Conditions: Obesity
Keywords: obesity growth hormone
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 44
Healthy volunteers: yes

Brief summary:
This study tests the following hypotheses:

Aim 1: Test the hypothesis that acute dipeptidyl peptidase 4 (DPP4) inhibition with the currently available anti-diabetic drug, sitagliptin, will increase stimulated growth hormone (GH) secretion in healthy lean adults by decreasing the degradation of growth hormone releasing hormone (GHRH).

Aim 2: Test the hypothesis that decreased degradation of GHRH during acute DPP4 inhibition will result in an increase in endothelium-dependent vasodilation mediated by GH and independent from GLP1 (glucagon like peptide-1) in healthy lean adults.

This study promises to provide novel data regarding how this increasingly used class of anti-diabetic drugs affects the pituitary GH axis and could affect blood vessel relaxation. Growth hormone levels are low in the setting of obesity and pre-diabetes. A further study may evaluate the effect of chronic DPP4 inhibitor therapy in a population of patients with obesity and pre-diabetes.

Detailed description:
Growth hormone secretion is low in patients with obesity, insulin resistance, and hyperlipidemia. GH therapy in these populations and others has been limited by side effects which include hyperglycemia. Another strategy to increase GH secretion is to enhance pulsatile GH secretion by growth hormone releasing hormone. Growth hormone releasing hormone (GHRH) has a half life of 5 minutes due to its rapid inactivation by DPP4. An alternative strategy to increase endogenous GH secretion is by inhibiting degradation of GHRH by DPP4. DPP4 inhibitors are currently approved therapies for the treatment of hyperglycemia in patients with type 2 diabetes mellitus. They additionally cause blood vessel relaxation. We therefore propose to test the hypothesis that DPP4 inhibition simultaneously enhances GH secretion while improving blood glucoses and vascular function in patient populations with low GH and increased cardiovascular risk. These preliminary aims serve primarily as a novel "proof of concept" study to define the effect of acute pharmacologic DPPIV inhibition on stimulated GH secretion.

Arms:
arm 1: In Aim 1: Healthy Lean adults are randomized in a double-blinded cross over fashion to sitagliptin versus placebo.

In Aim 2: Healthy Lean adults receive sitagliptin and are randomized in a double-blinded cross over fashion to pre-treatment with either LNMMA (L-N-Monomethyl-arginine) versus placebo. arm 2: In Aim 1: Healthy Lean adults are randomized in a double-blinded cross over fashion to sitagliptin versus placebo.

In Aim 2: Healthy Lean adults receive sitagliptin and are randomized in a double-blinded cross over fashion to pre-treatment with either pegvisomant versus placebo. arm 3: In Aim 1: Healthy Lean adults are randomized in a double-blinded cross over fashion to sitagliptin versus placebo.

In Aim 2: Healthy Lean adults receive sitagliptin and are randomized in a double-blinded cross over fashion to pre-treatment with either Exendin 9-39 versus placebo.

Interventions: intervention 1: Sitagliptin intervention 2: Pegvisomant intervention 3: Placebo intervention 4: L-NMMA intervention 5: Exendin 9-39
Intervention descriptions: intervention 1: During Aim 1, given on one of two study days (other study day subjects receive placebo.) During Aim 2, given during both of two study days. intervention 2: During Aim 2, given 72 hours prior to one of two study days (Group B subjects only) intervention 3: During Aim 1, given on one of two study days (other study day subjects receive sitagliptin.) During Aim 2, given on one of two study days (other study day subjects receive either L-NMMA, pegvisomant, or Exendin 9-39 pending their group assignment) intervention 4: During Aim 2, given during one of two study days (Group A subjects only) intervention 5: During Aim 2, given during one of two study days (Group C subjects only)

Protocol document:
Prot_SAP_000.pdf: 
 
9/8/16 
1 
 
 
 
 
 
 
 
 
 
The Effect of Dipeptidyl Peptidase IV Inhibition on Growth Hormone-
Mediated Vasodilation 
 
Jessica K. Devin, MD MSCI 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
9/8/16 
2 
 
Table of Contents: 
 
1.0 
Background and Previous Human Studies 
2.0 
Rationale and Specific Aims 
3.0 
Inclusion/Exclusion Criteria 
4.0 
Enrollment/Randomization 
5.0 
Study Procedures 
6.0 
Risks 
7.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to 
Participants or Others 
8.0 
Study Withdrawal/Discontinuation 
9.0 
Statistical Considerations 
10.0 Privacy/Confidentiality Issues 
11.0 Follow-up and Record Retention 
 
 


