Conditions: Insulin Sensitivity
Keywords: Insulin signaling and glucose disposal in muscle Hepatic insulin sensitivity TLR4 signaling and inflammatory gene expression Plasma cytokine concentration Intramyocellular diacylglycerol and ceramide content
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 15
Healthy volunteers: yes

Brief summary:
To determine whether pharmacologic inhibition of Toll-like receptor 4 (TLR4) with eritoran for injection (E5564) will reduce inflammation and improve glucose metabolism in insulin resistant (obese and T2DM) subjects.

Detailed description:
E5564 = Eritoran

Arms:
arm 1: Obese subjects receive IV administration of 5% Dextrose in water/D5W (vehicle) 12 mg every 12 hours arm 2: Obese subjects receive IV administration of Eritoran 12 mg every 12 hours arm 3: T2DM subjects receive IV administration of 5% Dextrose in water/D5W (vehicle) 12 mg every 12 hours arm 4: T2DM subjects receive IV administration of Eritoran 12 mg every 12 hours

Interventions: intervention 1: Eritoran intervention 2: D5W
Intervention descriptions: intervention 1: Pharmacologic inhibitor of TLR4 receptors. intervention 2: 5% Dextrose Water = Vehicle

Protocol document:
Prot_SAP_000.pdf: 
 
14-498H, Musi, Form BB v2.0, 02-10-16, AMD 
CONFIDENTIAL 
[Type here] 
COVER PAGE FOR PROTOCOL AND STATISTICAL ANALYSIS 
PLAN 
 
 
 
 
 
 
Official Study Title: The Effect of TLR4 Inhibition in Obese and 
Type 2 Diabetic Subjects (Eritoran2) 
 
NCT number: NCT02267317 
 
IRB Approval Date: 02-10-2016 
 
Unique Protocol ID: HSC20140498H 
 


NIH_ERIT-002 
 
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CLINICAL RESEARCH PROTOCOL 
 NIH_ERIT-002 
 
 
 
THE EFFECT OF TLR4 INHIBITION IN OBESE AND  
TYPE 2 DIABETIC SUBJECTS (NIH_ERIT-002) 
 
 
 
 
 
 
Regulatory Sponsor: 
Nicolas Musi, MD (Sponsor Investigator) 
Professor, Department of Medicine, Diabetes Division 
University of Texas Health Science Center at San Antonio 
       7703 Floyd Curl Dr. 
      Mail Code 7886 
      San Antonio, Texas 78229-3900 
      (210) 562-6140 
 
Funding Sponsor: 
US National Institutes of Health/National Institute of Diabetes 
and Digestive and Kidney Diseases (NIH-NIDDK)  
 
GRANT TITLE: ROLE OF TLR4 ON INSULIN RESISTANCE 
IN HUMAN SUBJECTS (R01DK080157) 
 
 
 
Study Product: 
ERITORAN (eritoran for injection, 6.5 mg) 
Protocol Number: 
NIH_ERIT-002  
IND Number: 
123955-0007 
IRB Tracking Number: 
20140498H 
NCT#: 
NCT02267317 
 
 
 
 
 
 
 
Initial version: 
v1.0_8.27.2014 
Amended  
v2.0_02-10-2016 
 
 
 
 


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                                                           TABLE OF CONTENTS 
 
STUDY SUMMARY ........................................................................................................................................... 1 
1. INTRODUCTION ........................................................................................................................................... 2 
1.1. 
BACKGROUND ..................................................................................................................................... 2 
1.2. 
INNOVATION ........................................................................................................................................ 4 
1.4. 
INVESTIGATIONAL AGENT..................................................................................................................... 9 
1.5. 
DOSE RATIONALE AND RISK/BENEFITS ................................................................................................. 9 
2. 
STUDY OBJECTIVE ............................................................................................................................ 9 
3. 
STUDY DESIGN ................................................................................................................................. 10 
3.2. 
GENERAL DESIGN ............................................................................................................................. 10 
3.3. 
STUDY ENDPOINTS ........................................................................................................................... 11 
3.4. 
POTENTIAL RISKS TO SUBJECT SAFETY ............................................................................................. 11 
4. 
SUBJECT SELECTION AND WITHDRAWAL .................................................................................. 12 
4.1. 
INCLUSION CRITERIA ......................................................................................................................... 12 
4.2. 
EXCLUSION CRITERIA ........................................................................................................................ 12 
4.3. 
SUBJECT RECRUITMENT AND SCREENING .......................................................................................... 13 
4.4. 
EARLY WITHDRAWAL OF SUBJECTS ................................................................................................... 13 
4.4.1. 
When and How to Withdraw Subjects ................................................................................... 13 
4.4.2. 
Data Collection and Follow-up for Withdrawn Subjects ......................................................... 13 
5. 
STUDY DRUG .................................................................................................................................... 13 
5.1. 
DESCRIPTION ................................................................................................................................... 13 
5.2. 
TREATMENT REGIMEN ....................................................................................................................... 14 
5.3. 
METHOD FOR ASSIGNING SUBJECTS TO TREATMENT GROUPS ............................................................ 14 
5.4. 
PREPARATION AND ADMINISTRATION OF STUDY DRUG ........................................................................ 14 
5.5. 
SUBJECT COMPLIANCE MONITORING ................................................................................................. 14 
5.6. 
PRIOR AND CONCOMITANT THERAPY ................................................................................................. 14 
5.7. 
PACKAGING ...................................................................................................................................... 15 
5.8. 
BLINDING OF STUDY DRUG ................................................................................................................ 15 
5.8.1. 
Receipt of Drug Supplies ....................................................................................................... 15 
5.8.2. 
Storage................................................................................................................................... 15 
5.8.3. 
Dispensing of Study Drug ...................................................................................................... 16 
5.8.4. 
Return or Destruction of Study Drug ...................................................................................... 16 
6. 
STUDY PROCEDURES ..................................................................................................................... 16 
6.1. 
VISIT 1 ............................................................................................................................................. 16 
6.2. 
VISIT 2 ............................................................................................................................................. 16 
6.3. 
VISIT 3 – INFUSION PROTOCOL .......................................................................................................... 16 
6.4. 
VISIT 4 ............................................................................................................................................. 16 
6.5. 
VISIT 5 ............................................................................................................................................. 18 
6.6. 
VISIT 6 ............................................................................................................................................. 18 
7. 
STATISTICAL PLAN .......................................................................................................................... 18 
7.1. 
SAMPLE SIZE DETERMINATION ........................................................................................................... 18 
7.2. 
STATISTICAL METHODS ..................................................................................................................... 19 
8. 
SAFETY AND ADVERSE EVENTS ................................................................................................... 19 
8.1. 
DEFINITIONS ..................................................................................................................................... 19 


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8.2. 
RECORDING OF ADVERSE EVENTS ..................................................................................................... 20 
8.3. 
REPORTING OF SERIOUS ADVERSE EVENTS AND UNANTICIPATED PROBLEMS ...................................... 21 
8.3.1. 
Investigator reporting: notifying the study sponsor ................................................................ 21 
8.3.2. 
Investigator reporting: notifying the UTHSCSA IRB .............................................................. 23 
8.3.3. 
Sponsor reporting: Notifying the FDA .................................................................................... 23 
8.4. 
MEDICAL MONITORING ...................................................................................................................... 24 
8.5. 
UNBLINDING PROCEDURES ................................................................................................................ 24 
8.5.1. 
Designated Safety Officer ...................................................................................................... 24 
8.5.2. 
VA Research Pharmacist ....................................................................................................... 24 
8.5.3. 
Sponsor Investigator .............................................................................................................. 24 
8.5.4. 
Clinical Study Coordinator ..................................................................................................... 24 
8.6. 
STOPPING RULES ............................................................................................................................. 25 
9. 
DATA HANDLING AND RECORD KEEPING ................................................................................... 25 
9.1. 
CONFIDENTIALITY .............................................................................................................................. 25 
9.2. 
SOURCE DOCUMENTS ....................................................................................................................... 25 
9.3. 
CASE REPORT FORMS ...................................................................................................................... 25 
9.4. 
DATA MANAGEMENT ......................................................................................................................... 26 
9.5. 
RECORDS RETENTION ....................................................................................................................... 26 
10. 
STUDY MONITORING, AUDITING, AND INSPECTING ................................................................... 26 
10.1. 
STUDY MONITORING PLAN ................................................................................................................ 26 
10.2. 
AUDITING AND INSPECTING ................................................................................................................ 27 
11. 
ETHICAL CONSIDERATIONS ........................................................................................................... 27 
12. 
STUDY FINANCES ............................................................................................................................ 28 
12.1. 
FUNDING SOURCE ............................................................................................................................. 28 
12.2. 
CONFLICT OF INTEREST ..................................................................................................................... 28 
12.3. 
SUBJECT STIPENDS OR PAYMENTS .................................................................................................... 28 
13. 
PUBLICATION PLAN ......................................................................................................................... 28 
14. 
REFERENCES ................................................................................................................................... 28 
15. 
ATTACHMENTS ................................................................................................................................. 28 
 
