Effect of Inhibition Soluble Epoxide Hydrolase on Insulin Sensitivity in Humans
Phase: PHASE2
Conditions: Diabetes Mellitus, Endocrine System Diseases, Glucose Metabolism Disorders, PreDiabetes, Obesity
Sponsor: Vanderbilt University Medical Center

 
 
 
 
 
 
Soluble Epoxide Hydrolase Inhibition and Insulin Resistance 
NCT03486223 
Protocol adapted from IRB approved version dated Date 8/11/2021 
 
 

 
 
 
 
 Variation in Soluble Epoxide Hydrolase Activity   
and Insulin Sensitivity in Humans  
 
 
 
 
James M. Luther, M.D. 
536 Robinson Research Building 
Vanderbilt University Medical Center 
 
Nancy J. Brown, M.D. 
D-3100 Medical Center North 
Vanderbilt University Medical Center 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Table of Contents: 
 
Study Schema 
1.0 
Background 
2.0 
Rationale and Specific Aims 
3.0 
Animal Studies and Previous Human Studies 
4.0 
Inclusion/Exclusion Criteria 
5.0 
Enrollment/Randomization 
6.0 
Study Procedures 
7.0 
Risks of Investigational Agents/Devices (side effects) 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to 
Participants or Others 
9.0 
Study Withdrawal/Discontinuation 
10.0 Privacy/Confidentiality Issues 
11.0 Follow-up and Record Retention 
 

1.0 
Background 
 
An estimated 422 million adults are living with diabetes worldwide.1 The majority (95%) 
have T2DM. Patients with T2DM are four times more likely to suffer from cardiovascular and 
renal disease than those without diabetes. Despite the development of new classes of anti-
diabetic medications,4 T2DM is uncontrolled in one-third of patients.5 In addition, while 
controlling blood glucose with anti-diabetic agents reduces microvascular disease, many 
commonly prescribed anti-diabetic agents do not reduce risk of cardiovascular events.6-9 
There is a continued need to develop therapies to prevent and to treat T2DM while 
decreasing end-organ damage. Data from rodents, outlined below, suggest that the EET/sEH 
pathway (Figure 1) represents a potential new target for the prevention or treatment of 
diabetes. The well-described beneficial effects of this pathway on endothelial function, 
inflammation, and end-organ damage make it a particularly attractive target.10 This proposal 
expands on preliminary data on the role of EETs and sEH in glucose metabolism in humans. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tissue-specific sEH expression and/or activity are increased in rodent models of 
diabetes and obesity. sEH activity is increased more than two-fold in microsomes prepared 
from livers of rats treated with alloxan or streptozotocin (STZ).30 sEH expression is also 
increased in the heart and gastrocnemius muscle of Akita mice, a murine model of T1DM, 
but not in liver.31 In T2DM, sEH expression is increased 2.6-fold in the liver of C57BL/6 mice 
after long-term (16 weeks), high-fat (HF) feeding, but not after shorter duration (8 weeks) HF 
feeding.26 sEH activity is increased approximately 35% in the liver regardless of the duration 
of HF feeding.26 Schäfer et al. reported that a HF diet significantly decreases protein 
expression of the epoxygenases that form EETs in the liver and up-regulates sEH three-
fold.32 sEH activity is also increased in epididymal fat in mice fed a HF diet.27 In addition, sEH 
expression increases with differentiation of preadipocytes into mature adipocytes.27 
Conversely, EET concentrations are higher in preadipocytes or mesenchymal stem cells 
(MSCs) compared to mature adipocytes.27,33  
 
Loss of Ephx2 and pharmacological sEH inhibition enhance insulin sensitivity in 
rodent models of insulin resistance. Ephx2 deficiency or pharmacologic inhibition of sEH 
increases endogenous EETs and enhances insulin signaling in liver and in epididymal 
adipose tissue in HF mice.19 Insulin-induced tyrosyl phosphorylation of the insulin receptor 
EETS
DHETS
Arachidonic Acid
muscle
adipose
liver
sEH
DiHOME
EpOME
CYP2C/J
IRS→PI3K→AKT
AMPK
glut 4
glucose
endothelium
↑microvasculature
↑vasodilation
↑insulin and glucose delivery
triglycerides
↑
glucose
I
I
PDE←AKT←PI3K
lipolysis
cAMP
free fatty acids
I
AKT
G6Pase
Figure 2: Possible mechanisms through which 
epoxyeicosatrienoic acids (EETs) improve 
glucose homeostasis. EETs are produced from 
arachidonic acid by epoxygenases CYP2C and 
CYP2J. EETs improve insulin signaling in 
muscle, liver, and adipose tissue.  EETs also 
increase capillary volume and microvascular 
blood flow in insulin-sensitive tissues. Actions of 
EETs are limited by hydrolysis by soluble 
epoxide hydrolase (sEH) to 
dihydroxyeicosatrienoic acids (DHETs). sEH 
activity is often measured as the ratio of 
dihydroxyoctadecenoic acid (DiHOME) to 
epoxy-9Z-octadecenoic acid (EpOME). AKT, 
protein kinase B; IRS, insulin receptor substrate; 
G6Pase, glucose 6 phosphatase; glut 4, glucose 
transporter type 4; PDE, phosphodiesterase; 
PI3K, phosphoinositide 3-kinase 