 
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3 
 
1. Background and Previous Human Studies 
Dipeptidyl peptidase IV (DPPIV) is a ubiquitously expressed cell surface serine 
protease which cleaves a dipeptide from the N-terminus of its polypeptide substrates 
containing a proline or alanine at the second residue. Interest in this peptidase and its 
pharmacological inhibition has arisen due to its proteolytic inactivation of the incretin 
hormones and the role this plays in glucose homeostasis. Selective DPPIV inhibitors 
improve glycemic control in patients with type 2 diabetes mellitus by decreasing the 
degradation of the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-
dependent insulinotropic peptide (GIP).(1;2) The DPPIV inhibitor sitagliptin was 
approved by the FDA in 2006 as a once daily oral medication to lower blood sugar 
levels in patients with type 2 diabetes mellitus. Two additional DPPIV inhibitors have 
since received FDA approval: saxagliptin (2009) and linagliptin (2011). The direct and 
indirect effects of these inhibitors on cardiovascular function remain an active area of 
investigation. 
GLP-1 also promotes endothelium-dependent vasodilation in the human vasculature 
in subjects with type 2 diabetes and stable coronary artery disease. In addition to 
preventing the degradation of the incretin hormones, DPPIV inhibitors prevent the 
degradation of other peptides with a penultimate proline or alanine including but not 
limited to the vasodilators substance P as well as BNP and the neurotransmitter 
neuropeptide Y.(3;4) Our group has previously demonstrated that five day treatment 
with sitagliptin significantly decreased mean arterial pressure in subjects with metabolic 
syndrome.(5)  Others have reported that acute sitagliptin treatment reduces 24 hour 
ambulatory blood pressure in non-diabetic patients with mild to moderate 
hypertension.(6) It has recently been demonstrated in an animal model and in vitro that 
acute DPPIV inhibition leads to vascular relaxation via a GLP-1 independent 
mechanism involving nitric oxide release mediated by Akt dependent phosphorylation of 
endothelial nitric oxide synthase.(7) 
DPPIV inhibitors could affect blood pressure and vascular function in part by their 
effect on the somatotropic axis. Pituitary growth hormone releasing hormone (GHRH), 
also known as growth hormone-releasing factor (GRF) is a substrate of DPPIV, which 
rapidly cleaves GRF(1-44)-NH2 to the biologically inactive GRF (3-44)-NH2 at Ala2-
Asp3.(1;2) Conversion to GRF (3-44)-NH2 is blocked in vitro by diprotin A, a DPPIV 
competitive inhibitor.(8) Human GRF is produced in the hypothalamus and stimulates 
the release of the growth hormone (GH) from somatotrope cells within the anterior 
pituitary gland; the active portion of the molecule resides in the first 29 amino acid 
residues. Opposing the actions of GRF, the hypothalamic hormone somatostatin inhibits 
the release of GRF.(9) Growth hormone circulates throughout the body exerting its 


 
9/8/16 
4 
 
numerous effects both directly and indirectly via the hepatic production of insulin-like 
growth factor-1 (IGF-1). DPPIV proteolysis plays a major role in the degradation and 
inactivation of human GRF in vivo.(2) Thus, we hypothesize that inhibition of DPPIV will 
result in decreased GRF degradation and augmentation of the somatotropic axis in vivo 
in humans. 
The direct vascular effects of GH and its downstream mediator IGF-1 are well-
established.  Systemic and arterial GH infusion increases forearm blood flow in healthy 
subjects independent of systemic and local IGF-1 production.(10;11)  This vasodilation 
is reversed by infusion of the nitric oxide inhibitor, L-N-monomethylarginine (L-
NMMA).(10) Furthermore, growth hormone receptors are present in abundance on 
human endothelial cells. Incubation of endothelial cells with GH in vitro increases the 
phosphorylation and activity of endothelial nitric oxide synthase (eNOS) resulting in 
increased formation of nitric oxide (NO).  NO is formed by endothelial nitric oxide 
synthase (eNOS) within the endothelial cells of the vasculature and then diffuses to 
underlying smooth muscle cells where it increases vasodilation via cGMP production 
and decreases inflammation and platelet aggregation.(12-14) Growth hormone 
treatment also contributes to vascular integrity by enhancing NO-mediated mobilization 
of endothelial progenitor cells in healthy individuals.(15;16) The exact mechanism(s) 
through which GH regulates eNOS remain unclear. Evidence exists for both an IGF-1 
mediated activation of eNOS involving the serine/threonine Akt phosphorylation-
dependent kinase as well as a direct GH-mediated activation of both Akt and JAK-STAT 
(Janus kinase-signal transducer and activator of transcription) pathways.(17) 
Interestingly, individuals with hypopituitarism experience increased cardiovascular 
and cerebrovascular morbidity and mortality as well as an increased prevalence of 
cardiovascular risk factors, despite adequate steroid and thyroid hormone replacement 
though lacking in growth hormone replacement.(18-20) The PI has demonstrated that 
adults with growth hormone deficiency also demonstrate alterations in plasma 
fibrinolytic balance, as compared to gender, age, and body mass index-matched 
controls.(21) Several prospective studies in adults with hypopituitarism have 
demonstrated that GH replacement improves cardiac function, intima-media thickness, 
endothelial function, and vascular reactivity as well as various biochemical markers of 
cardiovascular and metabolic disease including fat distribution and inflammatory 
cytokines.(22-27) Furthermore, GH deficient adults exhibit low levels of metabolites of 
systemic NO production; and GH replacement therapy normalizes biomarkers of 
NO.(16;28;29)  Many of the adverse cardiovascular factors observed in GH deficient 
individuals are accordingly attributed to inadequate vascular NO bioavailability which 
can be reversed with GH treatment.   


