will not be notified about these results.  
 
If you do not wish to partake in this sub-study then you can simply circle “no” at the end of this consent 
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form. This will not affect your care or involvement in the Linagliptin study. 
 
WHAT RISKS OR PROBLEMS COULD YOU HAVE BY BEING IN THIS STUDY?  
You may experience risks or discomfort as a result of being in this study. As with any research study 
there may be risks to you that are not know at this time. The known risks are described below. 
Having your blood drawn can be uncomfortable and can sometimes cause a bruise. In some people, it 
can cause fainting. In very rare cases, an infection may occur. Only trained people will draw your blood. 
Let your doctor or study coordinator know if you have had any problems with blood draws. 
 
The Tanita Body Composition Scale may cause a pacemaker or similar device to malfunction. It should 
not be used by people with pacemakers or other similar devices. It is important that you tell the study 
doctor if you have a pacemaker or other similar devices. 
 
Linagliptin is a medication that is on the market now and has FDA approval. However, like all 
medications it may have side effects or risks from taking it. These risks include: 
• Low blood sugar (Hypoglycemia), particularly with taken with insulin or other insulin secreting 
medications. 
• Acute pancreatitis (pain in your stomach area that is severe and will not go away. The pain 
may be felt going from your abdomen through to your back. The pain may happen with or 
without vomiting. Pancreatitis could be potentially fatal.  
• Bullous Pemphigoid, a rare skin condition which involves fluid filled blisters forming on the 
skin 
• Mouth ulceration (like canker sores), or stomatitis (an inflamed mouth) 
 
If you have any of these symptoms you should contact your study doctor or study staff immediately. 
In less than 5% of patients (taking linagliptin alone or in combination with insulin) 
• Upper respiratory tract infection (naso-pharyngitis) 
• Diarrhea 
• C o u gh  
• Constipation 
• Swelling of your ankles (angio-edema), pain in joints (arthralgia), pain in muscles and 
extremities, which could be severe and disabling. 
 
Allergic reactions 
Include: skin rash, itching, flaking or peeling, raised red patches on your skin (hives), swelling of your 
 
face, lips, tongue and throat that may cause difficulty in breathing or swallowing, difficulty with
 
 swallowing or breathing. 
 
If you have any of these symptoms, stop taking the study medication and contact your doctor or go to 
the nearest hospital emergency room right away. 
 
General Statement 
Tell your study coordinator if you have ever had inflammation of your pancreas (pancreatitis), stones in 
your gallbladder (gallstones), a history of alcoholism and high blood triglyceride levels. 
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You need to supply us the entire list of your medications, importantly other diabetes medications. Please 
mention if you are taking any medication for tuberculosis such as Rifampicin. You should immediately 
contact your study coordinator if you start any prescription drug or other medication (including over-
the-counter drugs and herbal supplements) not prescribed by the study physician-investigator. 
You must not be pregnant or breastfeeding, and you should not become pregnant or breastfeed while 
you are taking the study treatments. You must use an adequate method to avoid pregnancy for the 
duration of this study. You should immediately contact your study coordinator if there is a change in 
your method to avoid pregnancy. 
 
 Risks to Reproduction, Unborn Babies and Nursing Infants Unforeseeable Risks 
There may be unknown risks to you, your unborn baby or nursing infant if you are or become pregnant 
during this study or are breastfeeding during this study.  
 
Pregnancy 
Pregnant subjects are advised to not use Linagliptin. Suitable contraceptives, such as the double barrier 
method of contraception, are suggested while the female patients of child bearing age are enrolled in this 
study. Hormonal preparations of contraception should be avoided. Estrogen and progesterone may have 
an effect on EPCs or mature endothelium or there may be a possible interaction between estrogen 
preparations and endothelium. If you have any questions about methods of birth control, please discuss 
them with your study doctor. 
 
Use of a Study-Prohibited Contraceptive Method 
You should notify your study physician/ coordinator if hormonal preparations are started as a form of 
contraception during the course of this study.  
 
Requirements for Pregnancy Testing 
During this study you may have a number of pregnancy tests. Pregnancy will be determined on the basis 
of a urine test. 
 
