Clinical trial NCT03012386
Conditions: Insulin Resistance Endothelial Dysfunction
Keywords: CD36 Endothelial dysfunction African Americans
Sponsor: Vanderbilt University Medical Center
Start date: 2017-01-01; Completion date: 2021-07-31
Enrollment: 26 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This proposal will test the hypothesis that chronic treatment with sildenafil with and without the use of nitric oxide substrate, L-arginine, protects against fatty acid induced impairment of endothelial function, improves insulin-stimulated microvascular recruitment, insulin sensitivity and glucose uptake in CD36 rs3211938 G-allele carriers.

Detailed description:
Subjects carrying the G-allele of CD36 coding SNP rs3211938 that results in 50% reduction of CD36 levels in \~25% of African Americans have endothelial dysfunction. Endothelial dysfunction results in impairment of insulin's vascular actions and eventually reduced insulin sensitivity. Insulin induces microvascular recruitment via stimulation of nitric oxide(NO)-cGMP pathway, which facilitates nutrient flux, e.g., glucose to skeletal muscle. Elevated fatty acids impair insulin-stimulated microvascular recruitment and reduce insulin sensitivity. Chronic treatment with sildenafil increases vascularity and muscle glucose uptake in high fat fed mice. In humans, Drs. Shibao (PI) recently reported that a 3-month treatment with sildenafil improves insulin sensitivity in patients with impaired glucose tolerance. More relevant to this project, endothelial dysfunction improved after 4-week treatment with sildenafil in G-allele carriers. This proposal will test the hypothesis that chronic treatment with sildenafil with and without the use of NO substrate, L-arginine, protects against fatty acids induced impairment of endothelial function, improves insulin-stimulated microvascular.

The protocol design was changed to single arm design.

Arms: arm 1: Sildenafil citrate 20 mg three times a day
Interventions: intervention 1: Sildenafil Citrate in G allele carrier intervention 2: Sildenafil Citrate in non G allele carrier
Intervention descriptions: intervention 1: chronic use of phosphodiesterase 5 inhibitor in G allele carrier intervention 2: chronic use of phosphodiesterase 5 inhibitor in Non G allele carrier

Protocol documents:
Prot_SAP_000.pdf: 
 
 
1 
8/28/19 
 
 
 
 
 
 
 
 
Role of CD36 in Nutrient Delivery and its Dysfunction in 
African Americans 
 
 
Principal Investigator: Cyndya A. Shibao, MD 
Division of Clinical Pharmacology/ 
Department of Medicine 
 
 
 
 
 
 
 
 
 
 
 
 
Jorge Celedonio, Research Fellow, Clinical Pharmacology,  
Naji N Abumrad, Sawyers Professor of Surgery, Department of Surgery, 
Robin Tamboli, Associate Professor of Surgery, Department of Surgery 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
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8/28/19 
Table of Contents: 
 
Study Schema 
1.0 
Background 
2.0 
Specific Aims 
3.0 
Inclusion/Exclusion Criteria 
4.0 
Enrollment/Randomization 
5.0 
Study Procedures 
6.0 
Risks of Investigational Agents/Devices (side effects) 
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 
References 
Addendum – Blinded Medications (Placebo and Sildenafil, 20mg) 
 


 
 
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8/28/19 
1.0 
Background 
 
CD36 is highly expressed in skeletal muscle, heart and adipose tissue of mice 1 both on tissue cells and in 
the endothelium of blood vessels.2, 3 Endothelial cells (EC) of large arteries have low CD36 while 
expression is high in EC from medium arteries, arterioles and capillaries. RNAseq data in humans show 
that aorta CD36 expression is 1/3 that of coronary arteries. CD36 content of the capillary endothelium is 
highest in heart, muscle and adipose tissues compared to other tissues.2, 4 CD36 has a regulatory role in 
Fatty acids (FA) uptake and oxidation by heart and muscle 5-7 and its expression in EC could contribute 
to this role. Our hypothesis is that EC CD36 is involved in vascular maintenance and regulation of 
“nutritive blood flow”.  
Preliminary data 
Endothelial dysfunction in humans carrying the minor allele (G) of coding CD36 SNP rs3211938  
(Figs. 1-4): We recently examined effect of treatment with sildenafil, which blocks cyclic GMP (cGMP) 
degradation on insulin sensitivity in pre-diabetic subjects.8 In another trial (NCT01334554) we examined 
effect on endothelial function of a 4-week sildenafil treatment in 
obese African American (AA) women with metabolic syndrome 
(MetS). Genotyping for CD36 single nucleotide polymorphism 
(SNP) rs3211938 was done retrospectively at the end of the trial 
using genomic DNA extracted from blood.9, 10 Samples had 100% 
concordance rate and included validated controls for each group 
(G/G, G/T, T/T). Endothelial function was assessed with brachial 
artery flow-mediated dilation (FMD). Linear regression was used 
to examine differences between treatment groups and effect of 
genotype.  
G-allele of coding SNP rs3211938 reduces CD36 (Fig. 1): The 
minor allele (G) of CD36 rs3211938 introduces a stop codon in 
exon 10 and was reported to have a frequency of ~9% in AA,10, 11 
and was ~13% in our recruited AA women. Genotype frequency (% 
subjects with G-allele) was ~25% of those screened. The G-allele 
reduces CD36 protein by ~50% in monocytes and platelets10.  
G-allele reduces cyclic GMP levels (Fig. 2): To test if the lower CD36 
level in G-allele carriers impacts the NO-cGMP pathway, cGMP levels 
were measured in blood. G-allele carriers (n=12) vs. non-carriers 
(n=31) had 30% less cGMP suggesting lower NO-cGMP activity.  
G-allele reduces endothelial function (Fig. 3): Endothelial dysfunction 
associates with less vasodilatory response.12-14 We examined the 
impact of rs3211938 on FMD in GT and TT subjects matched for age 
and BMI and with no metabolic syndrome (MetS) according to ATP 
criteria15 since MetS reduces endothelial function. FMD was 40% 
lower in GT (p=0.035) vs. TT indicating that the G-allele reduces the 
vasodilatory response to 5 min ischemia.  
G-allele impacts response to sildenafil therapy (Fig. 4): AA women 
(n=46) with high insulin or 3 of 5 MetS traits participated in a trial 
that examined potential benefits of 4-week treatment with sildenafil 
vs. placebo in a double blind randomized study. We confirmed 
compliance by detecting sildenafil levels in subjects assigned the drug 
but not placebo. Forty women completed the study and 34 were 
genotyped for rs3211938 (6 withheld consent). We found a significant 
genotype/drug response interaction: Sildenafil improved FMD in G-
FFA (mEq/L)
T/T
G/-
0.0
0.5
1.0
1.5
P= 0.01
cGMP (fmol/well)
T/T
G/T
0
500
1000
1500
2000
P= 0.01
N=103 
N=43 
Fig. 2: Rs3211938 G-allele 
lowers cGMP level. 
B. 
A. 
   2   2   1   2   1   2   1   2   
  TT     GT  TT  GT  TT  GT  TT 
   2     2     2     1    1     2     2    
    TT            GT         TT      
T/T
G/-
0.0
0.5
1.0
1.5
FFA (mEq/L)
p= 0.0003
cGMP (fmol/well)
T/T
G/T
0
500
1000
1500
2000
p= 0.013
rs3211938
A.
B.
rs3211938
 FMD,%
placebo sildenafil 
placebo sildenafil 
0
1
2
3
4
5
T/T
G/T
*
rs32
 RHI
placebo sildenafil 
-1.0
-0.5
0.0
0.5
1.0
1.5
T/T
D. 
C. 
E. 
TT
GT
TT
GT
P =0.003 
P =0.01 
Fig. 1: rs3211938 G-allele lowers 
CD36: G carriers have 50% less CD36 
on monocytes and platelets (also WB). 
N=12/group. 
Fig. 3: G-allele reduces endothelial 
function. Left: Changes in brachial 
artery diameter before and after 5 min 
of ischemia. Right: Flow mediated 
dilation (FMD) is 44% lower in GT, n=8, 
than in T/T, n=9, p=0.03.  