 
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5 
 
Patients with the cardiovascular risk factors of hypertension, hyperlipidemia, 
diabetes mellitus or tobacco use demonstrate endothelial dysfunction characterized by 
the inability of the endothelium to generate adequate amounts of NO.(14) Basal NO-
mediated dilatation in the forearm arterial bed of patients with insulin-dependent 
diabetes is impaired as assessed by venous occlusion plethysmography.(30) The 
impairment in endothelial function in experimental animals and humans with diabetes 
mellitus is attributed to reduced NOS activity and/or NO availability as well as the 
accompanying increased production of reactive oxygen species. This characteristic 
endothelial dysfunction has been implicated in the pathogenesis of diabetic vascular 
disease.(31)  
This aim promises to provide important new data regarding how DPPIV inhibition 
affects the somatotropic axis in healthy adults.  Given the known effects that growth 
hormone and IGF-1 have on endothelial function as well as cardiovascular and 
metabolic markers, an augmentation of this axis may positively impact vascular function 
in the diabetic population. None of our currently available anti-glycemic agents offer this 
benefit. Data from the Framingham Heart Study demonstrates a negative correlation 
between circulating IGF-1 and insulin resistance in a community based sample.(32) 
GRF-induced GH secretion is blunted in premenopausal women with hyperlipidemia 
and insulin resistance.(33) Thus derangements in the somatotropic axis have previously 
been described in individuals at risk for the development of diabetes.  
Adults with diabetes demonstrate heart disease death rates two to four times higher 
than adults without diabetes (“Diabetes Statistics” American Diabetes Association; 
www.diabetes.org); thus DPPIV inhibitors may potentially represent a novel therapy that 
is simultaneously able to confer both a glycemic and vascular benefit in this at-risk 
population. We therefore expect that findings from this study will lead to further 
investigations regarding currently unrecognized but significant vascular effects of this 
relatively new class of medications increasingly used in the diabetic population.  
2. Rationale and Specific Aims 
This study tests the hypothesis that DPPIV inhibition will favorably augment the 
somatotropic axis in healthy individuals by decreasing the degradation of GRF by 
DPPIV. This study specifically establishes the effect of acute DPP4 inhibition on 
arginine-stimulated GH secretion and GH-mediated blood flow in healthy humans. 
Arginine decreases inhibitory hypothalamic somatostatin secretion, allowing the primary 
stimulus of GH secretion to be GHRH.  We will study healthy adults to obtain maximal 
GH signal in this proof-of-concept initial aim, given that obesity diminishes the GH 
response to pharmacologic stimuli.  


 
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6 
 
-Aim 1A tests the hypothesis that acute DPP4 inhibition will increase stimulated 
GH secretion in healthy lean adults by decreasing the degradation of GHRH. 
-Aim 1B tests the hypothesis that decreased degradation of GHRH during acute 
DPP4 inhibition will result in an increase in endothelium-dependent vasodilation 
mediated by GH, and independent from GLP-1. 
 
 
This study promises to provide novel data regarding how this increasingly used class 
of anti-diabetic drugs could modulate vascular function. Given the known changes 
within the somatotropic axis in the setting of insulin resistance, further studies may wish 
to evaluate the effect of DPPIV inhibition in individuals with the metabolic syndrome. 
 
 
 
3. Inclusion/Exclusion Criteria 
Forty-two (fourteen per group)  healthy, non-smoking subjects (31 females and 11 
males), age 18 through 40 years of age will participate in this randomized, double-blind, 
placebo-controlled crossover study. Subjects taking medications other than 
multivitamins will be excluded.  Pregnancy will be excluded in women of child-bearing 
age by serum β-HCG. 