Occurrence of Pregnancy or Suspected Pregnancy 
If you become pregnant, suspect pregnancy or if you missed your period or it is late, or if you have a 
change in your usual menstrual cycle (e.g., heavier bleeding during your period or bleeding between 
periods), you should immediately contact your study doctor.  
 
Discontinuation from the Study 
Should you become pregnant during this study, you will be immediately withdrawn from the trial or 
have the investigational product(s) discontinued (as stated in the protocol) and will be referred for 
obstetric care. The sponsor has not set aside any funds to pay for any aspects of obstetric, child or related 
care and does not plan to pay for them.  
 
Pregnancy Reporting 
In case of a pregnancy, your pregnancy and its outcome will be reported to the study sponsor. 
 
Information for Men with Partners of Childbearing Potential 
Linagliptin, as per currently available medical literature does not appear to pose a risk to a woman who 
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becomes pregnant while her male partner is a study subject, using linagliptin. However, you are asked to 
inform your study doctor if your partner becomes pregnant while you are enrolled in this clinical trial, 
and you and your partner will be asked to provide information about the pregnancy outcome. The 
sponsor has not set aside any funds to pay for any aspects of obstetric, child or related care and does not 
plan to pay for them. 
 
WILL YOU BENEFIT FROM BEING IN THIS STUDY? 
• You may benefit from the nutritional counseling and exercise motivation 
• You may have improved cardiovascular health and possibly a decrease in weight and/or blood 
Pressure 
• Your diabetes could improve, stay the same or get worse 
• You may not benefit directly but your participation could benefit society. What we learn from 
this research study may help other people with pre-diabetes or diabetes in the future. 
 
WHAT OPTIONS OTHER THAN THIS STUDY ARE AVAILABLE TO YOU?  
If you do not want to be in this study, other treatments may be available to you such as: 
• You could take part in another research study 
• You could choose to exercise and diet on your own, or with your own doctor 
• You could choose no treatment 
 
WILL THERE BE ANY COSTS TO YOU?  
Clinical services provided during a research study are either research-related or related to usual medical 
care. 
Research-related services are not the responsibility of you or your insurance. Your medical care at these 
visits for this study is research-related and you will not have to pay for it. 
 
WILL YOU RECEIVE ANY COMPENSATION?  
You will receive $50 per visit to cover your time and expenses such as transportation, parking and 
babysitting. 
You will receive up to $200 for all 4 visits. You will be paid with a VISA gift card (or similar). 
 
WHAT HAPPENS IF YOU HAVE A COMPLICATION?  
It is possible that you could have complications or injuries that are directly related to your participation in 
this study. If this happens, you will be offered medical treatment at the responsibility of yourself or your 
insurance company. GWU Medical Faculty Associates, GWU, GWU Hospital or the sponsor does not 
have a program to provide compensation or free medical treatment for research-related injury. You do 
not lose any of your legal rights by signing this form. 
 
HOW WILL YOUR PRIVACY BE PROTECTED?  
We will protect your privacy as a participant in this research study and the confidentiality of your 
research information. Information obtained from the research will be recorded in a separate file from 
your Hospital Medical Record and confidentiality will be maintained by assigning you a code number. 
Your study file will be stored in a secure area in the Clinical Research Division, GWU Medical Faculty 
Associates. While the study is ongoing, an electronic database of your study data will be kept using 
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RedCap, which is an online protected database that is stored at Children’s National. The information 
that identifies you will be kept separate from the RedCap database, and will be kept in a password 
protected excel document (only accessible to the study staff) that is stored on the MFA network. We 
may be required by law to report some information (for example; certain infectious diseases, suspected 
abuse) to a state agency for public health or safety reasons. 
If we publish information from this research study or use it for teaching, your name will not be used. 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required by U.S. 
Law. This Web site will not include information that can identify you. At most, the Web site will 
include a summary of the results. You can search this Web site at any time. 
 
INFORMATION ABOUT THE PRIVACY OF PROTECTED HEALTH INFORMATION  
GWU Medical Faculty Associates, its employees, and its affiliates are required by law to protect the 
privacy of information that identifies you. If you enroll in this research study, your protected health 
information (referred to as PHI in the rest of this section) may be used and shared with others as 
explained below. 
 