 
 
4  
8/ 2 8/ 1 9  
allele carriers b ut n ot i n T/ T c o ntr ols, p = 0. 0 1 8.   
 
I n c o ncl usi o n a ge netic varia nt t hat re d uces C D 3 6 le vel t hat is 
pre vale nt i n A A, di mi nis hes e n d ot helial f u ncti o n a n d i m pacts res p o nse 
t o treat me nt wit h P D E 5 i n hi biti o n. T he pri mar y g oal of t his pr oject is 
t o st u d y t he r ole of E C C D 3 6 i n vasc ular re g ulati o n. O ur st u d y will be 
t he first t o assess t he i m pact of t he C D 3 6 ge ne o n t he ris k of 
e n d ot helial d ysf u ncti o n a n d its r ole i n me diati n g vasc ular effects of 
i ns uli n a n d F A s. T he w or k will pr o vi de f u n da me ntal i nf or mati o n wit h 
healt h rele va nce c o nsi deri n g t he hi g h pre vale nce of o besit y a n d is 
partic ularl y rele va nt i n A A w here o besit y is a b o ut 1. 5 f ol d hi g her t ha n 
i n w hites; 6 8 % of A A me n a n d 8 2 % of A A w o me n bei n g o ver wei g ht 
or o be se. 
 
Si g nific a nce  
Vasc ular stiffe ni n g a n d h y pert e nsi o n are maj or healt h care pr o ble ms i n Wester n s ocieties a n d ris k fact ors 
f or str o ke, m y ocar dial i nfarcti o n, a n d ki d ne y a n d heart fail ure. O ne t hir d of t he a d ult p o p ulati o n is 
h y perte nsi ve wit h a n ot her ha vi n g “ pre -h y perte nsi o n” t hat c o m m o nl y de vel o ps wit hi n fe w years i nt o 
h y perte nsi o n. 1 6 -1 8 Vasc ular stiffe ni n g is als o a n earl y e ve nt i n o besit y -i n d uce d dia betes a n d car di o vasc ular 
disease a n d ass ociates 
wit h 
w orse l o n g -ter m o utc o mes. 
Africa n 
A merica ns ( A A) are ge neticall y 
pre dis p ose d t o e n d ot helial d ysf u ncti o n, w hic h c o ntri b utes t o t he dis pr o p orti o natel y hi g her b ur de n of 
car di o vasc ular disease. 1 9 -2 1 C o m pare d wit h w hites, healt h y a d ults a n d a d olesce nt A A ha ve i m paire d 
micr o vasc u lar vas o dilat or y f u ncti o n a n d greater lar ge arterial stiff ness.1 9, 2 2I de ntif yi n g ne w tar gets a n d 
t hera pe utic a p pr oac hes f or pre ve nti n g a n d treati n g e n d ot helial d ysf u ncti o n is of critical i m p orta nce i n t he 
ge neral p o p ulati o n a n d es peciall y i n t he A A s u b p o p ulati o n. O ur st u d y will be t he first t o assess i m pact of 
t he C D 3 6 ge ne o n t he ris k of e n d ot helial d ysf u ncti o n a n d its r ole i n me diati n g vasc ular effects of i ns uli n 
a n d F As . T he w or k will pr o vi de f u n da me ntal i nf or matio n wit h healt h rele va nce c o nsi deri n g t he hi g h 
pre vale nce of o besit y a n d is partic ularl y rele va nt i n A A w here o besit y is a b o ut 1. 5 f ol d hi g her t ha n i n 
w hites; 6 8 % of A A me n a n d 8 2 % of A A w o me n bei n g o ver wei g ht or o bese. 2 3 
 