 
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7 
 
Inclusion Criteria 
 Age 18 to 40 years inclusive 
 BMI ≤ 25 kg/m2 
 For female subjects: 
o Status-post surgical sterilization, or 
o If of child-bearing potential, utilization of a barrier method of birth 
control following negative serum β-HCG at screening visit and on 
every study day 
Exclusion Criteria 
 Smoking 
 Type 1 or Type 2 Diabetes Mellitus, as defined by a fasting glucose of 126 
mg/dL or greater at the time of screening visit or the use of anti-diabetic 
medication 
 Hypertension, as defined by an untreated seated SBP greater than 140 
mmHg and/or an untreated DBP greater than 90 mmHg at the time of 
screening visit or the use of anti-hypertensive medication 
 History of reported or recorded hypoglycemia (plasma glucose < 70 
mg/dL) 
 Pregnancy and/or Breast-Feeding 
 Use of any medication other than multivitamin, including use of 
transdermal as well as oral hormone replacement therapy  
 Anemia defined as hematocrit <35% at screening visit 
 Cardiovascular or cerebrovascular disease, including history of myocardial 
infarction, history of congestive heart failure, history of stroke 
 Pulmonary Hypertension 
 Abnormal thyroid hormone levels (TSH) at the time of screening visit 
 Abnormal serum IGF-1 at the time of screening visit 
 Impaired renal function, defined as eGFR <60 mL/min/1.73M2 
 Impaired hepatic function (AST or ALT > 2 X upper limit of normal range) 
 Treatment with an investigational drug in the 1 month preceding the study 
 
 
4. Enrollment/Randomization 
Subjects will be randomly assigned to treatment order (see below table) for each pair 
of visits using a block randomization algorithm with a block size of 2. 
 study 
biostatistician, will provide an allocation schedule.  The Vanderbilt Investigational Drug 
Service will be responsible for the storage, preparation, and labeling of all 


 
9/8/16 
8 
 
investigational agents and for maintaining accurate drug storage and dispending logs. A 
Clinical Research Pharmacist in the Vanderbilt Investigational Drug Service will devise 
standard operating procedures for the pharmacy to follow with regard to preparing, 
labeling, blinding, and dispensing study drug.  The Investigational Drug Service will 
retain a secure set of sealed envelopes containing the treatment assignment.  
Randomized subjects who do not complete the whole protocol for any reason will be 
replaced. 
Aim 1A 
Randomization  
Anticipated 
Result 
Aim 1B Randomization 
Anticipated Result 
 
 
 
Sitagliptin vs. 
Placebo 
(N=42) 
 
 
 
Sitagliptin 
increases 
stimulated 
GH secretion 
and FBF 
Group A  (N=14) 
Sitagliptin + 
[L-NMMA vs. Placebo] 
Endothelium-dependent 
vasodilation mediated by 
Arginine, GH, and GLP-1 
blocked 
Group B (N=14) 
Sitagliptin + 
[GH Receptor Antagonist vs. 
Placebo] 
GH mediated vasodilation 
blocked 
Group C (N=14) 
Sitagliptin + 
[GLP-1 Receptor Antagonist vs. 
Placebo] 
Endothelium-dependent and 
independent vasodilation 
mediated by GLP-1 blocked 
 
 
 


 
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9 
 
 
5. Study Procedures 
 
 
 
The figure above illustrates the Aim 1A study protocol for Groups A and C (Group B 
has completed Aim 1A).  Each subject will undergo two study days separated by at least 
a 2 week wash-out.  Seventy-two hours prior to each study day, subjects will start once 
daily study drug (sitagliptin 100 mg by mouth or matching placebo), following a negative 
pregnancy test obtained in the Vanderbilt Clinical Research Center (CRC), as 
applicable.  This duration and dose of sitagliptin was chosen to achieve steady-state 
with significant inhibition of DPP4 activity.(34) On each day, subjects will report to the 
Vanderbilt Clinical Research Center (CRC) in the morning after an overnight fast and 
having abstained from exercise that morning.  Subjects will be studied in the supine 
position in a temperature-controlled room. All subjects will receive a peripheral venous 


 
9/8/16 
10 
 
catheter in the non-dominant arm for infusion of arginine as well as a peripheral venous 
catheter in the contra-lateral arm for repeated blood sampling. Normal saline will be 
infused into the catheter in the non-dominant arm prior to arginine administration for 
purposes of keeping the vein open. Subjects will then be given their 4th dose of study 
drug. One hour after administration of study drug we will measure baseline forearm 
blood flow (FBF, see below) and obtain baseline venous blood samples (see below). 
We will infuse arginine (30 grams) intravenously over 30 minutes. We have chosen this 
dose of arginine, previously used in the clinical setting to diagnose GH deficiency, to 
yield a peak plasma GH level of 10 ng/mL 30minutes after completion of arginine 
infusion.(35;36) .  Immediately following arginine administration we will repeat 
measurements of forearm blood flow every 30 minutes and venous blood sampling 
every 15 minutes up to 150 minutes after arginine infusion. The protocol will be 
repeated following a 2-week washout using the opposite study drug (sitagliptin or 
matching placebo). Both blood pressure and heart rate will be continuously monitored 
throughout each study day. At the completion of each study day subjects will be fed 
lunch prior to discharge from the CRC. 
 