What PHI will be used and shared with others for this research study? 
• Information gathered from your medical records. 
• New information created as a result of this study (for example, from study exams, tests, 
procedures, surveys, etc.). 
Why will your PHI be used and shared? 
• We need to use and share your PHI in order to conduct this research study, monitor your safety 
and the safety of the study as a whole, and to ensure that the research meets legal, institution, and 
accreditation standards. 
• We may also need to use and share your information for treatment purposes, payment, or for 
health care operations. 
Who may use and share your PHI? 
• The study doctors and the staff helping them conduct the research. 
• Other people within GWU Medical Faculty Associates and its affiliates such as those who 
oversee research, process bills and payments, conduct quality assurance, and provide legal 
advice. 
• Other doctors, medical centers, and research staff taking part in this study as necessary. 
• Insurers and their agents as necessary. 
• Organizations that accredit hospitals and research programs. 
• Boehringer Ingelheim (BI), the funding source of the research study. 
In order to check that we are conducting research properly, government agencies may access 
information that could identify you. For example, the following people/groups may inspect research 
records: 
• The Office of Human Research Protections in the U.S. Department of Health and Human 
Services. 
• State agencies such as the Department of Public Health. 
• Other domestic and foreign government agencies if required. 
• Representatives of Federal regulatory agencies such as the U.S. Food and Drug 
Administration (FDA) Once your PHI has been released it may no longer be protected by 
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federal law.  
 
Can you see your research records? 
• You can ask to see your research records but sometimes that can only happen after the research 
study is completed. If you would like to see your research records please discuss this with your 
study doctor or call: Sabyasachi Sen, MD at 202-994-8560.  
How long will your PHI be used and shared for this research study? 
• Since research is an ongoing process, there is no scheduled date at which your PHI will be 
destroyed.  
What if you decide that you no longer want your PHI used or shared for this research study? 
• You can withdraw your permission at any time for us to use and share your PHI by contacting 
your study doctor. We will not be able to take back any information that has already been used 
or shared. You will not be able to continue in the research study once you withdraw your 
permission. 
 
If you have any questions about this study, please contact: Dr. Sabyasachi Sen, MD at 202-994-
8560 or Fiona Dore at, GWU Medical Faculty Associates, 2150 Pennsylvania Ave NW, Suite 3-325, 
and Washington, D.C. 20037, e-mail- fdore@mfa.gwu.edu 202-741-2489 
If you experience a complication or injury that you believe may be related to this study, please contact 
Dr. Sabyasachi Sen at 202-994-8560. Indicate you are participating in a research study and suspect you 
have a complication for which you need to speak directly to Dr. Sen, the Principal Investigator. 
 
If you would like to discuss your rights as a research participant, or wish to speak with someone 
not directly involved in the study, please contact the George Washington university Office of 
Human Research at 202-994-2715, this is your representative. 
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OPTIONAL URINE-EXOSOME SUB STUDY 
 
I would like to partake in the optional urine exosome sub-study. I understand that I do not need to undergo 
any additional procedures in order to partake, and that it is just my excess urine sample that will be analyzed. 
I understand that there will be no additional compensation provided, and that I will not get the results from 
this sub-study.  
 
I would like to participate in the OPTIONAL urine exosome sub-study:  
 
YES _______   
 
 
NO _______  
(Initials)  
 
 
 
(Initials) 
 
 
 
STATEMENT OF VOLUNTARY CONSENT  
By signing below, I am volunteering to be in this research study. 
 
 
Participant's Name (Print): _______________________________________________________  
 
Signature:   __________________________________________________ Date:  ___________  
 
 
STUDY REPRESENTATIVE STATEMENT  
I have explained the purpose of the research, the study procedures, the possible risks and discomforts, 
the possible benefits, and have answered all questions to the best of my ability. 
 
Study Representative's Name (Print):  ______________________________________________  
 
Signature:  ___________________________________________________________________  
 
Date: ______________ 
 
 
 
Time: ____________________ 
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