2. 0  
S pecific Ai ms  
O ur preli mi nar y data s h o w e vi de nce of e n d ot helial d ysf u ncti o n i n s u bjects carr yi n g t he G -allele of C D 3 6 
c o di n g S N P rs 3 2 1 1 9 3 8 t hat res ults i n 5 0 % re d ucti o n of C D 3 6 le vels i n ~ 2 5 % of A A . S u bjects wit h 
e nd ot helial d ysf u ncti o n de vel o p i m pair me nt of i ns uli n’s vasc ular acti o ns a n d e ve nt uall y re d uce d i ns u li n 
se nsiti vit y. I ns uli n i n d uces micr o vasc ular recr uit me nt via sti m ulati o n of N O-c G M P pat h wa y, w hic h 
facilitates n utrie nt fl u x, e. g., gl uc ose t o s keletal 
m uscle. Ele vate d F As  i m pair i ns uli n-sti m ulate d 
micr o vasc ular recr uit me nt a n d re d uce i ns uli n se nsiti vit y. As o ur Va n der bilt c ollea g ue a n d c olla b orat or 
( Dr. 
Wasser ma n) s h o we d, c hr o nic treat me nt wit h sil de nafil i ncreases vasc ularit y a n d m uscle gl uc ose 
u pta ke i n h i g h fat fe d mice. I n h u ma ns, Drs. S hi ba o (PI) a n d Br o w n rece ntl y re p orte d t hat a 3 -m o nt h 
treat me nt wit h sil de nafil i m pr o ves i ns uli n se nsiti vit y i n patie nts wit h i m paire d gl uc ose t olera nce.8 M ore 
rele va nt t o t his pr oject, we s h o we d t hat e n d ot helial d ysf u ncti o n i m pr o ves after 4 -wee k treat me nt  wit h 
sil de nafil onl y i n G -allele carriers ( Fi g. 2 ). I n t his pr o p os al, we will test t he h y p ot hesis t h at c hr o nic 
tre at me nt wit h sil de n afil wit h a n d wit h o ut t he use of N O s u bstr ate, L -ar gi ni ne , pr otects a g ai nst F A 
i n d uce d i m p air me nt 
of 
e n d ot heli al f u ncti o n, i m pr o ves i ns uli n-sti m ul ate d 
micr o v asc ul ar 
recr uit me nt, i ns uli n se nsiti vit y a n d gl uc ose u pt a ke i n C D 3 6 rs 3 2 1 1 9 3 8 G -allele c arriers.  
 
3. 0  
I ncl usi o n/ E xcl usi o n Criteri a 
 
F i g. 4: G -all el e  i nfl u e n c es  r e s p o n s e 
t o sil d e n afil. A A w o m e n w er e giv e n  
dr u g or pl ac e b o f or 4 w e eks . 
En d ot h eli al f u ncti o n ( % c h a n g e i n 
F M D) o nly i m pr ov e d i n G -all el e  ( G T) 
c arri ers, ( p = 0. 0 1 8). N: pl ac e b o = 2 1, 
S C = 1 9.  
r s 3 2 1 1 9 3 8
 D  F M D, %
pl a c e b o sil d e n afil 
pl a c e b o sil d e n afil 
0
1
2
3
4
5
T/ T
G/ T
*


 
 
5 
8/28/19 
We will screen and genotype 120 subjects. We will study 40 subjects (30-G allele carriers and 10 non-
carriers). We will conduct the study in AA population only. The characteristics of the subject population 
are detailed under the inclusion and exclusion criteria.  
 
Inclusion criteria 
• African American men and women. 
• Age 18-50 years 
• BMI 25-40 kg/m2 
 
Exclusion criteria: Subjects presenting with any of the following will not be included in the study: 
• 
Diabetes type 1 or type 2, as defined by a FPG > 126 mg/dL or history of abnormal OGTT (two-
hour plasma glucose > 200 mg/dL), or the use of anti-diabetic medication  
• 
The use of nitrates or any disease that might require the use of nitrates  
• 
Pulmonary hypertension  
• 
Hypertension, defined as SBP/DBP >140/90 mm Hg or the use of anti- hypertensive agents  
• 
Use of a PDE5 inhibitor for erectile dysfunction 
• 
Use of an alpha blocker  
• 
Pregnancy or breast-feeding. Women of child-bearing potential will be required to have undergone 
tubal ligation or to be using an oral contraceptive or barrier methods of birth control.  
• 
The use of any potent CYP3A4 inhibitor. 
• 
Cardiovascular disease such as myocardial infarction, presence of angina pectoris, significant 
arrhythmia, congestive heart failure (left ventricular hypertrophy acceptable), deep vein thrombosis, 
pulmonary embolism, second or third degree heart block, mitral valve stenosis, aortic stenosis or 
hypertrophic cardiomyopathy  
• 
Allergies to eggs, soy products and Intralipid® infusion 
• 
History of serious neurologic disease such as cerebral hemorrhage, stroke, or transient ischemic 
attack  
• 
History or presence of immunological or hematological disorders  
• 
Impaired hepatic function (aspartate amino transaminase [AST] and/or alanine amino transaminase 
[ALT] > 3.0 x upper limit of normal range) 
• 
Impaired renal failure (eGFR < 60 mL/min/1.73m2) 
• 
Hematocrit < 34%  
• 
Any underlying or acute disease requiring regular medication which could possibly pose a threat to 
the subject or make implementation of the protocol or interpretation of the study results difficult  
• 
Treatment with chronic systemic glucocorticoid therapy (more than 7 consecutive days in 1 month)  
• 
History of alcohol or drug abuse  
• 
Treatment with any investigational drug in the one month preceding the study  
• 
Mental conditions rendering a subject unable to understand the nature, scope and possible 
consequences of the study  
• 
Inability to comply with the protocol, e.g., uncooperative attitude, inability to return for follow-up 
visits, unlikelihood of completing the study, and investigator discretion  
 