 
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11 
 
 
The figure above illustrates Aim 1B. If an effect of sitagliptin on FBF is observed, 
subjects will be invited to return for 2 additional study visits (Study Visits 3 and 4) in 
order to elucidate the mechanism underlying the increase in FBF. Study Visit 3 will 
occur at least 8 weeks after Study Visit 2. Subjects will be given DPP4 inhibitor as well 
as double-blinded antagonist vs. placebo (see previous table) on each study days. In 
Group B, DPP4 inhibition will be given as a single oral dose (sitagliptin 200 mg) on 
each study day; a minimum of 4 weeks will separate study days. In Groups A and C, 
DPP4 inhibition will be given as 100 mg sitagliptin by mouth daily, starting 72 hours prior 
to each study day with the 4th dose given on the study day; a minimum two-week 
washout will separate study days. Venous sampling will occur following assessment of 
forearm blood flow.  All other study procedures will take place as previously described.  
Details regarding the dose and administration of each antagonist are provided below.   
Forearm Blood Flow Measurements 
FBF will be measured using mercury-in-silastic strain-gauge plethysmography. The 
wrist is supported in a sling to raise the level of the forearm to above the level of the 


 
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12 
 
atrium, and a strain gauge is placed in the widest part of the forearm.  The strain gauge 
is connected to a plethysmograph (model EC-4, D.E. Hokanson, Issaquah, WA), 
calibrated to measure the percent change in volume and connected to a chart recorder 
to record flow measurements.  For each measurement, a cuff placed around the upper 
arm is inflated to 40 mmHg with a rapid cuff inflator (model E-10, Hokanson) to occlude 
venous outflow from the extremity.  The hand is excluded from the measurement of 
blood flow by inflation of a pediatric sphygmomanometer cuff to 200 mmHg around the 
wrist during measurement of FBF.  Flow measurements are recorded for approximately 
7 secs/15 secs, and the slope is derived from the first 3-4 pulses; 7 readings are 
obtained for each mean. 
Venous Blood Sampling 
Following measurement of FBF, venous samples will be obtained prior to arginine 
administration and immediately as the completion of arginine 30 minute infusion.  
Venous samples will then be obtained every 15 minutes up to 150 minutes following 
completion of arginine infusion in Aim 1A.   In Aim 1B, venous samples will be obtained 
before and following arginine infusion at 8 designated timepoints.  Samples may be 
analyzed for ADMA, arginine, GH, insulin, glucose, GLP-1, DPP4 activity and antigen, 
cGMP, PAI-1 antigen, tPA activity and IGF-1.   
The dose of GH receptor antagonist (pegvisomant 80 mg given subcutaneously 72 
hours prior to each study day in Aim 1B) was chosen as 80 mg of pegvisomant 
produces maximal drug levels 72 hours following administration and a reduction in free 
IGF-1 comparable to that observed in GH deficiency, thus indicative of efficient GH 
receptor blockade and a state of acute GH deficiency.(37;38) Pegvisomant is a GH 
analog that disables signal transduction through the GH receptor and thereby functions 
as a receptor antagonist. This selective blockade allows us to examine the effects of GH 
secretion in the absence of cofounders such as changes in IGF-1 secretion and body 
composition which may accompany chronic GH deficiency. This method of achieving 
acute GH blockade has been reported to safely and effectively study GH secretion in a 
variety of metabolic conditions and to evaluate the effect of GH secretion on 
cardiovascular risk markers.(37-39) 
The dose of GLP-1 receptor antagonist (Exendin-(9-39) IV bolus of 7500 pmol/kg 
over 1 minute at time 0 (Aim 1B) followed by continuous infusion of 750 pmol/kg/min 
for 150 minutes) was chosen as this dose elicits blockade of GLP-1 action at its 
receptor in healthy volunteers during a fixed blood glucose, as measured by 
augmented post-prandial glucagon secretion, a blunted increase in post-prandial 
insulin secretion, and a significant increase in post-prandial GLP-1 secretion.(40-


 
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13 
 
42)The timing of Exendin-(9-39) infusion may be adjusted pending results of Aim1A 
to coincide with the timing of peak FBF response. 
 
The dose and timing of the nitric oxide synthase inhibitor, L-NMMA (3mg/kg priming 
IV infusion over 15 minutes given prior to Arginine infusion in Aim 1B and then 
continued at 6 mg/kg over an additional 120 minutes) has been selected to achieve 
peak hemodynamic response 15 minutes after priming dose and maintain 
hemodynamic effects for up to one hour after completion of infusion.(43;44) Doses up 
to 13.5 mg/kg IV have safely been given to healthy adults. (44-46)  The elimination 
half-life of L-NMMA is approximately 60 minutes.(43) Co-infusion of a total dose of 6.5 
mg/kg L-NMMA with L-arginine has not been shown to influence L-arginine-stimulated 
GH secretion.(47)  
 
 
6. Risks 
1. Repeated blood sampling from venous catheters may cause bleeding or 
infection.   
2. Frequent blood draws can cause anemia. 
3. Insertion of venous catheters may cause bleeding, bruising, or infection.  
 