(IR) is increased, as is tyrosyl phosphorylation of insulin receptor substrate (IRS)-1. Insulin-
stimulated association of IRS-1 and the P85 subunit of phosphoinositide 3-kinase is 
enhanced in Ephx2-null and inhibitor-treated mice, resulting in increased phosphorylation of 
Ser473 and mitogen-activated protein kinase (MAPK). Ephx2 deficiency and sEH inhibition 
also reduce HF-induced endoplasmic reticulum (ER) stress in the liver and subcutaneous 
adipose tissue (SAT).21 Iyer et al. reported that sEH inhibition decreases glucose after an 
oral glucose load, decreases non-esterified fatty acids and total cholesterol, and decreases 
steatosis in the liver of high carbohydrate-, HF-fed rats.20 
Beneficial effects of sEH inhibition are likely due to decreased degradation of 
endogenous EETs. In heme oxygenase (HO)-2 deficient mice, a model of obesity and insulin 
resistance, treatment with an EET agonist reduces weight gain, subcutaneous and visceral 
fat, and glucose.23 The EET agonist increases vascular adiponectin, phosphorylation of 5' 
adenosine monophosphate-activated protein kinase (AMPK), nitric oxide synthase (eNOS), 
and improves endothelial function.23 The EET agonist increases circulating adiponectin and 
decreases tissue necrosis factor (TNF)-α and monocyte chemotactic protein (MCP)-1. 
EETs and sEH inhibition enhance insulin signaling in vitro. In HepG2 cells, sEH 
inhibition prevents palmate-induced ER stress.21 sEH inhibition or treatment with EETs or 
epoxy-12Z-octadecenoic acid (EpOME), the epoxygenase product of linoleic acid, increases 
insulin-stimulated IR and protein kinase B (AKT) phosphorylation, whereas DHET and 
dihydroxy-12Z-octadecenoic (DiHOME) reduce insulin signaling.21 EETs prevent inactivating 
phosphorylation of IRS-1 at S312 and increase insulin-stimulated AKT phosphorylation and 
glucose 6 phosphatase (G6P) expression in HepG2 cells.34 Schäfer et al. also observed an 
effect of EETs on insulin-stimulated phosphorylation of AKT in primary mouse hepatocytes 
but did not detect an effect on phosphorylation of IR or IRS-1.35  
In cultured MSCs, multi-plural stromal cells that can differentiate into adipocytes, EETs 
suppress adipogenesis and induction of fatty acid synthase and increase phosphorylation of 
AKT as well as adiponectin levels.36 These effects are enhanced by sEH inhibition. Treatment 
with an EET agonist also decreases adipocyte generation and increases HO-1, adiponectin, 
and glucose uptake by MSCs.36,37 MSCs derived from HO-2 null mice demonstrate 
decreased HO-1 activity and increased adipogenesis.38 An EET agonist increases HO-1, 
reduces adipogenesis, increases adiponectin, and decreases inflammatory cytokine 
production.38  
In vivo, EETs can enhance insulin sensitivity not only through direct effects on insulin 
signaling, but also by increasing blood flow and insulin and glucose delivery. In wild-
type and obese insulin-resistant  mice (db/db), administration of an sEH inhibitor increases 
muscle capillary blood volume and microvascular blood flow as measured by contrast-
enhanced ultrasound; in db/db mice this is accompanied by a decrease in glucose.22 
Conversely, insulin-induced increases in muscle capillary blood volume and microvascular 
blood flow are prevented by administration of an epoxygenase inhibitor.22 
EETs or sEH inhibition may enhance insulin secretion. Studies in islets provide 
conflicting data regarding the effect of EETs on insulin release.39-41 Luo et al. reported that 
Ephx2-null mice and sEH inhibitor-treated mice demonstrate preserved glucose-stimulated 
insulin secretion and are protected against islet cell apoptosis following STZ treatment.24,25 
Insulin release in response to glucose or potassium channel inhibition is increased in isolated 
islets from Ephx2-null mice and mice treated with an sEH inhibitor.24 EETs may also influence 
pancreatic islet cell number or size in models of T2DM. In HF-fed mice, for example, targeted 
deletion of Ephx2 or selective sEH inhibition increases islet size and vascular density 
measured by staining for CD31.19  Other investigators report that sEH inhibition increases 
the number of β cells without increasing islet size in high carbohydrate- and HF-fed rats.20 
We have not observed an effect of EPHX2 genotype on glucose-stimulated insulin 

secretion in humans, and for this reason focus this proposal on insulin sensitivity. 
Soluble epoxide hydrolase inhibitors are under development in humans. Among the 
pharmacological inhibitors of soluble epoxide hydrolase tested in rodents, the first to be 
tested in humans was 1-(1-acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea or AR9281.17 A 
study of the effects of AR9281 in patients with hypertension and impaired glucose tolerance 
(NCT00847899) was completed, but not published. The sEH inhibitor GSK2256294 [(1R,3S)-
N-(4-cyano-2-(trifluoromethyl)benzyl)-3-((4-methyl-6-(methylamino)-1,3,5-triazin-2-
yl)amino)cyclohexanecarboxamide] has been studied safely in Phase I and II studies.18 The 
tmax is 0.5 hrs, and t1/2 is 48 hrs. With multiple dosing in overweight smokers, steady-state 
was achieved within seven days. GSK2256294 10 mg achieved more than 95% inhibition of 
sEH throughout the dosing interval.18  
While sEH inhibition may have beneficial cardiovascular and renal effects in T2DM by 
improving endothelial function,11,29 there could also be adverse consequences of long-term 
sEH inhibition. In pre-clinical models, EETs increase vascular endothelial growth factor 
(VEGF), increase proliferation and survival of endothelial cells, and promote angiogenesis, 
leading to metastasis of existing tumors.42 The effect of sEH inhibition on angiogenesis and 
tumor growth is complex, however. While EETs promote angiogenesis, other fatty acid 
substrates of sEH, such as the epoxydocosapentaenoic acids (from omega-3 fatty acids) 
reduce angiogenesis.43 In some models, dual cyclooxygenase and sEH inhibition decreases 
angiogenesis and tumor growth.44 In addition, chronic inflammation contributes to the 
development of many cancers, and the anti-inflammatory effects of EETs may prevent these. 
For example, sEH expression is increased in colon cancer; Ephx2-null mice are protected 
against dextran sulfate sodium-induced colitis and colon cancer.45 There was no effect of 
sEH inhibitor GSK2256294 on circulating VEGF in Phase II studies.18 In this proof-of-concept 
study, we will treat patients with an sEH inhibitor for one week to avoid any risk of long-term 
effects.   
 
 
 