4.0 
Enrollment/Randomization 


 
 
6 
8/28/19 
Recruitment and informed consent: We will recruit from the Nashville AA community for this 
planned study. The genotype frequency of the G allele in AA is ~ 25%, we conservatively estimate that 
we need to screen around 120 subjects to find 30 G allele carriers. As in previous studies, we will use 
radio public service announcements, word of mouth, social media, and distribution of ads to local 
municipal sites previously approved by the Vanderbilt Institutional Review Board. We will also use 
established Internet resources maintained by the Vanderbilt CTSA such as ResearchMatch and 
SubjectLocator. We may use a screening electronic survey to determine if the volunteer qualifies for the 
study. All subjects that we screen and genotype for this study will be offered the possibility to participate 
in a CD36 registry. We will use e-consent to consent the subjects. 
 
5.0 
Study Procedures 
 
In our preliminary data, (Fig. 4) we observed improvement in endothelial function after a 4-week 
treatment with sildenafil only in G-allele carriers with metabolic syndrome (MetS). It is well established 
that MetS is associated with endothelial dysfunction.  
In the proposed study, we will recruit AA subjects G-allele carriers that have no evidence of MetS, but 
have evidence of endothelial dysfunction (Fig. 3). We will assess the effect of the PDE5 inhibitor on 
insulin-stimulated microvascular recruitment and insulin sensitivity after 4 weeks  on sildenafil treatment 
under conditions of elevated blood FA levels to produce insulin resistance. We will also explore the 
added effect of the NO substrate, L-arginine. The study is designed to obtain information on the potential 
of enhanced NO-cGMP pathway to rescue endothelial function, insulin-stimulated microvascular 
recruitment, and to mitigate the deleterious effects of elevated FA on insulin sensitivity in G-allele 
carriers. 
 
 
 
 
Figure 5 - Experimental Protocol: Panel A; study design, and panel B; infusion protocol. Each 
subject will undergo 3 insulin clamps.  
 


 
 
7 
8/28/19 
 
Study Design:  
We will invite subjects to participate in the following visits: 
Pre-screening (this visit is optional, separate consent). We will collect a blood sample for DNA and other 
labs. 
 
Screening visit: We will perform a history and physical, routine labs, including cell blood count, 
comprehensive metabolic panel, lipid profile, single ECG, and pregnancy test. Labs will be repeated if 
visit 1 is scheduled more than 3 months after the screening visit. 
Visit 1: If the subjects meet inclusion criteria, this visit will occur after overnight fasting. They will 
receive a ~6-hour saline infusion in conjunction with approximately 3-hour “insulin clamp”. L-arginine 
will be infused during the last 30 min of the clamp (see infusion protocol). Contrast enhanced 
ultrasonography (CEU) will be performed. Blood pressure (BP) and heart rate (HR) will be monitored 
throughout the study.  
Visit 2 (will occur at least 2 weeks after visit 1): This visit will occur in the CRC. Subjects are asked to 
fast overnight. In each study day, a catheter is placed into the antecubital vein for blood sampling. A 
second one is placed in the other antecubital vein for infusions (glucose, insulin, 20% Intralipid®, heparin 
and L-arginine, US contrast agent, (see infusion protocol). Blood pressure (BP) and heart rate (HR) will be 
monitored throughout the study. The Vanderbilt Investigational Pharmacy will be responsible for drug 
storage and dispensing logs.  
Visit 3 (will occur after ~ 4-week treatment with sildenafil 20 mg po three times a day).The last dose of 
sildenafil 20 mg po will be provided the morning of the study and prior to initiation of the “insulin clamp”. 
 
Drug infusion protocol (visits 1, 2 and 3, Fig. 5 B): In visit 1, subjects will receive i.v. infusion of 
saline (45ml/h) for 6 hours. During visits 2 and 3, subjects will receive i.v. 20% Intralipid® (45 ml/hr.) 
and heparin (200 units prime followed by 200 units/hr) for a total of 6 hours as previously described26. 
Heparin is given to activate endothelial lipoprotein lipase and to facilitate the conversion of circulating 
lipids to FA. The “insulin clamp” will start at minute 180 min after initiating the continuous infusion of 
Intralipid® + heparin and will last for 3 additional hours. During the “insulin clamp”, plasma glucose will 
be clamped at the same basal levels (~90-95 mg/dl) using variable infusion of IV glucose. Blood samples 
for plasma FA and insulin will be collected at -10, 180, 300, 315, 330, 340, 350 and 360 min. Plasma FAs 
will be collected with 1 mg/L of tetrahydrolipstatin to prevent in vitro lipolysis27 and measured using the 
NEFA-HR(2) Kit (Wako). We will measure microvascular 
recruitment (MBV) in the forearm brachioradialis muscle with 
contrast 
enhanced 
ultrasonography 
(CEU, 
Fig. 
6), 
and 
measurements of brachial artery diameter and flow as previously 
described, see techniques. Insulin infusion (up to 80 mU/m2.min-1) 
will be started at time 180 min and continued for 3-hours, with 
simultaneous 
variable 
infusion 
of 
glucose. 
Ultrasound 
measurements will be carried out between 300-330 min to examine 
the effect of sildenafil or placebo. During the last 30 min, we will 
infuse i.v. L-arginine (10 mg/kg/min for 30 minutes) 24. Then, we 
will repeat ultrasound measurements at the end of the L-arginine 
infusion to examine effect of L-arginine + sildenafil or L-arginine + 
intralipids alone on these endpoints. 
 