4. Arginine has previously been used to diagnose growth hormone deficiency (IV, 
30 grams).(36) After completion of infusion, L-arginine plasma concentration 
rapidly decreases within 30 minutes to 47% of the maximum level. (43) 
Possible side effects include flushing, nausea and vomiting, headache, 
numbness and local venous irritation.  Rarely, a burn-like reaction or injury to 
the skin requiring surgery has been reported. Rarely, allergic reaction has 
been reported. 
5. Sitagliptin - Drugs like sitagliptin can cause lowered blood sugar (common) in 
individuals with high blood sugar (diabetes). This can result in dizziness, 
nausea, shaking, sweating, fast heartbeat, vision changes, headache, anxiety, 
tiredness, or confusion. It can rarely cause fainting, seizures, or coma. Other 
side effects are pancreatitis (damage to the pancreas), kidney damage (which 
can be life threatening), allergic reaction (which can be life threatening), rash, 
and liver damage (which can be life threatening). The FDA is warning that 
dipeptidyl peptidase-4 (DPP-4) inhibitors such as sitagliptin, saxagliptin, 
linagliptin, and alogliptin may cause joint pain that can be severe and 


 
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14 
 
disabling. After the patients discontinued the DPP-4 inhibitor medicine, their 
symptoms were relieved, usually in less than a month. Some patients 
developed severe joint pain again when they restarted the same medicine or 
another DPP-4 inhibitor. 
 
6. The dose of GH receptor antagonist (Pegvisomant 80 mg given 
subcutaneously 72 hours prior to each study day in Aim 1B, Group B only) was 
chosen as 80 mg of pegvisomant produces maximal drug levels 72 hours 
following administration and a reduction in free IGF-1 comparable to that 
observed in GH deficiency, thus indicative of efficient GH receptor blockade 
and a state of acute GH deficiency.(37;38) Pegvisomant is a GH analog that 
disables signal transduction through the GH receptor and thereby functions as 
a receptor antagonist. This selective blockade allows us to examine the effects 
of GH secretion in the absence of cofounders such as changes in IGF-1 
secretion and body composition which may accompany chronic GH deficiency. 
This method of achieving acute GH blockade has been reported to safely and 
effectively study GH secretion in a variety of metabolic conditions and to 
evaluate the effect of GH secretion on cardiovascular risk markers.(37-39) 
Side effects in patients taking this medication on a daily basis may include 
pain and reaction at the injection site, flu-like symptoms, nausea, and diarrhea 
(common). Rarely, swelling, dizziness, sinusitis and elevations in liver tests 
have been reported with repeated use. 
 
 
7. Exendin-(9-39) (IND 
)-is an investigational agent (Clinalfa®, Bachem 
Distribution Services; Weil am Rhein, Germany) that will be infused 
intravenously following dissolution in 0.25% human serum albumin. Salehi et. 
al. have not experienced any untoward effects with the use of this antagonist 
in healthy participants, those with Type 2 diabetes mellitus, or history of 
gastric bypass.(40;42;48) Deane et. al. reported that blockade of GLP-1 
action was associated with accelerated gastric emptying and larger glucose 
excursions in the hour following meal intake(49). The dose of GLP-1 receptor 
antagonist (Exendin-(9-39) IV bolus of 7500 pmol/kg over 1 minute at Time 0 
(Aim 1B) followed by continuous infusion of 750 pmol/kg/min for 150 minutes) 
was chosen as this dose elicits blockade of GLP-1 action at its receptor in 
healthy volunteers during a fixed blood glucose, as measured by augmented 
post-prandial glucagon secretion, a blunted increase in post-prandial insulin 
secretion, and a significant increase in post-prandial GLP-1 secretion.(40-42) 
The timing of Exendin-(9-39) infusion may be adjusted pending results of 
Aim1A to coincide with the timing of peak FBF response. 
8. L-NG-monomethyl arginine citrate (L-NMMA) (IND #
)- is an 
investigational agent (Clinalfa®, Bachem Distribution Services; Weil am Rhein, 
Germany) that will be infused intravenously. Side effects of systemic 