ATTACHMENT A. MATERIAL TRANSFER AGREEMENT ........................................................................... 30 
ATTACHMENT B. EISAI, INC., SERIOUS ADVERSE EVENT REPORTING FORM .................................... 31 
ATTACHMENT C. STUDY EVENT SCHEDULE ............................................................................................ 32 
ATTACHMENT D. SAMPLE CONSENT FORM ............................................................................................. 33 
ATTACHMENT E. REFERENCES .................................................................................................................. 34 
ATTACHMENT F. INVESTIGATOR BROCHURE .......................................................................................... 39 
ATTACHMENT G. GRADING SCALE FOR PHLEBITIS ............................................................................... 40 
ATTACHMENT H. SUMMARY OF PROTOCOL CHANGES ......................................................................... 41 
 


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List of Abbreviations 
 
 
ERK -  extracellular-signal related kinase  
FFA - free fatty acid(s) 
IKK - IB kinase  
JNK -  c-Jun N-terminal kinase 
LPS - Lipopolysaccharide 
MAPK - mitogen-activated protein kinases 
NFB – Nuclear Factor  B 
OGTT – Oral Glucose Tolerance Test 
T2DM – Type 2 Diabetes Mellitus 
TLR4 – Toll like receptor 4 
 
 
 


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       Study Summary 
Title 
The Effect of TLR4 Inhibition in Obese and Type 2 Diabetic Subjects 
Protocol Number 
NIH_ERIT-002 
Phase 
Phase II 
Methodology 
This is a double-blind, placebo-controlled, cross-over study.  Obese 
nondiabetic, obese type 2 diabetic, and lean normal glucose tolerant (control 
group) will receive the TLR4 inhibitor eritoran (E5564/eritoran for injection) or 
placebo (vehicle) I.V. for 72 h.  Insulin sensitivity will be assessed with insulin 
clamps.  Measurements of inflammation will be performed in blood, skeletal 
muscle and adipose tissue. 
Study Duration 
This study is funded by a 4 year NIH grant.  Each subject will undergo 6 study 
visits over a period ranging between 4 and 6 months.   
Study Center(s) 
Single-center: Audie L. Murphy VA Medical Center in San Antonio, TX 
Objective 
To determine whether pharmacologic inhibition of TLR4 with eritoran (E5564) 
improves/ameliorates baseline (pre-existing) measurements of inflammation 
and insulin resistance in obese nondiabetic and type 2 diabetic subjects. 
Number of Subjects 
Three groups: (i) Obese nondiabetic; (ii) Obese type 2 diabetic; and (iii) lean 
normal glucose tolerant (NGT). 
20 completers per group; (30 consented per group assuming ~30% screen 
failure or withdrawals) 
Inclusion Criteria 
1) One of these 3 groups: (i) obese (BMI = 30-37 kg/m2) normal glucose 
tolerant; (ii) obese (BMI = 30-37 kg/m2) T2DM subjects; or (iii) lean (BMI <26 
kg/m2) normal glucose tolerant; 2) Both genders; 3) Age = 18-65 years; 4) All 
ethnic groups; 5) Premenopausal women , non-lactating, and with a negative 
pregnancy test.  Postmenopausal women on stable dose of or not exposed to 
hormone replacement for ≥6 months; 6) Hematocrit ≥ 34%, serum creatinine ≤ 
1.4 mg/dl, normal electrolytes, urinalysis, and coagulation tests; Liver function 
tests up to 2X; In T2DM subjects, hemoglobin A1c should be ≤ 8.5%; 7) Stable 
body weight (±1.5%) for ≥ 3 months; 8) One or less sessions of strenuous 
exercise/wk for last 6 months. 
Exclusion Criteria 
1) Presence of glucose intolerance based on ADA criteria (lean/obese NGT); 
diabetics with HgA1c ≥8.5% will be excluded.  2) Current treatment with drugs 
known to affect glucose and lipid homeostasis; The only allowed antidiabetic 
agents will be sulfonylureas and metformin. Subjects on a stable dose of statin 
(> 3 months) are eligible; 3) Non-steroidal anti-inflammatory drugs or systemic 
steroid use for more than a week within 3 months; 4) Current treatment with 
anticoagulants (warfarin).  Aspirin (up to 325 mg) and clopidogrel will be 
permitted if they can be held for seven days prior to the biopsies, in 
accordance with the primary physician; History of coagulopathy will be 
excluded. 5) History of heart disease (New York Heart Classification greater 
than class II; more than non-specific ST-T wave changes on the ECG), 
peripheral vascular disease, pulmonary disease, smokers; 6) Poorly controlled 
blood pressure (systolic BP>160, diastolic BP>90 mmHg); 7) Active 
inflammatory, autoimmune, infectious, hepatic, gastrointestinal, malignant, 
and/or psychiatric disease. 
Study Product, Dose, 
Route, Regimen 
Eritoran for injection, 12 mg intravenously (I.V.) every 12 h. Total of 6 doses 
(72 mg) 
Duration of 
administration 
72 h  
Statistical 
Methodology 
Means between treatment groups will be compared using a repeated-
measures General Linear Model (GML) analysis. 