 
1
2
3
0
2
4
6
MBV (dB)
N=7 AA woman
Skeletal  
muscle 
A
B
C
Fig. 6: Contrast 
Enhanced US. Image 
of forearm muscle 
before (A) and during 
contrast infusion (B). 
Microvascular Blood 
Volume (MBV, C) 
measured thrice in 7 
AA subjects


 
 
8 
8/28/19 
Endpoints: The primary endpoint is the change in MBV (ΔMBV) during insulin infusion from baseline, 
an index of insulin-stimulated microvascular recruitment. We will compare ΔMBV in CD36 G-allele 
carriers with and without chronic sildenafil treatment (primary analysis) and with and without chronic 
treatment with sildenafil + L-arginine infusion. Secondary measurements are insulin sensitivity as 
measured by glucose infusion rate (M), insulin sensitivity index (M/I), and hemodynamics (BP and HR).  
We hypothesize that chronic treatment with sildenafil will improve insulin-mediated increases in MBV in 
the CD36 G-allele carriers compared with placebo. We do not expect that sildenafil will affect insulin-
mediated increases in MBV in non-carriers. Our preliminary data showed that 4-week treatment with 
sildenafil had no effect on flow-mediated dilation, a measurement of NO-dependent endothelial function 
in non-carriers. We expect that chronic treatment with sildenafil would improve insulin sensitivity as 
measured by the glucose infusion rate during the insulin clamp in G-allele carriers. Furthermore, we will 
determine if changes in insulin-stimulated microvascular recruitment can be potentiated with the addition 
of NO substrate such as L-arginine in G-allele carriers.  
We do not anticipate difficulties in the conduct of the human studies. Dr. Shibao (PI) has experience 
infusing Intralipid® and heparin in humans (NCT02365285) and in assessing microvascular recruitment 
(Fig.6) and blood flow in skeletal muscle 30. Dr. Abumrad NN has > 25-year experience in the conduct of 
the “insulin clamp” for evaluating insulin resistance and skeletal muscle uptake31. Further, we do not 
anticipate problems with recruitment of AA volunteers or with compliance as we previously reported 32. 
Compliance will be monitored by measuring sildenafil concentration in plasma at the end of the study to 
prevent un-blinding.  
 
Standard Techniques  
 
Hyperinsulinemic euglycemic clamp (“insulin clamp”): The insulin infusion rate (up to 80 mU/m2.min-
1) chosen will suppress hepatic glucose production and maximally stimulate peripheral glucose utilization 
allowing measurements of glucose utilization and insulin sensitivity 33.   
Contrast-enhanced ultrasound (CEU): Images will be obtained using a linear-array transducer 
connected to an ultrasound (L9-3 mm transducer, iU22; Phillips). This equipment allows real-time 
imaging using low (0.08) and high (1.2) mechanical index as the contrast (microbubbles; Definity; 
Bristol-Myers Squibb) is infused (1.5 ml/min) for 10 min. At steady state (~4 min) the high mechanical 
index (1.2) used will destroy the microbubbles at the start of video recording. Switching to the low index 
(0.08) will let microbubbles resonate allowing real-time recording of vascular replenishment. Data are 
analyzed in our lab using QLAB software34. Our preliminary data in 7 AA (Fig. 14) show baseline MBV 
of 4.0±2.1 (3.8-4.3, SD 1.1 to 3.2).  
 
 
Optional procedure 
Harvesting of endothelial cells: A 3 J-shaped vascular guide wire will be advanced into antecubital vein 
and ECs will be retrieved from the wire tips by washing with cell dissociation buffer (Invitrogen) at 4 C. 
Endothelial identity is verified by staining for endothelial antigens (CD31) and nuclear integrity by DAPI. 
We can safely recover ~800 EC/procedure. We will quantify protein levels of eNOS and p-eNOS by 
immunofluorescence. Two-control human umbilical vein EC slides will be included. Captured Images 
will be analyzed using Metamorph. EC protein expression/HUVEC will minimize the possible confound 
of differences in intensity of staining among different sessions (see letter of collaboration from Dr. A. 
Gamboa). For newly consented subjects, this procedure will be performed at the screening visit, if they 
provide verbal consent. Patients who are already enrolled and have completed the screening visit may 
return at any time to the clinic to have this procedure. 
 
6.0 
Risks 


 
 
9 
8/28/19 
 
Blood Draws: Subjects may experience discomfort, bruising, and/or bleeding, or infection at the needle 
insertion site after a blood draw.  Rarely, some people faint. Frequent blood sampling may cause anemia. 
We will exclude subjects with hematocrit <34%.  
 
12-hour fast:  Participants may experience hunger during a 12-hr fast, however this is a standard 
requirement by clinicians for accurate blood testing. 
 
Ultrasound: The ultrasound test is a safe procedure that uses low-power sound waves. There are no 
direct risks from the ultrasound test. 
 
PDE-5 inhibitors: 
Sildenafil can cause severe hypotension when taken together with nitrates or sGC stimulator. We have 
included the use of this medication class as an exclusion criterion. We will monitor BP and HR 
throughout the study. Subjects with BP<90/50 mmHg and symptoms of hypotension at any pressure will 
be discontinued. 
PDE5- inhibitors may cause prolonged erection (4 hours) or priapism (painful erections > 6 hours). In 
addition, PDE-5 inhibitors have been reported to cause headaches, dyspepsia, back pain, flushing, nasal 
congestion, and myalgia or limb pain. In prior studies by our group, headaches were more common 
among subjects randomized to a PDE-5 inhibitor than placebo. 
Reports of changes in color vision (an effect of PDE-5 inhibition) are observed with sildenafil citrate. 
An association of PDE-5 inhibitor use with acute hearing loss and non-arteritic ischemic optic neuropathy 
(rare) has been reported. The causal relationship between PDE-5 inhibitor use and hearing loss or AION 
is not clear, but subjects will be advised of a potential link. 
We have reported edema as an unexpected, related AE in the first subject that we studied. 
 