 
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15 
 
administration include increase in blood pressure and decrease in heart rate. 
(3mg/kg priming IV infusion over 15 minutes given prior to Arginine infusion in 
Aim 1B and then continued at 6 mg/kg over an additional 120 minutes) has 
been selected to achieve peak hemodynamic response 15 minutes after 
priming dose and maintain hemodynamic effects for up to one hour after 
completion of infusion. (43;44)The elimination half-life of L-NMMA is 
approximately 60 minutes.(43) Co-infusion of a total dose of 6.5 mg/kg L-
NMMA with L-arginine has not been shown to influence L-arginine stimulated 
GH secretion.(47) Doses up to 13.5 mg/kg IV have safely been given to 
healthy adults.(44-46) 
 
7. Reporting of Adverse Events or Unanticipated Problems Involving Risk to 
Participants or Others 
 
.  
All adverse events and instances of non-compliance with the protocol, subject 
withdrawals, and subject complaints will be formally summarized and reviewed by the 
Data and Safety Monitoring Board (DSMB) bi- annually. The DSMB will receive these 
in written format and review the study for appropriate and timely subject accrual, 
adherence to the protocol, and data accuracy and completeness. The DSMB will 
provide the PI (Dr. Devin) with written confirmation of receipt of this bi-annual 
summary as well as a recommendation with accompanying rationale for study 
termination or continuation. This written confirmation will be provided to the IRB and 
include results of the review and concerns, if any, regarding subject safety or study 
drug tolerability. The DSMB will ensure that all adverse event reporting to the IRB is in 
accordance with current policies. The members of the DSMB will be available for 
discussion of any questions regarding protocol adherence, consent issues, or adverse 
events. The DSMB will review final results of the study. 
 
The DSMB is comprised of Drs. 
, 
, 
, and Dr. 
 
(Group C only). Dr. 
 is a translational researcher and has experience with 
the use of sitagliptin to treat patients with diabetes.  Dr. 
 is a translational 
researcher and has experience with the administration of vasoactive substances.  All 
adverse events specifically related to the use of arginine and pegvisomant will be 
graciously reviewed as they occur with Dr 
, who as Director of Vanderbilt’s 
Pituitary Center has extensive experience in the use of these agents to diagnose and 
treat patients with pituitary disease in the clinical setting.  Dr. 
 is a clinical 
endocrinologist and translational researcher at the University of Cincinnati who has 
experience with the infusion of exendin (9-39) in clinical research protocols. Dr. 
 will serve as the DSMB chair. 
 
All protocols will be reviewed and approved by the Vanderbilt IRB before any subject 
is enrolled.  Dr. Devin, her mentor (Dr. 
) and her co-investigators will 


 
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16 
 
closely oversee the protocol. Any serious adverse events or toxicities will be reported 
to the Vanderbilt IRB as per policy and will immediately be reviewed with the DSMB. 
Any noncompliance with the IRB-approved protocol that increases risk or affects 
participants’ rights, safety or welfare will be reported to the IRB within 10 working 
days of the investigators’ knowledge and immediately reviewed by the DSMB. 
 
Any untoward medical event will be classified as an adverse event, regardless of its 
causal relationship with the study. An adverse event will be classified as serious if it a) 
results in death, b) if life-threatening, c) requires inpatient hospitalization or 
prolongation of existing hospitalization, d) results in persistent or significant disability or 
incapacity, e) is a congenital anomaly or birth defect. All non-serious adverse events 
and instances of non-compliance with the protocol will be reported annually to the IRB 
at the time of continuing review.  
 
 
Dr. Devin will review enrollment progress, all adverse events, protocol adherence 
data, and data quality entry at a minimum of every two weeks with her mentor (Dr. 
 Dr. Devin will review with Dr. 
 each enrolled subject’s chart 
every two weeks for side effects and tolerability of the investigational drug. These 
findings will be included in the DSMB bi-annual report unless dictated otherwise 
above. 
 
Summary Reports will be submitted to the IRB at least annually and will contain a) the 
number of adverse events and an explanation of how each event was handled, b) the 
number of complaints and how each complaint was handled, c) the number of subject 
withdrawals and an explanation of why the subject withdrew or was withdrawn, and d) 
the number of protocol violations (non-compliance with protocol) and how each was 
handled. 
 
 
8. Study Withdrawal/Discontinuation 
 
If at any time during the study, a subject develops any symptoms related to study 
participation that subject will be withdrawn from the study. If, in the opinion of the 
investigator, a subject is non-compliant, that subject will be withdrawn from the study. 
Subjects who are withdrawn will be followed until symptoms have resolved. 
  