 
2.0 
Rationale and Specific Aims 
 
The World Health Organization estimates that 422 million adults or 8.5% of the world’s 
adult population was living with diabetes in 2014, compared to 108 million or 4.7% of the 
adult population in 1980.1  Exercise, weight loss, and drugs like metformin can reduce the 
incidence of type 2 diabetes (T2DM), but lifestyle changes are difficult to maintain, and not 
all patients can take metformin.2,3 Several exciting new classes of drugs have been 
developed for the treatment of diabetes,4 yet diabetes remains uncontrolled in at least  one-
third of patients.5 In addition, controlling glucose with anti-diabetic agents may not reduce 
macrovascular events.6-9 
This proposal addresses the potential 
of small molecule inhibitors of soluble 
epoxide hydrolase (sEH) to improve insulin 
sensitivity in humans. Epoxyeicosatrienoic 
acids (EETs) are formed from arachidonic 
acid by the action of P450 epoxygenases 
(CYP2C and CYP2J) (Figure 1).10  EETs 
act as potent vasodilators and have been 
identified 
as 
endothelium-derived 
hyperpolarizing factors.11 In the kidney, 
EETs 
promote 
sodium 
excretion 
by 
inhibiting the translocation of the Na+/H+ 
exchanger (NHE3) in the proximal tubule.12 
EETs reduce inflammation by preventing 
NFκB activation.13 It follows that increasing 
the actions of EETs in rodent models 
protects against hypertension, endothelial 
dysfunction, cardiovascular remodeling, 
and renal injury.10,14,15 The effects of EETs 
are limited by hydrolysis by sEH to the less active dihydroxyepoxyeicosatrienoic acids 
(DHETs), and potential strategies to increase EETs include either increasing epoxygenase 
expression or reducing sEH activity.16 Thus, specific pharmacological sEH inhibitors have 
been under development in clinical studies for the treatment of hypertension and lung 
disease.17,18 
Studies in rodents point to an important role for EETs and sEH in the regulation of glucose 
homeostasis and insulin sensitivity (Figure 2). Specifically, inhibiting sEH or disrupting 
Ephx2, the gene encoding sEH, increases insulin sensitivity in rodent models of T2DM and 
insulin resistance.19-21 These effects have been attributed to enhanced insulin signaling in 
liver, muscle, and adipose tissue, as well as to beneficial effects on muscle capillary blood 
volume and microvascular blood flow.19-22 Administration of exogenous EETs replicates 
these effects.23 Other studies have identified a beneficial effect of EETs or sEH inhibition on 
islet cell function or apoptosis.19,20,24,25 Unfortunately, studies in rodents also suggest that 
tissue sEH activity increases with obesity.26,27 
Our group has begun to elucidate the role of EETs and EPHX2 in the regulation of 
glucose homeostasis in humans. We have found that circulating EETs correlate with insulin 
sensitivity and are significantly decreased in individuals with insulin resistance.28 In addition, 
we have found that insulin sensitivity is increased in overweight carriers of a loss-of-function 
variant in EPHX2 (rs751141 or Arg287Gln), but not in obese carriers of the variant.28 The 
287Gln allele is also associated with decreased vascular resistance.29 We propose to study 
the mechanism of these effects and to test the overarching hypothesis that genetic or 
Figure 2: Epoxyeicosatrienoic acid 
(EET)/soluble epoxide hydrolase (sEH) 
pathway. sEH is encoded by Ephx2 in mice and 
EPHX2 in humans. 
 

pharmacological factors that decrease sEH activity will improve insulin sensitivity by 
increasing blood flow and/or by increasing tissue insulin signaling. We will test the 
hypothesis that treatment with a specific sEH inhibitor decreases sEH activity and 
increases insulin-stimulated vasodilation and insulin sensitivity, via effects on tissue 
(muscle or adipose) insulin signaling and/or increased microvasculature.  
 
 

 
3.0 
Animal Studies and Previous Human Studies 
 
Circulating EET concentrations are decreased in obesity or insulin resistance. EET 
concentrations are decreased in obese patients with coronary artery disease (CAD) 
compared to non-obese patients with CAD, and body mass index (BMI) correlates inversely 
with plasma EETs.46 We have found that plasma EETs are lower in individuals with metabolic 
syndrome than in those without metabolic syndrome (Figure 3A).28 Similarly, EET 
concentrations correlate with insulin sensitivity calculated in two independent studies in 
subjects undergoing hyperglycemic clamps (Figure 3B) or frequently sampled intravenous 
glucose tolerance tests (Figure 3C). The relationship between plasma EET concentrations 
or sEH activity and EET concentrations or sEH activity in insulin-sensitive tissues is not 
known.  
 
 
 
Our group and others have studied 
the relationship between functional polymorphisms in the gene encoding for sEH, 
EPHX2, 
and 
obesity, 
insulin 
sensitivity, 
or 
diabetes. 
The 
Arg287Gln variant encodes for an 
sEH enzyme with lower hydrolase 
activity, while Lys55Arg encodes 
for an sEH enzyme with increased 
hydrolase 
activity.47 
Both 
polymorphisms 
have 
lower 
phosphatase activity.48 We have 
assessed the relationship between 
both variants and the ratio of 
DiHOME to EpOME in the plasma, 
as a marker of sEH activity. Like 
EETs, EpOMEs are substrates for 
hydrolysis by sEH to DiHOMEs. 
The ratio of DiHOME to EpOME is 
 
Figure 3: (A) Plasma epoxyeicosatrienoic acids (EETs) in hypertensive patients with (N=12) and without 
(N=28) metabolic syndrome. Relationship between total EETs and insulin sensitivity index (ISI) 
calculated in subjects undergoing hyperglycemic clamps (B) and IV glucose tolerance tests (C). *p<0.05 
versus without metabolic syndrome 
Arg/Arg Arg/Gln
0.0
0.5
1.0
1.5
2.0
N=10
N=7
EPHX2 Arg287Gln genotype
DiHOME/EpOME
Lys/Lys Lys/Arg
0.0
0.5
1.0
1.5
2.0
2.5
N=12
N=6
*
EPHX2 Lys55Arg genotype
DiHOME/EpOME
 
Figure 4: Plasma ratio of DiHOME to EpOME, as a 
marker of sEH activity, in a small number of subjects of 
known EPHX2 loss-of-function 287Arg genotype (Left) 
or gain-of-function Lys55Arg genotype (Right). *p=0.05 
versus Lys/Lys 
 
Figure 5: Effect of EPHX2 Arg287Gln genotype on the 
relationship between body mass index (BMI) and insulin 
sensitivity index (ISI) calculated from hyperglycemic 
clamps.  
p=0.02 for genotype, p=0.03 for genotype x BMI in all 
subjects.  

used in vivo because the linoleic acid metabolites circulate at concentrations ten-fold higher 
than EETs and DHETs.49 In Figure 4, preliminary data in individuals undergoing metabolic 
studies in whom we obtained DNA and measured plasma EpOME and DiHOME activity 
indicate that the loss-of-function 287Gln variant is associated with a non-significant (in this 
small number) 22% decrease in plasma sEH activity while the gain-of-function is associated 
with a significant 45% increase in activity compared to wild-type.  
 