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1. Introduction 
 
This document is a protocol for a human research study. This study is to be conducted according to Good 
Clinical Practice guidelines as adopted by FDA, applicable government regulations, and Institutional 
research policies and procedures.  
1.1. 
Background 
Molecular biology of insulin resistance. Insulin resistance at the level of skeletal muscle is one of the 
earliest abnormalities in the pathogenesis of type 2 diabetes (T2DM). A wide array of abnormalities 
distinguishes muscle of obese and T2DM subjects from muscle of lean non-diabetic individuals. Defects 
include reductions in insulin-stimulated IRS-1 tyr phosphorylation, PI 3-kinase activation, Akt and AS160 
phosphorylation, and GLUT4 translocation (1, 2). These molecular abnormalities are strongly correlated 
with reduced glucose disposal (1, 3). Therefore, interventions designed to improve muscle insulin 
resistance are likely to be effective in preventing and treating T2DM. The liver also is a key organ for 
maintenance of normal glucose homeostasis, and hepatic insulin resistance is an early event in the natural 
history of T2DM (4). Improving hepatic insulin sensitivity also is a key strategy for diabetes prevention and 
treatment. 
TLR4 signaling. Toll-like receptors (TLRs) are cell-surface receptors that generate immune responses to 
pathogens by activating a cascade of inflammatory events (5). TLR4, one of the best characterized TLRs, 
is highly expressed in immune cells (monocytes, macrophages), and insulin target cells including 
myocytes, adipocytes and hepatocytes. Lipopolysaccharide (LPS or endotoxin), an outer membrane 
component of Gram (-) bacteria is a potent agonist of TLR4 (6). The soluble protein LPS-binding protein 
(LBP) binds LPS in plasma and regulates LPS-dependent responses. As discussed in more detail below, in 
addition to LPS, saturated free fatty acids (FFA) from a nutritional/metabolic source also can function as 
TLR4 agonists (7, 8).   
       The structure of LPS is shown below (Figure 1). The lipid A moiety within LPS is a 1-6-linked 
disaccharide of glucosamine, acylated with R-3-hydroxylaurate or myristate. The 3-hydroxyl groups of these 
saturated fatty acids are further 3-O-acylated by lauric acid, myristic acid, or palmitic acid (6); if lipid A is 
deacylated it loses its endotoxic properties and acts as an antagonist against lipid A (9).  This implies that 
fatty acids acylated in lipid A are key for ligand 
recognition and TLR4 activation.    
Upon stimulation of TLR4 and its coreceptors, CD14 
and MD-2, the adaptor proteins TIRAP and MyD88 are 
recruited to the TIR domain of TLR4 (10). This 
association leads to the stimulation of inflammatory 
kinases, including the mitogen-activated protein kinases (MAPK); c-Jun 
N-terminal kinase (JNK), p38, and extracellular-signal related kinase 
(ERK), and the IB kinase (IKK) complex, which then results in the activation of transcription factors such 
as nuclear factor-B (NFB), c-jun, and activator protein-1 (AP-1) (10). These inflammatory pathways have 
been linked to insulin resistance at a number of levels. Activation of JNK (11) and IKK (12) serine 
phosphorylates IRS-1, inhibiting its capacity to associate with PI-3 kinase. IKK also phosphorylates IB, 
causing translocation of NFB into the nucleus and gene transcription of inflammatory proteins such as 
tumor necrosis factor-α (TNFα), inducible nitric oxide synthase (iNOS), monocyte chemotactic protein 
(MCP)-1 and interleukin (IL)-6 (13). Concordantly, inhibition of either JNK or IKKβ/NFB can improve 
insulin sensitivity (14-16). These findings provide evidence that the insulin signaling cascade is negatively 
regulated by TLR4. 
Role of TLR4 on lipid-induced insulin resistance. Several lines of evidence implicate a deleterious 
effect of lipids (i.e. FFA) on insulin sensitivity. For example, most obese and T2DM subjects have elevated 
plasma FFA level that correlate with the severity of insulin resistance (17). The increase in plasma FFA is 


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caused by enhanced lipolysis, decreased lipid oxidation, and increased fat mass. In insulin resistant 
subjects, an acute reduction in plasma FFA with an antilipolytic drug (acipimox) significantly improves 
insulin sensitivity (18, 19), whereas an experimental elevation of plasma FFA in normal glucose-tolerant 
subjects rapidly induces insulin resistance (20, 21). Despite the considerable evidence implicating 
nutritional FFA in the pathogenesis of insulin resistance, how FFA impair insulin action, particularly in 
humans, is not well understood.    
     Based on the finding that saturated FA contained in lipid A moiety of LPS are essential for its biologic 
activity (9), Lee et al tested whether FFA from a nutritional/metabolic source have an LPS-like effect to 
directly activate TLR4. This group demonstrated that similar to lipid A, saturated FFA are potent activators 
of TLR4 in monocytes (7). In contrast, unsaturated FFA antagonize the effect of saturated FFA on TLR4 
(22). In L6 myotubes, inhibition of TLR4 protects against the detrimental effect of FFA on insulin action (23, 
24). During this funding cycle, our group showed that acute palmitate treatment induces robust NFB 
activation via TLR4 in human myotubes (25). Studies in mice have shown that disrupted TLR4 function 
protects against acute and chronic fat-induced impairments in insulin action in vivo (26, 27). In general, 
studies have shown a protective effect of TLR4 inhibition against lipid-induced insulin resistance in muscle 
(26, 28). Some (26, 28), albeit not all (29) mouse studies that investigated the effect of TLR4 disruption 
also have revealed protection at the level of the liver. 
     The findings that saturated FFA can mimic the effect of LPS and activate TLR4 led us to evaluate during 
this funding period whether TLR4 expression and function are altered in insulin resistant subjects. We 
observed increased gene expression and protein content of TLR4 in muscle of obese and T2DM subjects, 
which inversely correlated with insulin sensitivity (25). We and others also demonstrated that TLR4 
expression is elevated in mononuclear cells from insulin resistant subjects (30, 31). These data, along with 
the findings that insulin resistant subjects have increased plasma FFA concentration (17), and that FFA 
can stimulate TLR4 (7), suggest that TLR4 activation may be an important mechanism by which nutritional 
FFA impair insulin action.  
Metabolic regulation by the microbiome. Initial research regarding the role of TLR4 in the pathogenesis 
of insulin resistance was conducted under the premise that saturated FFA from a nutritional/metabolic 
origin have an LPS-like effect to induce an inflammatory response. Notably, in the last ~3 years an 
increasing number of studies (some from our group) suggest that LPS produced by the gastrointestinal 
flora, per se, also could be a causative factor in human metabolic disease. The human gut hosts an 
enormous number and variety of microorganisms. This microbial community (microbiome) is considered to 
be a possible causative factor of metabolic disorders (32). In line with this, administration of non-
absorbable antibiotics, which reduce gut flora number, improves systemic inflammation and glucose 
metabolism in ob/ob and high fat fed mice (33, 34). The mechanism by which gut microbiota regulates 
glucose metabolism is not clear. One hypothesis, discussed in more detail below, involves bacterial 
production of LPS (i.e. metabolic endotoxemia). Other potential mechanisms include the production of 
substances that alter intestinal wall integrity, food energy extraction, energy expenditure, and appetite (34-
36),   
Metabolic endotoxemia and insulin resistance. Accumulating evidence suggests that chronic elevation 
of circulating intestinal-generated LPS (i.e. metabolic endotoxemia) could play an important role in the 
pathogenesis of insulin resistance. We (37) and others (38, 39) have demonstrated that obese and T2DM 
subjects have elevated LPS concentration in plasma, in association with the severity of insulin resistance 
(37).  Unlike other situations of endotoxemia, such as sepsis where plasma LPS level may increase ~100-
fold (40), in situations of insulin resistance (obesity, T2DM) LPS levels are only modestly increased by ~2-
3-fold. The cause of metabolic endotoxemia in obese and T2DM subjects is not clear. Ghanim et al. 
reported that a high calorie diet (high in fat and carbohydrate content) caused a significant elevation in 
plasma LPS, accompanied by increased TLR4 and NFB expression in mononuclear cells (41, 42). In 
addition, a dietary survey conducted in 1,015 subjects demonstrated that high fat intake was directly 
associated with plasma LPS level (43). Postulated mechanisms by which fat intake may increase plasma 
LPS include (i) changes in the composition of the microbiome (33, 34, 44, 45); (ii) lipid-induced damage of 
the intestinal barrier (44), (iii) and chylomicron-mediated transport of LPS (46).   


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       The observation that plasma LPS concentration is elevated during states of insulin resistance has 
prompted studies into the effect that LPS level modulation has on insulin sensitivity. As mentioned above, 
reducing LPS levels with non-absorbable antibiotics improves glucose metabolism in insulin resistant mice 
(33, 34). In line with this finding, intravenous low-dose LPS administration to healthy individuals rapidly 
induces insulin resistance (47). Collectively, these data suggest that metabolic endotoxemia could play an 
important role in the pro-inflammatory state and insulin resistance that occurs in obese and T2DM subjects.   
1.2. 
Innovation 
 Insulin resistant subjects have increased TLR4 expression and signaling in insulin sensitive tissues 
(muscle) (25) and peripheral mononuclear cells (30, 31). Moreover, the plasma concentration of the TLR4 
ligands, FFA (17) and LPS (37, 48, 49), is increased in these subjects. This suggests that activation of 
TLR4 by FFA and LPS may be a key mechanism underlying the inflammatory state and insulin resistance 
of obesity and T2DM. Yet, there is no direct in vivo data demonstrating a role for TLR4 in the 
pathogenesis of insulin resistance in human subjects. In this study we will examine the effect of a 
selective TLR4 antagonist, with the goals to (i) establish the role that TLR4 signaling plays in the 
pathogenesis of insulin resistance and T2DM in humans; and (ii) elucidate the mechanism by which TLR4 
mediates insulin resistance in human subjects. This is the first human study to determine the cellular 
and molecular effects of a TLR4 antagonist in insulin sensitive tissues (muscle, fat) in vivo, and to 
examine how TLR4 inhibition affects whole body glucose metabolism. Figure 2 below outlines the 
proposed model by which TLR4 mediates insulin resistance 
and summarizes the rationale behind this application. 
 