9. Statistical Considerations 
 
Anticipated Results 


 
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17 
 
DPPIV cleaves GRF at its penultimate alanine.(1;2) GRF stimulates endogenous GH 
secretion, potentially leading to an increase in the GH-mediated vasodilator 
response.(10;11) We expect that DPPIV inhibition (sitagliptin) will result in decreased 
degradation of GRF and an increase in stimulated GH secretion. (Aim 1A) We expect 
that this will increase forearm blood flow and that this effect will be prevented by pre-
treatment with a GH receptor antagonist, but remain unaffected by administration of L-
NMMA as well as GLP-1 receptor antagonist. (Aim 1B)  
 
Sample Size and Power Calculation 
Aim 1A. Our preliminary data in females (N=4) demonstrate a difference in mean peak 
GH following arginine stimulation of 6.79 ng/mL (mean 14.07 ± SD 4.73 ng/mL Drug A 
vs. 20.86 ± 13.23 ng/mL Drug B). Sample size was calculated with PS Software using 
the design for a paired t-test with a 0.05 two-sided significance level.(50) A sample size 
of 28 females will have 80% power to detect a difference in means of 6.79 ng/mL 
assuming a 12.25 ng/mL SD of the differences. Accounting for a 10% drop-out between 
the 2 study days, we will enroll 31 females in this study. Our preliminary data in males 
(N=2) demonstrates a difference in mean peak GH in the opposite direction. Pre-
menopausal women are more responsive to GHRH and secrete more GH per pulse 
than men, the latter is primarily a GHRH-mediated phenomenon.(51-53) Given the 
known sexual dimorphism in pulsatile GH secretion, we have chosen to study only 
enough men to allow us to estimate the difference in males. Studying 11 males will 
allow a half-width of the 95% confidence for the difference to be about 7.6 ng/mL. 
Enrolling forty-four subjects (32 females; 12 males) in Aim 1A divided amongst 3 groups 
in Aim 1B will allow each group to be adequately powered to detect a statistically 
significant difference in FBF (see below). 
Aim 1B. Prior data indicates that a difference in forearm blood flow of the following is 
statistically significant p<0.001: baseline forearm blood flow mean ±SEM 4.4 ±0.2 vs 2.7 
±0.3 ml/min/100mLfollowing L-NMMA in n=59 subjects. (54) Conservatively assuming a 
correlation coefficient of 0.6 for the two measures on the same subject, the SD for the 
within subject difference would be 1.8.  Assuming, for example, that sitagliptin+GH 
antagonist would achieve FBF of 2.7 ± 2.3 and sitagliptin+placebo would have FBF of 
4.4±1.5 (mean±SD), a sample size of 11 completed subjects would have 80% power to 
detect this difference. Sample size was calculated with PS Software using the design for 
a paired t-test.  Assuming a 10% drop-out in between the 2 study visits we will enroll 14 
patients in each Group; gender distribution of the groups will be determined by the 
results from Aim 1A.  


 
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18 
 
 
Data Analysis Plan 
The primary endpoints are: 
Aim 1A: Peak GH level following Arginine stimulation, assessed in a paired manner 
with and without sitagliptin. 
Aim 1B: Repeated measurements of FBF following sitagliptin at the specified times will 
be assessed in a paired manner with and without antagonist. 
 
Even though we do not expect any carry-over effect in this 2 by 2 crossover study with 
repeated measures based upon our prior experience with studies of sitagliptin, we will 
first test for a carry-over effect using the T-test approach proposed by Jones and 
Kenward.(55)  Mixed effect models will be used to analyze the data with a random 
subject effect and with the treatment (sitagliptin vs placebo or sitagliptin+GH antagonist 
vs sitagliptin+placebo) and the time trend as measured at the above time points as fixed 
effects.  The focus of this study will be the treatment effect and the time trend of the 
endpoints; however, mixed effect models also provide the flexibility of controlling for and 
evaluating covariates as needed by the study.  The mixed effect models are robust in 
the sense that they can include subjects with missing data at some time points but not 
all time points to estimate the effects of interest. The above specifies the general model 
for most of the endpoints, however, there will be no time trend in the model for peak GH 
level.   Besides the above evaluation of the treatment effect and the time trend through 
the regression models, specific inferences regarding effects of interest will be made by 
reporting a point estimate along with a 95% confidence interval. Hypotheses will be 
tested at the level of α=0.05. This data analysis plan will be carried out using statistical 
software SPSS for Windows (Version 19.0, SPSS, Chicago) and the open source 
statistical package R (version 2.12, R Development Core Team, 2006).  
10. Privacy/Confidentiality Issues 
 
Clinical data, including clinical laboratory, will be entered by a member of the Key Study 
Personnel in a protected source database (RedCap).  A unique identification case 
number will be used to protect the confidentiality of the study participants.  The case 
numbers and participants' names will be included in the protected source database, 
accessible only by Key Study Personnel, but only case numbers will be included in any 
spreadsheet used for the statistical analysis. 
 


 
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19 
 
 
11. Follow-up and Record Retention 
 
The total duration of enrollment will be 2 years, and we anticipate completion of a 
manuscript within 3 years of initiation. All records will be retained for 7 years following 
publication of the data.  After that time, records may be archived for an additional 5 
years and then shredded. 
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