We have observed an association between the loss-of-function EPHX2 287Gln variant 
and increased insulin sensitivity calculated in 
individuals undergoing hyperglycemic clamps 
(Figure 5).28 We found no relationship between 
EPHX2 genotype and glucose-stimulated insulin 
secretion. Interestingly, the relationship between 
the EPHX2 loss-of-function 287Gln variant and 
insulin sensitivity varied with BMI whether or not 
subjects had metabolic syndrome. These data 
suggest that decreasing sEH activity in patients at 
risk for T2DM could enhance insulin sensitivity. 
Nevertheless, questions remain to be addressed. 
Increased insulin sensitivity in EPHX2 287Gln 
carriers could result from enhanced insulin 
signaling in peripheral tissues, from increased 
capillary blood volume and microvascular blood 
flow or from enhanced endothelial function. We 
have found that basal forearm vascular resistance 
is decreased in EPHX2 287Gln carriers compared 
to Arg/Arg.29  
Loss of epoxygenase Cyp2c44 impairs muscle 
and adipose tissue insulin sensitivity in vivo in 
mice. In addition to EETs, other lipid mediators 
serve as substrates for hydrolysis by sEH. To 
determine whether EETs per se are involved in 
glucose 
homeostasis 
we 
assessed 
insulin 
sensitivity in mice lacking Cyp2c44, which demonstrate decreased EET production.50 We 
found that hepatic glucose production (Figure 6A, EndoRa) was increased and insulin-
stimulated glucose uptake in the gastrocnemius (not shown), vastus lateralis, and adipose 
tissues (Figure 6B-C) was significantly reduced in Cyp2c44(-/-) mice (manuscript in 
revision). This was accompanied by impaired vasodilation (Figure 6D), but not by changes 
in insulin-signaling in isolated muscles ex vivo (not shown). These data support a role for 
EETs in peripheral and hepatic insulin sensitivity and suggest that the effect of EETs may 
depend on enhanced endothelial function and glucose delivery to insulin-sensitive tissues. 
 
 
 
Figure 6: (A) Endogenous glucose 
production, muscle (B) and adipose tissue 
(C) glucose uptake was impaired in vivo in 
Cyp2c44(-/-) 
mice 
assessed 
during 
hyperinsulinemic-euglycemic clamps. (D) 
Mesenteric artery relaxation in response 
to pinacidil was impaired in Cyp2c44(-/-) 
mice. *p<0.05, **p<0.01, ***p<0.001 
 
 

 
4.0 
Inclusion/Exclusion Criteria 
 
Inclusion criteria  
 
1. Men and women,  
2. Age 21 to 60 years, and  
3. Pre-diabetes as defined by  
a. Fasting plasma glucose 100-125 mg/dL, or  
b. Two-hour plasma glucose 140-199 mg/dL, or  
c. HbA1c 5.7-6.4% 
4. BMI  ≥ 30 kg/m2, inclusive 
5. For female subjects, the following conditions must be met: 
a. Postmenopausal status for at least one year, or 
b. Status-post surgical sterilization, or 
c. If of childbearing potential, utilization of adequate birth control and willingness 
to undergo serum β-hcg testing prior to drug treatment and on every study 
day. 
 
Exclusion criteria  
Subjects presenting with any of the following will not be included in the study:  
1. Diabetes type 1 or type 2, as defined by a fasting plasma glucose of 126 mg/dL 
or greater, a two-hour plasma glucose of 200 mg/dL or greater, a HbA1c >6.4%, 
or the use of anti-diabetic medication  
2. Subjects who have participated in a weight-reduction program during the last 
six month or whose weight has increased or decreased more than two kg over 
the preceding six months 
3. Resistant hypertension, defined as hypertension requiring the administration of 
more than three anti-hypertensive agents including a diuretic to achieve control 
4. Use of spironolactone   
5. Pregnancy or breast-feeding  
6. Any significant history of smoking, as defined by smoking one pack-year over 
any cumulative time or smoking any amount within the last ten years  
7. Any history of cancer including skin cancer, any history of a precancerous 
lesion, abnormal PSA, or lack of screening adherent to American Cancer 
Society Guidelines for the Early Detection of Cancer  
8. Cardiovascular disease such as myocardial infarction within six months prior to 
enrollment, 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 
9. Abnormal corrected QT interval on screening ECG (QTc).  
10. Treatment with anticoagulants  
11. History of serious neurologic disease such as cerebral hemorrhage, stroke, or 

transient ischemic attack  
12. History or presence of immunological or hematological disorders  
13. Diagnosis of asthma requiring regular inhaler use  
14. Clinically significant gastrointestinal impairment that could interfere with drug 
absorption  
15. Impaired hepatic function (aspartate amino transaminase [AST] and/or alanine 
amino transaminase [ALT] >3.0 x upper limit of normal range) 
16. History of gastrointestinal bleed 
17. Estimated glomerular filtration rate (eGFR)<60 mL/min/1.73 m2 or with an 
albumin-to-creatinine ratio (UACR) >300µg/mg, where eGFR is determined by 
the four-variable Modification of Diet in Renal Disease (MDRD) equation, where 
serum creatinine is expressed in mg/dL and age in years: eGFR 
(mL/min/1.73m2)=186 • Scr-1.154 • age-0.203 • (1.212 if black) • (0.742 if female) 
18. Hematocrit <35%  
19. 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  
20. Treatment with chronic systemic glucocorticoid therapy  
21. Treatment with lithium salts  
22. History of alcohol or drug abuse  
23. Treatment with any investigational drug in the month preceding the study  
24. Mental conditions rendering a subject unable to understand the nature, scope, 
and possible consequences of the study 
25. Inability to comply with the protocol, e.g., uncooperative attitude, inability to 
return for follow-up visits, and unlikelihood of completing the study 
 
 
 

 
5.0 
Enrollment/Randomization 
 
Assignment to treatment order will be randomized and blinded to the investigators 
and subjects.  Subjects will be randomly assigned to drug order using a permuted-block 
randomization algorithm. Dr. Yu, study biostatistician, will provide an allocation schedule. 
Vanderbilt Investigational Drug Services (IDS) will be responsible for the storage, 
preparation, and labeling of all investigational agents and for maintaining accurate drug 
storage and dispensing logs. The Clinical Research Pharmacist will devise standard 
operating procedures for the pharmacy to follow with regard to preparing, labeling, blinding, 
and dispensing study drug.  
After subjects have been consented and screened, the investigator or research nurse 
will fax a copy of the consent form and a prescription containing check boxes for the inclusion 
and exclusion criteria to Vanderbilt IDS. The pharmacist will confirm that consent was 
obtained and the subject met entry criteria, assign the subject a randomization number, and 
provide the investigator with labeled study drug as indicated in the protocol. An extra label 
containing the randomization number will be attached to each study drug.  The investigator 
will affix this extra label to the subject’s records. The Investigational Pharmacy will retain a 
secure set of sealed envelopes containing the treatment assignment.  These will be opened 
in the event of a clinical scenario which necessitates unblinding, as determined by the PI and 
the DSMC (see Data and Safety Monitoring Plan).  Subjects randomized who do not 
complete the whole protocol for any reason will be replaced. 
 