Hyperinsulinemic clamp: 
There is a risk of hypoglycemia during the hyperinsulinemic euglycemic clamps. Glucose concentrations 
will be monitored every 5 minutes during the insulin clamp. We will infuse variable rate of 20% glucose 
to prevent hypoglycemia. Dr. Abumrad has more than 25 years’ experience using this technique to assess 
for insulin sensitivity in humans. A CRC nurse and a physician will be present at all times during the 
insulin clamps. 
 
Intravenous Cather Insertion:  
Inserting a plastic catheter into the vein may cause a brief amount of pain and possibly a small bruise at 
the site, but will allow us to draw blood without repeated sticks throughout the study. It is also possible to 
develop an infection at the location of the catheter. It is uncomfortable to have the intravenous catheter in 
place for any length of time because it causes discomfort when the patient moves their arm. 
 
Ultrasound contrast agent: 
There is small risk of arrhythmias, chest pain, hypotension, and dyspnea with use of ultrasound contrast 
agents (microbubbles; Definity; Bristol-Myers Squibb). Subjects will be informed about this risk. 
 
Intralipid®: 
In some people, headache, dizziness, flushing, drowsiness, nausea, vomiting, or sweating may occur after 
taking Intralipid®. Other serious side effects include signs of infection (fever, persistent sore throat), 
pain/swelling/redness at injection site, pain/swelling/redness of arms/legs, bluish skin, sudden weight 
gain, shortness of breath and back/chest pain. 


 
 
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We will instruct the subjects to immediately call their doctor or the PI, Dr. Shibao (or designee) if any of 
these rare but very serious side effects occur: mental/mood changes, bone pain, muscle weakness, 
yellowing skin/eyes, dark urine, easy bruising/bleeding, severe stomach/abdominal pain, trouble 
breathing, or any symptoms of a serious allergic reaction, including: rash, itching/swelling (especially of 
the face/tongue/throat), severe dizziness, trouble breathing. 
 
Heparin: 
In some people, heparin can cause abdominal or stomach pain or swelling, back pain or backaches, 
bleeding from the gums when brushing teeth, blood in the urine, constipation, coughing up blood, 
dizziness, headaches, heavy bleeding or oozing from cuts or wounds, joint pain, stiffness, or swelling, 
unexpected or unusually heavy menstrual bleeding, unexplained bruising or purplish areas on the skin, 
unexplained nosebleeds, or vomiting of blood or material that looks like coffee grounds. These side 
effects are uncommon. 
Rarely, heparin may cause blood under the skin (blood blister) at the place of injection, chest pain, chills 
or fever, fast or irregular breathing, irritation, pain, redness, or ulcers at the place of injection, itching and 
burning feeling, especially on the bottom of the feet, nausea or vomiting, numbness or tingling in the 
hands or feet, pain, coldness, or blue color of the skin on the arms or legs, peeling of the skin, puffiness or 
swelling of the eyelids or around the eyes, shortness of breath, skin color change, especially near the place 
of injection or in the fingers, toes, arms, or legs, skin rash, hives, or itching, tearing of the eyes, tightness 
in the chest, or trouble with breathing. 
 
L-arginine:  
Due to the hypertonicity of the solution, the medication needs to be administered via IV infusion. 
Extravasation has resulted in burn-like reactions and skin necrosis requiring surgical intervention. 
Excessive rates of infusion (e.g. < 30 minutes) may result in local irritation, flushing, nausea, or vomiting. 
Other side effects associated with this medication are headaches, numbness. 
 
Breach of Confidentiality: 
In the event of a security breach, confidential information may be stolen. We have strong security 
procedures in place to minimize the possibility of a breach.  Although we cannot provide a 100% 
guarantee that the participant’s data will be safe, our procedures minimize the chance that a breach would 
take place. 
 
There is no benefit of the proposed research to the subjects per se. AA are genetically predisposed to 
endothelial dysfunction. Our study will determine if a genetic variation in the CD36 receptor associated 
with a ~50% decreased expression is associated with a reduction in insulin vascular actions and/or 
negative metabolic outcome such as reduced insulin sensitivity and glucose uptake in the setting of 
elevated FA. Furthermore, our study will determine if these abnormalities could be prevented with the use 
of sildenafil citrate. 
 
7.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to Participants or 
Others 
 
All protocols will be reviewed and approved by the Vanderbilt Institutional Review Board (IRB) before 
any subject is enrolled. The PI will be responsible for ensuring both data integrity and for ensuring that 
study participants are safely cared for and that all adverse events (AEs) are noted, followed, and reported 
to the IRB. Any untoward medical event will be classified as an adverse event, regardless of its causal 
relationship with the study. An AE will be classified as serious if it a) results in death, b) is 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. Serious AEs 


 
 
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will be reported to Dr. Talat A. Ikizler, Catherine McLaughlin Hakim Professor of Medicine and 
Nephrology at Vanderbilt University, the Data and Safety Monitoring Officer (DSMO), and the IRB 
within 7 days of the PI’s notification of the event. Non-serious, unexpected adverse events will be 
reported to the IRB at the time of the annual continuing review.  
 