 
 
 
 
 
 
  
 
 
 
1.3.  Preliminary data. Below is a description of preliminary data which lay the foundation for the 
proposed research. 
a) Insulin resistant subjects have abnormal TLR4 expression and TLR4-driven signaling in skeletal 
muscle. Considering that animal models of insulin resistance are characterized by increased TLR4 
expression (26), we examined whether insulin resistant subjects also have alterations in the expression 
and function of this receptor. We demonstrated that obese and T2DM subjects have increased TLR4 gene 
expression (Fig. 3A), TLR4 protein content (Fig. 3B) and inflammatory gene (IL-6) expression (Fig. 3C) in 
muscle, indicative of increased TLR4-driven signaling (25). 
       
 
 
 
 
 
Fig. 3A 
Fig. 3B 
Fig. 3C 
Figure 2. Working Model of TLR4- mediated insulin resistance. 
In insulin-resistant states (obesity, T2DM) there is an increase in 
the plasma concentration of two TLR4 ligands, LPS and FFA. 
LPS is elevated due to increased intestinal permeability and  
chylomicron-mediated transport.  FFA are elevated due to  
lipolysis and adipose tissue expansion. 
 
UponTLR4 stimulation, the IKK/NFkB and MAPK pathways are 
stimulated.  These inhibit insulin signaling at the level of IRS-1 
and promote gene transcription of pro-inflammatory proteins that 
also impair insulin action. 
 
This protocol (NIH_ERIT-002) will test whether a selective TLR4 
antagonist will improve/ameliorate baseline (pre-existing) inflammation  
insulin resistance. 


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b) An experimental elevation in circulating FFA increases TLR4 expression/signaling in muscle. We 
showed that saturated FFA increase TLR4 expression and signaling in primary human myotubes (25). To 
determine whether lipids also promote an inflammatory 
response in humans in vivo, we administered a continuous (48 
h), low dose lipid infusion (Intralipid 30 ml/h) to 14 lean normal 
glucose-tolerant subjects. As a control experiment, on a 
different occasion the same subjects received saline. The lipid 
infusion raised mean plasma FFA concentration from 355 to 
484 M [a typical plasma FFA level observed in insulin 
resistant subjects (450-600 
M)] and reduced peripheral 
insulin sensitivity (assessed 
by 
hyperinsulinemic 
euglycemic clamp) by 17%. 
Molecular analysis of vastus 
lateralis 
muscle 
demonstrated that raising 
FFA 
upregulated 
the 
expression 
of 
TLR4 
by 
~50% (50). Lipid infusion also increased the expression of various other genes within the TLR pathway 
(Fig. 4A) and impaired insulin-mediated IRS-1 tyr phosphorylation (Fig. 4B) (50). Notably, mass 
spectrometry-based analysis revealed that the total (unfractionated) muscle content of several intracellular 
lipid species (ceramides, diacylglycerols, acylcarnitines) was unaffected by Intralipid (50). These data 
demonstrate that a modest increase in plasma FFA is sufficient to upregulate the TLR4 pathway (and 
induce insulin resistance) in healthy subjects. 
c) TLR4 mediates FFA-induced insulin resistance in myotubes. To evaluate whether the inflammatory 
response caused by FFA is mediated via TLR4, we tested the effect of the TLR4 inhibitor TAK-242 
(Takeda Pharm.) on FFA-induced NFB p65 and JNK phosphorylation, and IL-6 expression in L6 muscle 
cells (23). Stearate (400 M for 6 h), a saturated FFA, increased NFB p65 phosphorylation (Fig. 5A), JNK 
phosphorylation (Fig. 5B), and IL-6 mRNA level (Fig. 5C). These inflammatory responses were significantly 
reduced by TAK-242, although the inhibition of NFB and JNK phosphorylation was partial.  
                                 
    
           
 
 
 
 
 
 
 
 
 
Fig. 5A 
Fig. 5B 
Fig. 5C 
Fig. 4A  
Fig. 4B  


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Fig. 7A 
We also tested the effect of TAK-242 on the insulin transduction pathway and glucose transport in L6 
myocytes (insulin was dosed 10 nM for 5 min and 100 nM for 20 min, respectively) (23). Stearate (400 M 
for 6 h) reduced insulin-stimulated Akt phosphorylation (Figs. 6A and 6B). Notably, the reduction in Akt 
phosphorylation was no longer observed when cells were pre-treated with TAK-242. Stearate (400 M for 6 
h) also reduced insulin-stimulated glucose transport (Fig. 6C). TAK-242 restored glucose transport, such 
that the reduction by stearate was no longer statistically significant. These in vitro studies demonstrate that 
pharmacologic TLR4 inhibition ameliorates lipid-induced insulin resistance. 
 
 
 
 
 
d) Elevated TLR4 content in peripheral blood mononuclear cells (PBMC) from insulin resistant 
subjects. Immune cells can infiltrate insulin sensitive tissues 
(muscle, liver, fat) where they induce an inflammatory response that 
impairs insulin action. We examined whether TLR4 content/signaling is 
altered in PBMC from insulin resistant subjects. As shown in Figure 7, 
reproduced from (31), PBMC from obese and T2DM subjects have 
increased 
TLR4 
expression 
(Fig. 
7A) 
and 
signaling 
(ERK 
phosphorylation) (Fig. 7B).                                                 
 
 
e) Lipid infusion increases TLR4 expression/signaling and TLR4-regulated cytokines. We have 
demonstrated that exposure to lipids increases TLR4 expression/signaling in human muscle, both in vitro 
(25) and in vivo (50). Next, we examined whether the increases in TLR4 expression and signaling 
observed in immune cells from insulin resistant subjects also are caused by elevated FFA. Using flow 
cytometry we measured TLR4 cell surface expression and MAPK phosphorylation in blood monocytes from 
the lean normal-glucose tolerant subjects described above (Fig. 4)  who received Intralipid/saline for 48 h. 
Infusion of lipid (but not saline) significantly increased TLR4 expression (Fig. 8A) and the phosphorylation 
of JNK  (Fig. 8B) and p38 (Fig. 8C). Notably, Intralipid also enhanced LPS-induced JNK phosphorylation 
when blood monocytes were exposed to LPS (1 g/ml) ex vivo (Fig. 8D), suggesting that lipids “prime” the 
monocytes to endotoxin.   
 
 
 
 
Figure 8.  Effect of saline/lipid infusion on TLR4 cell surface expression and MAPK phosphorylation in monocytes. A: TLR4 cell surface     
expression. B: Basal JNK phosphorylation. C: Basal  p38 phosphorylation. D: LPS-stimulated JNK phosphorylation.  *P<0.05 vs. Saline 
f) Lipid infusion increases plasma cytokine concentration. As shown in Figure 9, the plasma TNF 
concentration was significantly elevated after the infusion of Intralipid, but not saline. Interestingly, the 
plasma level of other cytokines such as IL-6 and MCP-1 was not different after lipid infusion, suggesting a 
selective 
(i.e. 
regulated) 
effect 
of 
the 
lipid 
infusion 
on 
cytokine 
production/release. 
 
 
Fig. 6A 
Fig. 6C 
7B 
Fig. 8 
Fig. 9 
Fig. 6B 


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g) Plasma LPS concentration is increased in obese and T2DM individuals. We measured LPS 
concentration in plasma from 11 lean, 9 obese nondiabetic, and 10 obese T2DM subjects. Insulin 
sensitivity (M) was measured with a euglycemic clamp. As shown in Fig. 10A, plasma LPS concentration 
was significantly elevated in the obese and T2DM subjects. Furthermore, there was a negative correlation 
between LPS concentration and insulin sensitivity (10).        
 