 

 
6.0 
Study Procedures 
Rationale: Our preliminary studies indicate that the EPHX2 loss-of-function variant 
(Arg287Gln) is associated with a 22% decrease in plasma sEH activity and enhanced insulin 
sensitivity. Studies in animal models suggest that sEH activity is increased in insulin-sensitive 
tissues in obesity, whereas epoxygenase activity is decreased.26,27,32 A potent 
pharmacological inhibitor of sEH has the potential to decrease sEH activity in insulin-
sensitive tissue even in obese subjects. In this Aim we will determine whether 
pharmacological sEH inhibition reduces sEH activity in insulin-sensitive tissues and whether 
pharmacological sEH inhibition enhances insulin sensitivity through increased insulin 
signaling or increased blood flow. 
 
Subjects: 
We will study 34 obese (BMI  ≥ 30 kg/m2) and pre-diabetic subjects, age 21 through 50 years. 
We will study fifty percent black subjects, fifty percent white subjects, and fifty percent 
women. Subjects with any history of cancer including skin cancer, cardiovascular disease 
(other than hypertension), renal, pulmonary, endocrine (other than insulin resistance or 
hyperlipidemia), or hematologic disease will be excluded. Individuals with diabetes, resistant 
hypertension, or any history of smoking will be excluded. Pregnancy will be excluded. 
Detailed inclusion and exclusion criteria appear in Human Subjects.  
 
 
Protocol: 
Once informed consent has been obtained, subjects will report to the CRC after an overnight 
fast, and we will obtain a medical history and complete a physical examination. We will obtain 
screening laboratory and an ECG. Between screening and the first study day, subjects will 
return to the CRC in the fasting state to undergo measurement of waist and hip 
circumference, a 75-gram OGTT, and body composition measurement by Dual Energy X-ray 
Absorptiometry (DEXA, see Standard Techniques).Because this is a crossover study, 
hypertensive participants of this Aim will continue to take their anti-hypertensive medications, 
and the doses will be maintained constant throughout the study.  
Subjects will then be randomized within one month of screening using a permuted-block 
algorithm to treatment with the sEH inhibitor GSK2256294 (10 mg/d) or matching placebo for 
one week in a double-blind, crossover design study. The Vanderbilt Investigational Drug 
Service will be responsible for storage, preparation, labeling, and dispensing of medication. 
GSK is supplying study drug and matching placebo. GSK2256294 has been studied safely 
in Phase I and II studies.18 In multiple dosing studies in overweight smokers, steady state 
was achieved within seven days. The dose of GSK2256294 achieves >95% inhibition of sEH 
throughout the dosing interval. Subjects will also be provided a weight-maintenance diet 
containing 150 mmol/day sodium and 80 mmol/day potassium for one day prior to study. On 
the seventh day of drug treatment, subjects will report to the CRC in the morning after an 
overnight fast to undergo a hyperinsulinemic-euglycemic clamp. Urine will be collected 
overnight for 8 hours prior to the study day, and again after clamp completion. On the day of 
study, we will place IV catheters in both arms. We will obtain an adipose tissue biopsy 
(Standard Techniques) and optional muscle tissue biopsy if subject consents. We will 
administer paraminohippurate (PAH, 8 mg/kg) as a loading dose, followed by a 12 mg/min 
continuous infusion for measurement of renal blood flow. After the subject has been supine 
for 45 min (and PAH is at steady state), we will measure forearm blood flow (FBF), BP, and 

heart rate in triplicate (Standard Techniques). We will draw blood for measurement 
ofGSK2256294 concentrations, PAH, plasma EETs, DHETs, EpOME, DiHOME, interleukin 
6 (IL-6) and other inflammatory cytokines, adiponectin, VEGF, and potassium. Subjects will 
then undergo a hyperinsulinemic-euglycemic clamp (see Standard Techniques). We will 
infuse 6,6-dideuterated glucose to assess endogenous glucose appearance (EndoRa) under 
fasting conditions, and we will assess hepatic insulin sensitivity by the response to low 
(20mU/m2/min) and high (80mU/m2/min) insulin infusion rates (Standard Techniques). At 
the end of the clamp we will repeat FBF measurements, followed by adipose tissue and 
optional muscle biopsy. We have experience obtaining biopsies before and at the end of 
hyperinsulinemic clamps (Preliminary Studies). At the end of each study day, we will obtain 
safety laboratory including CBC with differential, complete metabolic profile, urinalysis, and 
ECG. 
After completion of the study day, subjects will undergo a seven-week washout from 
study drug and then receive the opposite drug for one week, while eating the same 
standardized diet. On the seventh day of treatment they will report to the CRC after an 
overnight fast and repeat the study day protocol.  
Standard Techniques 
BP Measurements: During screening, washout, and active treatment outpatient BP will 
be measured with an aneroid sphygmomanometer (Welch Allyn, Skaneateles Falls, NY), 
using the appearance and complete disappearance of the Korotokoff sounds (K1 and K5) as 
systolic blood pressure (SBP) and diastolic blood pressure (DBP). The mean of three seated 
measurements will be used. During study days, BP will be measured every five min using an 
automated oscillometric recording device (Dinamap, Critikon, Carlsbad, CA). 
 
Adipose harvest: Adipose tissue will be obtained from the periumbilical area using a 
TulipTM Medical closed syringe system for lipoaspiration (see Letter of Support). Under 
aseptic conditions and local lidocaine anesthesia, a small incision is made in the skin. The 
GEMS Johnnie Snap lock is placed into a 60-cc syringe, the Tulip liposuction cannula with 
60-cc syringe attached is inserted at an angle through the incision to below Scarpa’s fascia, 
and suction is applied until the syringe activates the clicker lock. The needle is moved in and 
out at a rate of approximately 1 Hz without breaking suction with a twisting motion.  The 
sampling continues until approximately 5 g of tissue is removed (approximately 5.5 cc at a 
specific gravity of 0.918 for human fat). The syringe containing the sample will be taken on 
ice to the lab where it will be separated into pieces for measurement of sEH activity, DiHOME, 
EpOME, EETs, DNA and RNA isolation, adipocyte size and number, cytokines, and 
adipokines.  
 