Data and Safety Monitoring Plan: Data and Safety Monitoring Plan: The protocol will be reviewed and 
approved by the Vanderbilt IRB before any subject is enrolled. The investigators will closely oversee the 
protocol in conjunction with the dedicated research fellow. A Data and Safety Monitoring Officer (DSMO) 
will provide objective review of treatment results as they relate to human safety and data quality. The 
officer will be Dr. Talat A. Ikizler. The DSMO will meet with the investigators prior to the initiation of the 
study to review the protocol and after one-third of the subjects have been enrolled in the study, to receive 
reports of the progress of the study. These reports will provide information regarding recruitment, safety 
reporting and data quality. No early stopping is planned. The DSMO will assess safety data including 
hypoglycemia, hypotension, common adverse events and other serious AEs. Interim data will be provided 
to the committee by Dr. Yu and the PIs (Drs. Shibao and Abumrad NN).  
The DMSO will have the authority to modify the protocol or to terminate the study if it deems such actions 
to be warranted. The DSMO will provide summary reports to the IRB and the investigators. 
The DSMO will review all serious AEs. Any serious AE will be reported to the DSMO, IRB, and NIH 
(and FDA if appropriate) as soon as possible, but not more than seven days from the investigators’ 
awareness of the event. Any untoward medical event will be classified as an AE, regardless of its causal 
relationship with the study. An AE will be classified as serious if it a) results in death, b) is 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  
Non-serious, unexpected AEs will be reported to the IRB at the time of annual review, unless we observe 
a pattern of such events, in which case we expect the DSMO will recommend earlier notification at the 
time of quarterly review. If warranted, appropriate changes will be made to the consent form. AEs will be 
graded as mild (no limitation of usual activities), moderate (some limitation), or severe (inability to carry 
out usual activities) and attributed according to the relationship to the study drug and/or procedures as Not 
related, Unlikely, Possible, Probable, or Definite. Any instance of non-compliance with the protocol will 
be reported at the time of annual review. Summary Reports will be submitted to the IRB at least annually 
and will contain a) The number of AEs and an explanation of how each event was handled, and 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 instances of non-
compliance with the protocol and how each was handled.  
 
8.0 
Study Withdrawal/Discontinuation 
 
Criteria for study withdrawal/discontinuation 
 
• 
Drug-related toxicity 
• 
Requirement for prohibited concomitant medications (see exclusion criteria) 
• 
Pregnancy 
• 
Request by subject to terminate treatment 
• 
Clinical reasons believed to be life threatening by the physician, even if not addressed on the 
potential risk section (Section 7.0) 
 
9.0 
Statistical Considerations  
 


 
 
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Data Analysis Plan: We have chosen a sample size based on the power to detect differences in our 
primary endpoint, insulin-stimulated MBV as measured by CEU. Our preliminary data showed baseline 
MBV measure with a mean in the range of 3.8 to 4.3 and an SD in the range of 1.1 to 3.2 (average 
mean±SD: 4.0±2.1, Fig.6). In our previous study (preliminary data, Fig. 4), we did not observe any 
improvement in endothelial function after a 4-week treatment with sildenafil in non-carriers whereas in 
G-allele carriers endothelial function as measured by FMD improved by 40% (mean±SD: from 4.8±2.2 to 
8.0±2.2). We assume sildenafil will have no effect on MBV among the non-carriers and sildenafil will 
increase CEU conservatively by about 30% among the G-allele carriers. A paired t-test will have 84% 
power to detect a 30% increase of CEU to 5.3 post-sildenafil treatment compared to placebo (4.0±2.1, 
mean±SD), assuming a within-subject correlation coefficient of 0.6 with a sample size of 24. The type I 
error rate is 0.05 and the study is on G carriers. We conservatively estimated 20% of dropout rate, and 
therefore we will enroll 30 subjects in this protocol. For insulin sensitivity (secondary endpoint), as 
previously shown25 Intralipid® + heparin infusion decreased glucose infusion rate from 5.6±2.3 to 
4.5±1.7mg/kg/min. Our study has 81% power to detect a similar difference using an SD for the within-
subject difference of 1.8 (calculated using the higher SD of 2.3 assuming a within-subject correlation 
coefficient of 0.7). 
 
Data Analyses: Standard graphing and screening techniques will be used to detect outliers and to ensure 
data accuracy. We will assess continuous outcomes for normality. If normality is violated, we will apply 
data transformation or consider non-parametric analysis methods. Summary statistics for both continuous 
and categorical variables will be provided by groups (intralipids, intralipids+sildenafil to describe the 
study sample.  Our analysis will use mixed effect models with a random subject effect and treatment 
(intralipids vs. intralipids+sildenafil), genotype (G-allele carriers vs. non-carriers), and their interaction as 
fixed effects.  
 
 
 
10.0 
Privacy/Confidentiality Issues 
 
All data will be collected specifically for the proposed research project. A unique identification case 
number will be used to protect the confidentiality of the study participants. Only case numbers will be 
included in spreadsheets used for the statistical analysis. PHI and access to the key for the ID numbers 
will only be viewable by members of the research team. Members of the research team will have access to 
the patient's medical record during the screening visit and throughout the study until the patient completes 
her participation in the study or meets any of the criteria for study withdrawal/discontinuation. 
 
11.0 
Follow-up and Record Retention 
 
Research records will be maintained for at least three (3) years from the date the research is closed with 
the Vanderbilt University IRB. All research records will be accessible for inspection and copying by 
authorized representatives of the IRB, federal regulatory agency representatives, and the department or 
agency supporting the research.  
 
All Health Insurance Portability and Accountability Act (HIPAA)-related documentation will be 
maintained for at least six (6) years from the date of the last use or disclosure of the Protected Health 
Information (PHI).  
 