 
 
 
 
 
 
h) LPS impairs insulin action ex vivo. We utilized an isolated muscle preparation to evaluate the effect of 
LPS on insulin signaling and glucose (2DG) transport ex vivo. Rat epitrochlearis muscles were incubated 
for 4 h with LPS (100 ng/ml or 1 μg/ml), followed by 50 mU/ml insulin (20 min). A shown in Figure 11, LPS 
reduced insulin signaling (Fig. 11A) and glucose transport (Fig. 11B) in a dose-dependent manner.   
 
 
 
 
 
 
 
 
 
 
 
We tested the effect of the TLR4 inhibitor TAK-242 on LPS-induced NFB p65 and JNK phosphorylation in 
L6 muscle cells (Fig. 12). LPS (100 ng/ml for 1 h) increased NFB (Fig. 12A) and JNK phosphorylation 
(Fig. 12B) and these responses were completely inhibited by TAK-242 (1 M) (23). In line with these 
findings, TAK-242 prevented LPS-mediated impairments in insulin-stimulated Akt phosphorylation (Fig. 
12C) and glucose transport (Fig. 12D) (23).                                 
    
           
 
 
 
 
 
 
 
i) Pharmacologic inhibition of TLR4 improves peripheral insulin resistance in vivo. To examine 
whether pharmacologic inhibition of TLR4 improves glucose metabolism in vivo, we tested the effect of the 
competitive TLR4 antagonist eritoran (E5564) (kindly provided by Eisai Pharmaceuticals, Andover, MA) on 
lipid-induced insulin resistance using a 2-step euglycemic clamp with tritiated glucose, as described (51). 
Fig. 10B 
Fig. 10A 
12B 
12C 
12D 
12A 
Insulin (50mU)  -
-
-
-
+    +    +    +    +
LPS  (1g/ml)    -
-
-
-
-
-
+    +    +
p-Akt
Akt
p-GSK3α
GSK3α
GSK3β
p-GSK3β
Insulin (50mU)  -
-
-
-
+    +    +    +    +
LPS  (1g/ml)    -
-
-
-
-
-
+    +    +
p-Akt
Akt
p-GSK3α
GSK3α
GSK3β
p-GSK3β
Fig. 11A 
Fig. 11B 


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Fig. 13 
Male Sprague-Dawley rats (~200 g) were pre-treated with eritoran (5 mg/kg) or vehicle through an arterial 
catheter.15 min after eritoran administration, Intralipid 20% (8.5 mg/kg.min) or saline were infused for 8 h. 
~4 h after the first dose, a second dose of eritoran (5 mg/kg) or vehicle was given. The clamp was started 
15 min after the second dose (time +4h) of eritoran/vehicle. During Step 1 (0-120 min), insulin was infused 
at 0.4 mU/kg.min to measure hepatic insulin sensitivity [suppression of endogenous glucose production 
(EGP)]. During Step 2 (120-240 min), insulin was infused at 4 mU/kg.min to measure peripheral (muscle) 
insulin sensitivity. A variable dextrose infusion was administered to maintain glucose at 5 mM. 
Somatostatin (3 g/kg.min) was infused during the clamp to suppress endogenous insulin release. The 
protocol is shown in Figure 13. 
  
Figure 14A below shows the glucose infusion rate (GIR) 
required to maintained euglycemia during the clamp. The mean GIR 
during the last 30 min of Phase 2 (210-240 min), an indication of peripheral (muscle) 
insulin sensitivity, was reduced by the lipid infusion, and the administration of eritoran 
significantly ameliorated the reduction in GIR caused by the lipid (Fig. 14A). Consistent with these findings, 
eritoran reversed lipid-induced reductions in peripheral (muscle) glucose disposal during Phase 2 of the 
clamp (Fig. 14B). 
 
 
 
 
 
 
 
 
 
j) Eritoran ameliorates lipid-induced insulin resistance in the liver. Figure 15 shows the effect of 
eritoran 
(E5564) 
on 
lipid-induced 
hepatic 
insulin 
resistance. As expected, in saline-infused rats insulin 
caused a dose-dependent suppression of EGP (from Step 
1 to Step 2). However, insulin was unable to inhibit EGP in 
lipid-treated rats. Notably, eritoran restored the ability of 
insulin to suppress EGP 
In summary, our preliminary data demonstrate that 
pharmacologic inhibition of TLR4 protects against lipid- 
and LPS-induced insulin resistance in vitro. Moreover, 
pharmacologic blockade of TLR4 with eritoran in rats 
partially reverses the deleterious effect of a lipid 
infusion in both the periphery (muscle) and liver. 
Based upon these exciting findings obtained from pre-
clinical studies, a proof-of-concept experiment is 
proposed to test the hypothesis that pharmacologic inhibition of TLR4 will reduce inflammation and improve 
baseline (i.e. pre-existing) impairments in glucose metabolism in insulin resistant (obese and T2DM) 
subjects. 
 
Saline-Vehicle   Intralipid-Vehicle   Intralipid+E5564 
Fig. 14A 
Fig. 14B 


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1.4. 
Investigational Agent 
 
Eritoran (E5564) is a drug originally developed by Eisai Pharmaceuticals (eritoran tetrasodium, currently 
labeled “eritoran for injection”) for the treatment of sepsis.  Eritoran is a synthetic analogue of Lipid A, the 
toxic part of endotoxin (also known as lipopolysaccharide, LPS). Eritoran has been shown to block the 
effects of LPS in vitro, in vivo, and in humans. The mechanism of action of eritoran is competitive inhibition 
of the binding of LPS to its functional receptor complex of TLR4 and co receptor MD2 (52-55).  The 
pharmacokinetic and pharmacodynamic properties and safety profile of eritoran have been studied 
extensively during phase 1, 2 and 3 experimental studies of endotoxemia in healthy volunteers, patients 
with severe sepsis, and patients with impaired hepatic function (55-58). Eritoran is safe and well tolerated, 
even at the highest doses tested (55).  We have confirmed the ability of eritoran to improve glucose 
metabolism in lipid-treated rats (Preliminary Data; Figures 14-15) and to suppress LPS- and stearate-
mediated MAPK activation in primary human myotubes (Figure 16). 
 
                                                                       
 
 
 
 
 
 
 
1.5. Dose Rationale and Risk/Benefits 
Eritoran (E5564) has a half-life of ~25-30 h and pharmacodynamic studies have shown that ~12 h after 
eritoran administration (12 mg), the inhibitory effect of the drug on TLR4 signaling is fully maintained (59).  
This dose (12 mg) is safe and well tolerated, and higher doses do not confer further inhibitory effect on 
TLR4 (59).   
This study is not intended to directly benefit the subject participants.  The proposed research is expected 
to expand our knowledge on the pathophysiology of insulin resistance and T2DM.  This enhanced 
knowledge may lead to the development of novel treatments for these disorders. 
The pharmacokinetic and pharmacodynamic properties and safety profile of eritoran have been studied 
extensively during phase 1, 2 and 3 experimental studies of endotoxemia in healthy volunteers, patients 
with severe sepsis, and patients with impaired hepatic function [57-60]. Eritoran is safe and well tolerated, 
even at the highest doses tested [57]. The only adverse event that occurred at a significantly higher rate 
with eritoran was phlebitis, although phlebitis occurred mainly at the highest doses tested (higher than 
what is proposed in this study) and during continuous infusion.  Phlebitis was minimal with intermittent 
administration (as given in this study) and was transient.  Risks will be minimized by a careful screening 
process including medical history, physical exam, and review of complete blood count, coagulation tests, 
chemistry and electrocardiogram.  The presence of experienced personnel, and of at least one physician, 
at all times is also critical to reducing risks. 
2. Study Objective 
 
To determine whether pharmacologic inhibition of TLR4 with eritoran will reduce 
inflammation and improve glucose metabolism in insulin resistant (obese and T2DM) 
subjects.  Rationale. Insulin resistant subjects have increased TLR4 expression and signaling (MAPK, 
NFB) in insulin sensitive tissues (25) and inflammatory cells (25, 30, 31). Moreover, the plasma 
concentration of two endogenous TLR4 ligands, FFA (17) and LPS (37, 48, 49), is increased in these 
Fig.16A 
Fig.16B 