PBMC harvest: PBMCs will be obtained through the peripheral IV into 3 EDTA tubes 
for total of 12 milliliters, and transported at room temperature to the lab for processing. 
Cells will then be passed through a Ficoll-Paque barrier, washed with PBS, and stored in 
DMSO. 
 
Muscle biopsy: Biopsies will be obtained from the vastus lateralis under aseptic 
conditions and local lidocaine anesthesia by percutaneous needle biopsy using the modified 
Bergström technique. Briefly, after proper aseptic technique and local anesthesia, a 5 mm 
incision is made in the skin. The needle is inserted through the incision to the skeletal muscle. 
The inner trocar of the needle is retracted and suction applied to pull muscle into the outer 
trocar. The inner trocar is then closed to cut the muscle. This needle biopsy is then rotated 
(90°) and the procedure is repeated twice. After removing the needle, tissue is immediately 

placed in ice-cold 0.9% NaCl, and the 
subcutaneous fat, if any, is separated from 
muscle. Muscle biopsies will be divided: a 
fresh sample will be taken on ice to the 
Eicosanoid Core for measurement of sEH 
activity, EETs, DHETs; two pieces will be 
frozen in liquid nitrogen and stored at -80ºC 
for DNA and RNA isolation and for Western 
blotting, one will be immersed in fixatives for 
electron microscopy.  
 
Body Composition Measurement:  Body 
composition will be measured by Dual 
Energy X-ray Absorptiometry (DEXA). The 
computer software permits measurements 
of the whole body as well as individual 
segments, this is an accurate, validated 
method with minimal radiation exposure 
(Mazess et al., 1990;Svendsen et al., 1993). 
A Lunar model DPX Absorptiometer (Lunar 
DPA software 3.6; GE Medical System, 
Madison, WI), a research scanner located in the Vanderbilt GCRC will be used to measure 
total and regional adiposity.  
 
FBF measurements: We will measure FBF before and at the end of hyperinsulinemia 
using venous occlusion mercury-in-Sialastic strain-gauge plethysmography.29,61,62 The wrist 
is supported in a sling to raise the forearm to above the level of the atrium, and the strain 
gauge is placed at the widest part of the forearm. The strain gauge is connected to a 
plethysmograph (model EX-5; D. E. Hokanson, Inc., Bellevue, WA), calibrated to measure 
the percentage change in volume and connected to a chart recorder. For each measurement, 
a cuff placed around the upper arm is inflated to 40 mm Hg with a rapid cuff inflator (model 
E-10; D. E. Hokanson, Inc., Bellevue, WA) to occlude venous outflow from the extremity. The 
hand is excluded from the measurement of blood flow by inflation of a pediatric 
sphygmomanometer cuff around the wrist to 200 mm Hg before and during measurements 
of FBF. Flow measurements are recorded for ∼7 of 15 s, and the slope is derived from the 
first three or four pulses; five to seven readings are obtained for each mean value. 
 
Hyperinsulinemic-euglycemic clamp: We will place a catheter in the antecubital vein for 
insulin and glucose infusion, and a catheter in a hand vein for glucose sampling. Subjects 
will rest at least 30 min before baseline measurements. We will infuse insulin in the arm 
contralateral to sampling. We have chosen insulin infusion rates to suppress hepatic glucose 
production partially during the low-dose infusion (20mU/m2/min), completely during the high-
dose infusion (80mU/m2/min), and to stimulate peripheral glucose utilization maximally in 
insulin-resistant subjects during the high-dose infusion.63-66 We will give a priming insulin 
dose at the time of each insulin infusion change. We will measure plasma glucose every 2.5 
to 5 min and give an exogenous infusion of 20% glucose to control the rate of fall of glucose 
and maintain the plasma glucose at a target of 90-95 mg/dL.67,68 We will administer potassium 
chloride (KCl) 40 mEq orally prior to hyperinsulinemia to maintain potassium levels.  Heart 
rate and ECG will be monitored continuously during all glucose clamp studies. Participants 
will  be instructed to empty their bladder prior to insulin infusion, at the end of the low dose 
Figure 8: (A) Hyperinsulinemic-euglycemic clamp 
protocol and results demonstrating (B) achieved 
plasma glucose, (C) glucose infusion rate, (D) plasma 
insulin, and (E) endogenous glucose production 
(EndoRa). Values are mean±SEM, n=67 for B-D; n=10 
for E.  