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induced during preadipocyte differentiation. Homology with human CD36. The Journal of 
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2. 
Greenwalt DE, Scheck SH,Rhinehart-Jones T. Heart CD36 expression is increased in 
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3. 
Cines DB, Pollak ES, Buck CA, Loscalzo J, Zimmerman GA, McEver RP, Pober JS, 
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Ramirez CE, Hui N, Yu C, Gamboa JL, Luther JM, Brown NJ,Shibao CA. Treatment 
with Sildenafil Improves Insulin Sensitivity in Prediabetes: A Randomized, Controlled Trial. The 
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9. 
Love-Gregory L, Sherva R, Schappe T, Qi JS, McCrea J, Klein S, Connelly 
MA,Abumrad NA. Common CD36 SNPs reduce protein expression and may contribute to a 
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Love-Gregory L, Sherva R, Sun L, Wasson J, Schappe T, Doria A, Rao DC, Hunt SC, 
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Ellis J, Lange EM, Li J, Dupuis J, Baumert J, Walston JD, Keating BJ, Durda P, Fox ER, 
Palmer CD, Meng YA, Young T, Farlow DN, Schnabel RB, Marzi CS, et al. Large multiethnic 
Candidate Gene Study for C-reactive protein levels: identification of a novel association at CD36 
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Barrett EJ,Rattigan S. Muscle perfusion: its measurement and role in metabolic 
regulation. Diabetes. 2012;61:2661-8. 
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Gruber HJ, Mayer C, Mangge H, Fauler G, Grandits N,Wilders-Truschnig M. Obesity 
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Kim JA, Montagnani M, Koh KK,Quon MJ. Reciprocal relationships between insulin 
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Evaluation, and Treatment of High Blood Cholesterol in Adults. The Expert Panel. Arch Intern 
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Grimm RH, Jr., Messerli FH, Oparil S, Schork MA,Trial of Preventing Hypertension Study I. 
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Narayan KM, Ali MK,Koplan JP. Global noncommunicable diseases--where worlds 
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Murray CJ, Atkinson C, Bhalla K, Birbeck G, Burstein R, Chou D, Dellavalle R, Danaei 
G, Ezzati M, Fahimi A, Flaxman D, Foreman, Gabriel S, Gakidou E, Kassebaum N, et al. The 
state of US health, 1990-2010: burden of diseases, injuries, and risk factors. Jama. 
2013;310:591-608. 
19. 
Hoffman RP. Metabolic syndrome racial differences in adolescents. Current diabetes 
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Kalinowski L, Dobrucki IT,Malinski T. Race-specific differences in endothelial function: 
predisposition of African Americans to vascular diseases. Circulation. 2004;109:2511-7. 
21. 
Morris AA, Patel RS, Binongo JN, Poole J, Al Mheid I, Ahmed Y, Stoyanova N, 
Vaccarino V, Din-Dzietham R, Gibbons GH,Quyyumi A. Racial differences in arterial stiffness 
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22. 
Duck MM,Hoffman RP. Impaired endothelial function in healthy African-American 
adolescents compared with Caucasians. The Journal of pediatrics. 2007;150:400-6. 
23. 
An R. Prevalence and Trends of Adult Obesity in the US, 1999-2012. ISRN obesity. 
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Creager MA, Gallagher SJ, Girerd XJ, Coleman SM, Dzau VJ,Cooke JP. L-arginine 
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25. 
Roden M, Stingl H, Chandramouli V, Schumann WC, Hofer A, Landau BR, Nowotny P, 
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26. 
Chai W, Liu J, Jahn LA, Fowler DE, Barrett EJ,Liu Z. Salsalate attenuates free fatty acid-
induced microvascular and metabolic insulin resistance in humans. Diabetes Care. 
2011;34:1634-8. 
27. 
Krebs M, Stingl H, Nowotny P, Weghuber D, Bischof M, Waldhausl W,Roden M. 
Prevention of in vitro lipolysis by tetrahydrolipstatin. Clin Chem. 2000;46:950-4. 
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Colombo PC, Ashton AW, Celaj S, Talreja A, Banchs JE, Dubois NB, Marinaccio M, 
Malla S, Lachmann J, Ware JA,Le Jemtel TH. Biopsy coupled to quantitative 
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Evaluation of Endothelial Function After a High-Fat Meal in African Americans. J Am Heart 
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Addendum 
SILDENAFIL 20 MG BLINDED CAPSULES  
#0 SIZE (PURPLE OPAQUE) 
IRB # 160955 
 
Ingredients: 
Sildenafil 20 mg tablets 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
         # 100 
 
(Teva, NDC 00093-5517-98) 
 
Microcrystalline Cellulose, NF 
          to cover 
 
(PCCA Part 30-1130) 
 
Capsule #1 Purple Locking (Gelatin)  #100 
 
(PCCA Part 30-3015) 
 
Yield 
100 capsules 
 
 
Starting Materials: 
Capsule machine  
 
Size “0” purple caps  
 
Compounding: 
 
1. Don appropriate attire for compounding. 
2. Place one sildenafil 20 mg tablet in each of 100 empty capsule bottoms. 
3. Cover tablet with microcrystalline cellulose, NF making sure capsule is completely 
full. 
4. Replace tops, clean capsules. 
5. Complete record in IDS log book. 
6. Package and label appropriately. 
 
References: 
USP/NF Chapter 795 Pharmaceutical Compounding  
 
Expiration Date: 
6 months 
 
Storage: 
 
Room temperature 
 
Sample Labels: 
 
 
Vanderbilt Investigational Drug Service 
 
Blinded Sildenafil 20 mg caps #100 
 
Dr. Cyndya Shibao 
 
Lot# IDSXXXX           Exp:  XX/XX/XXXX 
 
IRB # 160955      XX/XX 
 
 
 
 
 


 
 
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