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individuals. This suggests that activation of TLR4 by FFA and LPS may be a mechanism underlying the 
inflammatory state and insulin resistance of obesity and T2DM. Yet, the relevance of TLR4 to the etiology 
of human metabolic disease is not known. In this Aim, we will test the hypothesis that TLR4 plays a direct 
role in the pathogenesis of insulin resistance in humans. Thus, we will examine whether blocking TLR4 
with eritoran ameliorates the inflammatory state and improves glucose metabolism in insulin resistant 
(obese and T2DM) subjects. This is the first human study to determine the molecular effects of a 
TLR4 antagonist in insulin sensitive tissues (muscle, fat) in vivo, and to examine how TLR4 
inhibition affects whole body glucose metabolism. 
3.  Study Design 
3.1.     Subjects 
20 obese (BMI = 30-37 kg/m2) normal glucose-tolerant, 20 obese (BMI = 30-37 kg/m2) T2DM subjects, 
and 20 lean (BMI <26 kg/m2) normal glucose-tolerant (control) subjects will be enrolled (~50% males). 
Subjects will be recruited through local advertisement and through the Diabetes and General Medicine 
Clinics of the Audie L. Murphy Division of the STVHCS and the Texas Diabetes Institute in San Antonio, 
TX.  ~90 subjects will be consented assuming ~30% screen failure or withdrawals. 
3.2. 
General Design 
The study is designed as a Phase II, randomized, controlled, interventional study. Enrollment duration is 
planned over a 4-year period.  Each subject will undergo 6 study visits over a period of 4-6 months.    
 
Study Design.  
 
Following a screening period, which can be up to  three months, each subject will be admitted on 2 
different occasions (separated by 4-10 weeks each) to receive either eritoran or vehicle (in random order) 
for 72 h. Studies will be performed in the Bartter Research Unit (BRU), located in the Audie L. Murphy VA 
Medical Center in San Antonio, TX. 
 
 
 
 
 
 
 
 
 
 
 
 


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3.3. 
Study Endpoints 
Primary outcome: Effect of eritoran on muscle insulin action (insulin signaling and peripheral glucose 
disposal) and hepatic insulin sensitivity (insulin suppression of EGP) in obese and T2DM subjects. 
 
Secondary outcomes:  Effect of eritoran on:  
- Inflammatory signaling in muscle and adipose tissue: TLR4 signaling and inflammatory gene 
expression 
- Muscle and adipose tissue macrophage infiltration.   
- TLR4 expression, NFB and MAPK activation/phosphorylation on peripheral blood monocytes.  
- Plasma cytokine concentration.  
- Intramyocellular diacylglycerol and ceramide content (membrane and cytoplasm fractions). 
3.4. 
Potential Risks to Subject Safety 
 
a) Drug Administration. 
Insulin and Glucose:  Since the glucose infusion is designed to counterbalance the metabolic effects 
of insulin, the risk of hypoglycemia is very low.  No other side effects of insulin and glucose are 
known. Plasma glucose concentration will be determined at 5 min intervals throughout the period of 
insulin/glucose administration. 
Eritoran: Six (6) phase I studies evaluating the safety of eritoran in healthy subjects have been 
conducted (55-60).  The only adverse event that occurred at a significantly higher rate with eritoran 
was phlebitis, although phlebitis occurred mainly at the highest doses tested (higher than what is 
proposed in this study) and during continuous infusion. Phlebitis was minimal with intermittent 
administration (as given in this study) and was transient.   
The pharmacokinetic and pharmacodynamic properties and safety profile of eritoran have been 
studied extensively during phase 1, 2 and 3 experimental studies of endotoxemia in healthy 
volunteers, patients with severe sepsis, and patients with impaired hepatic function (55-58).  
Eritoran is safe and well tolerated, even at the highest doses tested (55). 
 
b) Blood withdrawal.  All studies involve the withdrawal of blood. Any subject who has donated blood in 
the previous two months will not be studied. The subjects will be instructed not to donate blood for 
two months after the study.  Any subject with a hematocrit of less than 34% will not be studied.  The 
maximal amount of blood to be drawn during the entire study for any given subject will be 
approximately  534 mL. Hematology comprehensive metabolic panel with lipids and liver function 
tests will be obtained at the Infusion Visit to assess effects on blood cell count, liver function and 
lipid metabolism. 
 
c) IV lines.  Intravenous catheters will be used for infusions and venipuncture.  Local hematomas 
occur in about 1% of catheterizations.  Infection is possible (<1%), but we have not experienced this 
complication.  One instance of thrombophlebitis has been observed (<1%).  The hand with the 
catheter will be placed in a warm (55o C) transparent plastic box to arterialize venous blood.  We 
have observed one instance of skin burning (2nd degree) using these boxes (<0.01%). 
 
d) Muscle biopsy.  At the time of biopsy, subjects may feel pain, discomfort, or pressure (variably 
described by different subjects) for about 5-10 seconds.  Pain or discomfort ceases as soon as the 
cannula is withdrawn.  Local hematomas occur in <2% of subjects.  One patient experienced a 
moderately painful hematoma that resolved within 2 weeks (0.1%).  About 1 in 50 subjects report 
non-clinically evident numbness or altered sensation at the biopsy site, which is transient.  
 


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There is the possibility that a future biopsy may not be done at the discretion of the PI in case the 
subject did not tolerate well a prior biopsy. 
 
e) Adipose tissue biopsy.  A burning discomfort will be felt for about 5-10 seconds during the 
application of lidocaine.  Mild wound infections occur in <2% subjects.   
 
f) Subjects will be exposed to radioactivity during these studies.  The radiation exposure from tritiated 
glucose and the DEXA is well within guidelines (Shreve WW et al. Proc 2nd International Conference 
on Peaceful Uses of Atomic Energy, Geneva, 1958; U.S. Department of Commerce National 
Bureau of Standards Handbook 69, 1969).  Subjects will also be exposed to a very small amount of 
radiation during the DEXA exam (0.04 mrem), which equals one day of natural background 
radiation.  The cumulative radiation exposure is well within the dose range (1,000 Ci) approved by 
the Hospital Radiation Committee. The local VA Radiation Committee will approve these studies 
before anyone is enrolled.  At these low exposures, risk is minimal. 
4. Subject Selection and Withdrawal 
4.1. 
Inclusion Criteria 
1) Subjects capable of giving informed consent. 
2) One of these 3 groups: (i) obese (BMI = 30-37 kg/m2) normal glucose tolerant; (ii) obese (BMI = 
30-37 kg/m2) T2DM; or (iii) lean (BMI <26 kg/m2) normal glucose tolerant. Lean subjects have 
no family history of diabetes. 
3) Both genders. 
4) Age=18-65 years. Older subjects are excluded because aging is a pro-inflammatory state. 
5) All ethnic groups. 
6) Premenopausal women, non-lactating, and with a negative pregnancy test. Postmenopausal 
women on stable dose of or not exposed to hormone replacement for ≥6 months. 
7) Lab: Hematocrit ≥34%, serum creatinine ≤1.4 mg/dl, normal electrolytes, urinalysis, and 
coagulation tests. Liver function tests up to 2X normal range. 
8) Stable body weight (±1.5%) for ≥3 months. 
9) One or less sessions of strenuous exercise/wk for last 6 months. 
4.2.      Exclusion Criteria 
1) For non-diabetic subjects, the presence of glucose intolerance on OGTT based on American 
Diabetes Association (ADA) criteria will be exclusionary 
2) Current treatment with drugs known to affect glucose and lipid homeostasis. Subjects on a 
stable dose of statin (>3 months) are eligible. Subjects on sulfonylureas and/or metformin are 
eligible.  
3) Hemoglobin A1c ≥ 8.5% (diabetic subjects only). 
4) Non-steroidal anti-inflammatory drugs or systemic steroid use for more than 1 week within 3 
months 
5) Current treatment with anticoagulants (warfarin). Aspirin (up to 325 mg) and clopidogrel will be 
permitted if these can be held for seven days prior to the biopsies. History of coagulopathy will 
be excluded. 
6) History of heart disease (New York Heart Classification greater than class II; more than non-


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specific ST-T wave changes on the ECG), peripheral vascular disease, pulmonary disease, 
smokers. 
7) Poorly controlled blood pressure (systolic BP>160, diastolic BP>90 mmHg). 
8) Active inflammatory, autoimmune, infectious, hepatic, gastrointestinal, malignant, and 
psychiatric disease. 
4.3. 
Subject Recruitment and Screening 
Subjects will be recruited by advertisement and through the outpatient clinics of the South Texas 
Veterans Health Care System and the Texas Diabetes Institute (TDI) in San Antonio, TX. Every month, 
~25 newly diagnosed T2DM patients are referred to the TDI.  
 