insulin infusion, and at the end of the high dose insulin infusion. 
EndoRa and utilization (Rd) will be determined by stable isotope enrichment.69 Total rates 
of appearance (Ra) and Rd will be calculated based on equations of Steele et al.70 as modified 
by Debodo.71 During hyperinsulinemic clamps, Ra and Rd, calculated using this approach, 
may be underestimated,72 particularly during states of high glucose flux.73 To avoid 
calculation errors due to glucose contamination, previously reported with 3H-glucose tracers, 
we will use 6,6-dideuterated glucose as the tracer and analyze enrichment using MS. To 
minimize the effect of rapidly changing enrichment,74 we will assess glucose turnover only 
when steady-state conditions exist, at the end of a 120-min tracer equilibration period and 
during the last 30 min of the clamp. (We define steady state as a period when the coefficient 
of variation of plasma glucose and enrichment are stable at <3%.) Insulin sensitivity will be 
estimated by the rate of glucose infusion (M) necessary to maintain euglycemia during the 
last 30 min of the clamp. To control for inter-individual variations in plasma insulin, the insulin 
sensitivity index will be calculated by dividing M by average steady-state insulin 
concentration. Tracer will be administered as a primed continuous infusion (3.6 mg/kg bolus 
followed by 0.06 mg/kg/min constant infusion) and will be added to the exogenous glucose 
infusion73 to maintain plasma glucose enrichment throughout the study. 
Laboratory Analyses 
Clinical assays will be run in Vanderbilt CLIA-approved laboratories. Blood for research 
assays will be centrifuged for 20 min at 0oC, and plasma/serum will be divided into aliquots, 
labeled, logged, and stored separately at -80oC until sampling. Standard quality measures 
will be applied to all analyses including (but not limited to) tracking of inter-assay variability 
and inclusion of standards and internal controls. 
EPHX2 genotyping: We will extract genomic DNA from whole blood using the AutoPure 
LS extraction system (Qiagen, Valencia, CA). EPHX2 rs751141 (Arg287Gln) and 
rs41507953 (Lys55Arg) will be genotyped using TaqMan assays (Applied Biosystems, Foster 
City, CA). SDS v2.4 (Applied Biosystems) will be used to create cluster plots and identify 
sample-associated fluorescent markers for determination of genotype.    
sEH activity and EETs: We will measure sEH activity in two ways. In tissue extracts, we 
will measure the rate of conversion of an added concentration of 14,15-EET (50 µM) to 14,15-
DHET,75 as well as the ratio of DiHOMEs to EpOMEs. The former is a true measure of 
enzyme activity, and we will compare the DiHOME/EpOME ratio to this gold standard. In 
plasma we will measure the molar ratio of DiHOME to EpOME. Plasma EETs, DHETs, 
EpOME, 
and 
DiHOMEs 
will 
be 
quantified 
using 
ultra-performance 
liquid 
chromatography/tandem 
mass 
spectrometry 
(UPLC/MS/MS).28 
Deuterated 
internal 
standards (Cayman Chemical, Ann Arbor, MI) will be added to each sample. For lipid 
extraction, three mL 0.15M KCl will be added to one mL plasma and extracted with eight mL 
of an acidified chloroform/methanol mixture at a two:one ratio. The organic phase will be 
collected and saponified (0.4M KOH in 80% methanol). EETs, EpOMEs, DHETs, and 
DiHOMEs will be separated by SiO2 chromatography. SiO2-purified EETs and EpOMEs will 
be hydrated to DHETs and DiHOMEs, respectively, in 9.5M acetic acid. EETs, EpOMEs, 
DHETs, and DiHOMEs will be analyzed by UPLC/MS/MS.  The UPLC is equipped with an 
Acquity BEH C18 column 1.0 x 100 mm, 1.7 µm (Waters). To separate analytes, we will 
perform gradient elution starting with a 70% mobile phase A (15 mM ammonium acetate, pH 
8.5) proceeding to a 60% mobile phase B (acetonitrile) over five min. Following gradient 
elution, columns will be washed for one minute with mobile phase B. The UPLC effluent will 
then be subjected to negative ion electrospray ionization using a triple quadrupole mass 
spectrometer, TSQ Quantum Vantage (Thermo Scientific, Hudson, NH), equipped with an 
electrospray source operating in negative ion-mode. Analytes and internal standards will be 
monitored using selected reaction monitoring.  

 
Glucose, C-peptide, and Insulin: Plasma glucose will be measured by the glucose 
oxidase method with a YSI glucose analyzer (YSI Life Sciences, Yellow Springs, OH). 6,6-
dideuterated glucose will be assessed using mass spectrometry. Plasma insulin 
concentrations will be determined by radioimmunoassay (RIA; Millipore, St. Charles, MO). 
Samples for C-peptide will be drawn into heparinized tubes containing 250 KIU Trasylol 
(Aprotinin) per mL of whole blood. C-peptide will be measured using RIA (Millipore).   
 
Western blot analysis of pAKT/AKT, CD31, VEGF: Adipose and muscle samples will 
be homogenized in the presence of proteases and phosphatase inhibitors. For adipose 
tissue, homogenate will be centrifuged for 10 min at 10,000 g; the stromal vascular fraction 
from the pellet at will be recovered and the adipocyte fraction (top of the tube) will be used 
for Western blots. Homogenates will be denatured after mixing with Laemmli buffer and 
heated at 95°C for 5 min. Samples (30 µg of protein) will be resolved electrophoretically in 4-
20% acrylamide gel and transferred to polyvinylidene difluoride membranes (Immobilon-FL, 
Millipore, Billerica, MA). Membranes will be blocked with Odyssey® blocking buffer and 
incubated overnight with antibodies: rabbit monoclonal antibody phospho-AKT, total AKT, 
(Cell Signaling, Danvers, MA), mouse monoclonal against CD31 (ThermoFisher Scientific, 
Rockland, IL), or mouse monoclonal against VEGF (Novus Biologicals, Littleton, CO). 
Membranes will then be incubated for one hour with Alexa Fluor 680-conjugated goat anti-
rabbit IgG (Invitrogen, Carlsbad, CA) or IRDye® 800CW donkey anti-mouse (LI-COR 
Biosciences, Lincoln, NE). Membranes used for phospho-AKT will be stripped and re-probed 
for total AKT to obtain the ratio of pAKT/AKT expression. Odyssey® Infrared Imaging System 
(LI-COR Biosciences) will be used to detect fluorescence intensity. Band densities will be 
analyzed using NIH Image J software. Coomassie blue staining will be used as the loading 
control for other analyses. 
 
Evaluation of PBMC immune cell populations: We will use flow cytometry to assess 
activated, memory, exhausted, and senescent CD4 and CD8 populations using surface 
markers CD38, HLA-DR, CD69, CD57, PD1, and CD45RO (as examples), iNKT cells using 
CD1d tetramer, as well as M1 (CD14+CD16+ with either CD86 or CD36) and M2 (CD163, 
CD301, and CD206) macrophage polarization. 
 
 
 

 
7.0 
Risks 
 
Potential Risks  
1. 
Frequent blood draws can lead to anemia. 
2. 
Withholding BP medications may result in elevation in BP. We will monitor BP every 
one to three days after stopping or decreasing these medications. If the BP is too high 
(see Protection against risk) or there are symptoms of high BP, the study will be 
stopped and prior medications will be restarted.  
3. 
Putting a catheter into a vein may cause bleeding, bruising, or infection (uncommon). 
We will use careful and sterile technique to minimize these side effects. 
 
4. 
Adipose and muscle tissue biopsies may cause bleeding, bruising, pain, or infection 
(uncommon). Only trained investigators will complete these procedures. We will use 
sterile technique to minimize the risk of infection, and local anesthesia (lidocaine) will 
be used to minimize pain. We will monitor subjects for one hour after the biopsies.  
 