Screening will be performed during Visits 1 and 2.  On Visit 1, following an overnight fast (no food after 9 
PM) subjects will come to the BRU at approximately 7 AM for consenting, followed by screening 
procedures including a medical history, physical exam, and screening tests (CBC, chemistry, lipids, 
HbA1c, PT, PTT, urinalysis and ECG). For subjects who are eligible based on these screening tests, an 
oral glucose tolerance test (OGTT) will be performed on Visit 2.   
4.4. 
Early Withdrawal of Subjects 
4.4.1. When and How to Withdraw Subjects 
Subjects have the right to withdraw fully or partially from the study at any time and for any reason without 
prejudice to his or her future medical care by the physician or at the institution. 
 
Withdrawal of full consent for a study means that the subject does not wish to receive further 
investigational treatment and does not wish to or is unable to continue further study participation 
including any follow-up in person, by phone, through third parties including relatives or friends, via 
discussion with other treating physicians, and by use of medical records; subject data up to withdrawal of 
full consent will be included in the analysis of the study. Any subject may withdraw full consent to 
participate in the study at any time during the study. The Sponsor Investigator or sub-investigator will 
discuss with the subject appropriate procedures for withdrawal from the study. 
 
4.4.2. Data Collection and Follow-up for Withdrawn Subjects 
If subjects are withdrawn prematurely from the study, appropriately designated research staff will make 
efforts to collect at least survival data throughout the protocol defined follow-up period for that subject.  
 
Sponsor Investigator will consult with Study Statistician with regard to any incomplete data set as 
compared to the full data set that fully supports the analysis.  If a subject withdraws consent to 
participate in the study, attempts will be made to obtain permission to record survival data up to the 
protocol-described end of subject follow-up period.  
 
Sponsor Investigator and designated research staff make it a high priority to obtain survival data on all 
subjects lost to follow up. Lost to follow up will be defined as a subject missing 2 or more consecutive 
visits, not answering or responding to 3 follow up phone calls to subject or emergency contacts, or 
returned receipt of 1 certified letter.  
5. Study Drug  
5.1. 
Description 
Eritoran (eritoran for injection) is a drug originally developed by Eisai Pharmaceuticals for the treatment 


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of sepsis.  Eritoran is a synthetic analogue of Lipid A, the toxic part of endotoxin (also known as 
lipopolysaccharide, LPS). Eritoran has been shown to block the effects of LPS in vitro, in vivo, and in 
humans. The mechanism of action of eritoran is competitive inhibition of the binding of LPS to its 
functional receptor complex of TLR4 and co receptor MD2 (52-55).  The pharmacokinetic and 
pharmacodynamic properties and safety profile of eritoran have been studied extensively during phase 
1, 2 and 3 experimental studies of endotoxemia in healthy volunteers, patients with severe sepsis, and 
patients with impaired hepatic function (55-58). Eritoran is safe and well tolerated, even at the highest 
doses tested (55). All studies on eritoran have been conducted using the intravenous route because 
this drug was developed for treatment of sepsis in critically-ill patients. 
 
Eritoran tetrasodium is a sterile, white freeze-dried cake or powder containing 7 mg of eritoran 
tetrasodium (as the tetrasodium salt) or 6.5 mg of eritoran (as the free acid) active packaged in a 10-mL 
glass vial. Since these will be a double-blind studies, matching placebo vials will be used. 
 
5.2. 
Treatment Regimen 
 
Following a screening period, which can be up to one month, each subject will be admitted on 2 
different occasions (separated by 3-6 weeks each) to receive either eritoran or vehicle (in random 
order) for 72 h.  Eritoran will be dosed 12 mg I.V. every 12 for 72 h.  A total of 6 doses (72 mg total) will 
be administered.  Each dose is diluted in 100 ml of 5% Dextrose (D5W) and infused over 4 hours. 
Studies will be performed in the Bartter Research Unit (BRU), located in the Audie L. Murphy VA 
Medical Center in San Antonio, TX. 
5.3. 
Method for Assigning Subjects to Treatment Groups 
The randomization key will be designed by the VA Research Pharmacy at the Bartter Clinical Research 
Unit (BRU) and provided in a blinded fashion to the Clinical Study Coordinator.  The Study Coordinator 
stores the Enrollment key, which is kept in a locked cabinet. Study staff maintains specimens in a de-
identified manner using only the unique Subject ID/Randomization number; source documents and data 
records are stored in electronic systems with restricted access and paper records in locked cabinets. 
5.4. 
Preparation and Administration of Study Drug 
The study drug will be provided by Eisai under Material Transfer Agreement (Attachment A., Section 
15) and shipped directly to the Audie L. Murphy Hospital Research Pharmacy. Upon receipt of 
physician’s order, the IV infusion at required dose will be prepared by the Research Pharmacist, 
according to VA policies and procedures and dispensed to designated study staff who will administer 
the infusion.  See also Blinding Study Drug, Section 5.8 and Study Procedures, Section 6.3, Visit 3 
Infusion Protocol for further details. 
5.5. 
Subject Compliance Monitoring 
Subject compliance is demonstrated by adherence to instructions in the consent and appearing for all 
study visits and procedures. Subjects receive a copy of the informed consent form at the time of 
consenting and will have an opportunity to review the consent document at every visit. 
 
Study staff will call via telephone 1-2 days before each visit and provide them with instructions (e.g. 
arrive fasting, bring concomitant medications, etc). 
5.6. 
Prior and Concomitant Therapy 
Exclusion criteria:  
Drugs that affect glucose and lipid homeostasis (subjects on a stable dose of statin >3 months are 


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eligible; subjects on sulfonylureas and/or metformin are eligible). 
Non-steroidal anti-inflammatory drugs or systemic steroid use for >1 week within 3 months; 
Treatment with anticoagulants (warfarin). Aspirin (up to 325 mg) and clopidogrel will be permitted if 
these can be held for seven days prior to the biopsies. 
5.7. 
Packaging    
Per MTA, Eisai will supply a total of 1,220 7mg vials of Eritoran for injection for use in the study. The 
Audie L. Murphy VA Hospital Research Pharmacy (“VA Research Pharmacy”) will keep all Study Drug 
in a locked, secure area at all times.  
 
Nature and Contents of the Container 
Eritoran for injection is packaged as unpreserved sterile lyophilized drug product in single-dose vials.  
VA Research Pharmacy will maintain complete, current records showing receipt, dispensing, and 
returns or destruction of Study Drug as required by Eisai’s written instructions, the Protocol, applicable 
federal, state and local laws, regulations and guidelines. 
 
VA Research Pharmacy will return to Eisai all unused Study Drug supplied by Eisai at the conclusion 
or early termination of the Study, or with prior written approval of Eisai, destroy all unused Study Drug 
and will provide certification to Eisai of such destruction. Sponsor Investigator or designated study 
monitor will review and approve VA Research Pharmacy reconciliation and destruction records before 
such records are submitted to Eisai. 
5.8. 
Blinding of Study Drug 
VA Research Pharmacy will be responsible for receipt, storage, dispensing, blinding/unblinding and 
destruction of Study Drug. 
5.8.1. Receipt of Drug Supplies 
 
Any damaged or unusable study drug in a given shipment will be documented by the VA Research 
Pharmacy.  The Research Pharmacist will notify the Sponsor Investigator and Eisai of any damaged 
study drug. 
 