5. 
Infusion of glucose may cause hyperglycemia. Symptoms of high blood sugar are 
hunger, thirst, frequent urination, dry mouth, and dry skin. Blood will be monitored 
during and for 2 – 3 hours after clamp completion to ensure blood sugar remains in 
the normal range.  IV glucose may cause irritation of the vein, including redness and 
pain.  We will give the glucose through a bigger vein to decrease this risk. 
6. 
Infusion of insulin may cause hypoglycemia.  Symptoms of low blood sugar are 
nausea, extreme hunger, feeling nervous or jittery, cold, clammy, wet skin and/or 
excessive sweating not caused by exercise, a rapid heartbeat, and numbness or 
tingling of the fingertips or lips, and trembling. Glucose (dextrose 20%) will be infused 
during periods of insulin administration, and glucose will be monitored every five min 
to maintain normal plasma glucose. After study completion, insulin will be stopped, a 
meal will be given, and intravenous glucose will be tapered.  Blood glucose will be 
monitored for 2-3 hours after the study until it stabilizes within the normal range. 
7. 
Eating a controlled diet for one day may be inconvenient. 
8. 
GSK2256294 is an investigational drug. To date there are no safety concerns based 
on Phase I and II trials in humans. There has been skin irritation at the site of ECG 
lead placement during GSK2256294. There may be risks that we do not know about 
at this time. There is a theoretical risk that inhibiting sEH and increasing EETs could 
promote angiogenesis in pre-existing cancer. 
 
 
Protections against risk 
 
1. 
A nurse or physician will be present at all times during the study days. 

2. 
Medication withdrawal (hypertensive subjects): Anti-hypertensive medications will 
be discontinued three weeks prior to the study day to avoid confounding effects on 
sEH activity or EETs. We have excluded individuals with resistant hypertension to 
minimize risk. Whenever appropriate, medications will be tapered before they are 
discontinued. During the washout period, BP will be measured every one to three 
days. If at any time the seated systolic BP is >170 mmHg or the seated diastolic BP 
>110 mm Hg or if a hypertensive subject develops symptoms of high BP regardless 
of the pressure, that subject will be discontinued from the study and his or her anti-
hypertensive medications will be restarted. In our experience, approximately five 
percent of enrolled subjects may be excluded by these BP criteria. Excluded 
subjects will be replaced.  
 
3. 
Subjects with a history of a hematocrit less than 35% will be excluded from study. 
4. 
Only trained investigators will complete adipose and muscle tissue biopsies. We will 
use sterile technique to minimize the risk of infection, and local anesthesia (lidocaine) 
will be used to minimize pain. We will monitor subjects for one hour after the biopsies. 
5. 
We will monitor glucose concentrations frequently during and immediately after 
hyperinsulinemic-euglycemic clamps. Should hypoglycemia occur, we will administer 
glucose. 
6. 
We have limited the duration of GSK2256294, and we are excluding participants with 
a history of cancer or precancerous lesions. We are requiring that subjects be current 
with American Cancer Society Guidelines for the Early Detection of Cancer and also 
have PSA screening. We are excluding smokers. 
7. 
We will monitor subjects actively for new adverse events using a standard 
questionnaire. 
Should any adverse effects of GSK2256294 become known during the study, we will notify 
the IRB, we will revise the protocol and consent form as appropriate, and we will advise 
and re-consent study participants 
 
 

 
8.0 
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 Principal Investigators will closely oversee the protocol in 
conjunction with the dedicated research nurse and Co-investigators.  
A DSMC will provide objective review of treatment results as they relate to human 
safety and data quality. The committee will be comprised of Naji Abumrad, M.D., The John L. 
Sawyers Professor of Surgery; Alvin C. Powers, M.D., Director of the Vanderbilt Diabetes 
Center, Professor of Medicine and Joe C. Davis Professor of Biomedical Science; and Dr. Yu 
Shyr, Harold L. Moses Chair in Cancer Research, Director of the Vanderbilt Center for 
Quantitative Sciences, and Director of the Vanderbilt Technologies for Advanced Genomics 
Analysis and Research Design (VANGARD). Dr. Abumrad will chair the committee. An 
important aspect of the membership of the DSMC is that all three members hold a primary 
appointment outside the Department of Medicine (Abumrad and Shyr) or lead a Research 
Center that reports directly to the Dean (Powers). 
 
The DSMC will meet at least twice, prior to the initiation of study procedures to review 
the protocol and after the first five subjects have been enrolled to receive a safety and progress 
report. These reports will provide information regarding recruiting, safety reporting, and data 
quality. No early stopping is planned. The committee will assess safety data including 
hypertension or hypotension, hypoglycemia, common adverse events, hospitalizations, and 
other serious adverse events. Interim data will be provided to the committee by Drs. Brown and 
Yu. The randomization will be blinded and presented on a coded basis unless the Committee 
votes to receive unblinded data. The Committee will have the authority to modify the protocols 
or to terminate the study if it deems such actions to be warranted. The DSMC will provide 
summary reports to the investigators. Dr. Luther will convey such reports to the NIH (or FDA) 
as appropriate. 
      The DSMC will also receive quarterly reports of enrollment, protocol adherence, data 
quality, and adverse events via e-mail. The DSMC will review all serious adverse events. Any 
serious adverse event will be reported to the DSMC, IRB, NIH, and FDA within 7 days of the 
PI’s notification of the event.  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) is life-threatening, c) requires inpatient hospitalization or 
prolongation of existing hospitalization, d) results in persistent or significant disability or 
incapacity, or e) is a congenital anomaly or birth defect. The DSMC may choose to become 
unblinded; however, it is expected that such unblinding would not occur without reasonable 
concern related either to patient safety or to data validity.   
Non-serious, unexpected adverse events will be reported to the IRB at the time of annual 
continuing review. If required, appropriate changes will be made to the consent form. Adverse 
events 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 protocol deviation will be reported at the time of annual continuing review.  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 and 
how each was handled. 
 
 

 
9.0 
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. 
 
 
10.0 Privacy/Confidentiality Issues 
 
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.   
We will use the web-based Vanderbilt Research Electronic Data Capture (REDCap) system to 
design electronic data collection forms. These forms will be pilot tested before use. Data will 
be input into a protected, web-based case report form (which can be readily downloaded 
into SAS, STATA, R, or SPSS). The form allows for direct data entry by investigators and is 
designed to minimize errors and erroneous values. Results from the Vanderbilt clinical 
laboratory can also be directly imported to REDCap, which further reduces typographical data 
entry errors. Expected ranges are pre-specified to prevent errors such as the shifting of 
decimal points. The program includes a computerized audit trail so that the identity of 
individuals entering or changing data and, in the case of changes, both original and revised 
data are saved. Data are backed up daily. Clinical data, including clinical laboratory, will be 
entered by the research nurse. Research laboratory data will be entered by a predoctoral 
student in the laboratory. 
 
 
11.0 Follow-up and Record Retention 
 
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.