Admixture incompatibility 
The diluted drug product is administered intravenously.  Because of the physico-chemical properties of 
eritoran, micelles (i.e., small aggregates) are formed in the reconstituted and admixed solutions.  
Since the micelle size might affect the disposition of eritoran after intravenous administration, the 
micelle size is controlled through the manufacture and formulation of the drug product. Specific 
admixing instructions are as follows: 
• 
Do not dilute or reconstitute eritoran with saline solutions or mix with other drugs or 
electrolytes. 
• 
The use of any solution other than those recommended may cause micelle size changes of 
eritoran, and may affect its disposition.  
• 
If administered through an existing intravenous line, flush with 5% Dextrose Injection, USP 
before and after infusion. 
• 
Chlorhexidine-treated lumens may cause precipitation and an increase in micelle size and may 
affect the rate of clearance from plasma.  
• 
Eritoran should only be admixed with 50 mL or 100 mL 5% Dextrose solution (D5W).  Eritoran 
should not be admixed with other drugs. 
5.8.2. Storage 
The drug product used for clinical studies has a shelf-life of 5 years.  The reconstituted drug product is 


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stable during storage at room temperature (or refrigerated) for up to 24 hours. 
 
Eritoran clinical trial supplies will be kept in the original packaging and stored in a secure location at a 
temperature between 15-30 °C (59 to 86 °F), protected from light. Additional site-specific guidelines for 
storage of investigational products, and any country-specific requirements specified in the product 
label must be followed. 
 
After reconstitution and admixture, storage of solutions of Eritoran will be limited to 4 hours at 15-30°C 
or up to 24 hours at 2-8 °C (36.5 to 46.4°F). Do not freeze.  Reconstituted Eritoran not 
administered within 24 hours should be discarded.  
 
For more details, please see also Section 15, Attachment F., Investigator Brochure. 
5.8.3. Dispensing of Study Drug 
Designated staff from the Audie L. Murphy Hospital Research Pharmacy maintain the Drug 
Administration Logs to track how, when and to whom the investigational drug was dispensed and 
assigned to subjects. Study clinical staff will keep administration records regarding dosing, unused 
drug, drug damaged, or wasted.   
 
Routine Study Drug reconciliation will be performed based on STVHCS Audie L. Murphy VA Hospital 
policy and standard operating procedures.    
5.8.4. Return or Destruction of Study Drug 
The procedures for final reconciliation of the site’s drug supply at the end of the study will be in 
accordance with VA Research Pharmacy standard operating procedures. 
 
There are no special precautions cited for disposal of eritoran for injection. 
 
6. Study Procedures 
 
Study visits. All studies will be performed in the clinical research center (named Bartter Research 
Unit; BRU) located in the Audie L. Murphy VA Hospital (part of the South Texas Veterans Health Care 
System) in San Antonio, TX.  
6.1. 
Visit 1 
Following an overnight fast (no food after 9 PM) subjects will come to the BRU at approximately 7 AM 
for consenting, followed by screening procedures including a medical history, physical exam, and 
screening tests (CBC, chemistry, lipids, HbA1c, PT, PTT, urinalysis and ECG). At the discretion of the 
PI, blood coagulation studies (platelets, PT, PTT) may be repeated every 4-6 months during the 
study.  
6.2. 
Visit 2 
Within 3- 45 days after Visit 1, eligible subjects will return to the BRU at 7 AM after an overnight fast for 
an oral glucose tolerance test (OGTT) and whole body DEXA. The DEXA is done to quantify fat and 
lean mass. Lean mass values are used to adjust measurements of insulin-mediated glucose disposal. 
6.3. 
Visit 3 – Infusion Protocol 
Visit 3:  Within 3 - 45 days  after Visit 2, subjects will return to the BRU for the first eritoran/vehicle 
infusion.  Hematology, comprehensive metabolic panel, including liver function tests and lipid panel 
will be performed prior to the infusion at Visits 3 and 5 (on Days 1, 2 and 3). If LDL cholesterol 


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increases more than 10% from Day 1 to Day 3 (on Visit 3 or 5), lipids will be re-assessed on the 
following visit. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
For visit 3, subjects will be admitted at approximately 7AM on the day of the eritoran/vehicle infusion. At 
approximately ~10AM subjects will receive eritoran (or vehicle) through an antecubital vein at a dose of 
12 mg every 12 h for 3 days (72 mg total). The half-lives of LPS (~5 min) (61) and FFA (~2-4 min) (62) 
are very short. Thus, blocking TLR4 for 72 h will be sufficient to protect the receptor from being 
activated by these agonists. While in the BRU, subjects are ambulatory and fed 3 times a day with an 
isocaloric diet based on their habitual energy intake and food preferences. 68 h after the first 
eritoran/vehicle dose, a 2-step euglycemic hyperinsulinemic clamp (63) with 3-3H glucose is started (at 
~5-6 AM). During Step 1 (0-120 min) insulin is infused at 20 mU/m2.min rate to measure hepatic and 
muscle insulin sensitivity. During Step 2 (120-240 min) insulin is infused at a 80 mU/m2.min rate to 
measure peripheral (muscle) insulin sensitivity (63). Indirect calorimetry is performed for 30 min 
immediately before the insulin clamp and at the end of each clamp step. A subcutaneous adipose 
tissue biopsy (64) and a vastus lateralis muscle needle biopsy (65) will be performed 45 and 30 min 
before the start of the insulin clamp, respectively. A second muscle biopsy will be performed on the 
contralateral leg at the end of the insulin clamp Subjects will be given lunch at ~1 PM, monitored for 
three hours, then discharged to home.  
 
Rationale behind dose. Eritoran has a half-life of ~25-30 h and pharmacodynamic studies have shown 
that ~12 h after eritoran administration (12 mg), the inhibitory effect of the drug on TLR4 signaling is 
fully maintained (59).  This dose (12 mg) is safe and well tolerated, and higher doses do not confer 
further inhibitory effect on TLR4 (59).   
 
 
Schema of Visit 3. Subjects will receive either placebo (vehicle) or eritoran, randomly assigned.  If on Visit 3 
they received placebo, on Visit 6 they will receive eritoran, and vice versa.  


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Analyses.   
Muscle: On pre-clamp samples we will perform TLR4 signaling assays [TLR4 protein, NFB and 
MAPK (JNK, p38, ERK) phosphorylation/activity; ~50 mg tissue], inflammatory gene (TLR4, IL-6, 
MCP-1, iNOS, TNF) expression (~20 mg), and intramyocellular lipid (ceramide, diacylglycerol) 
content (~20 mg), as described by our group (25, 59, 65, 66). On pre-clamp and clamp samples we 
will perform insulin signaling assays (~30 mg) (67). On pre-clamp samples macrophage infiltration 
and inflammatory state will be assessed by immunohistochemistry (IHC) (68-70) and real-time PCR 
(71) using macrophage markers (IHC: CD68 for total, CD40 for M1 and CD206 for M2 macrophages; 
PCR: EMR-1). 
 
Adipose tissue: We will perform assays of TLR4 signaling (TLR4, IKK/NFB and MAPKs) (~200 mg 
tissue) and inflammatory gene (TLR4, IL-6, MCP-1, iNOS, TNF) expression (~200 mg). Macrophage 
infiltration and inflammatory state will be assessed by IHC and FACS, as described by our group (72). 
 
Blood/plasma: Insulin, FFA and tritiated glucose are assayed 18 times during the clamp. Plasma 
cytokines (Inflammation Panel; Myriad RBM), LPS and LBP are assayed before the first eritoran dose 
and just before the clamp. Plasma LBP is measured because it is considered an integrated measure 
of plasma LPS level and indicator of subclinical endotoxemia (73). Measurement of TLR4 expression 
and TLR4 signaling (MAPK and NFB phosphorylation) in monocytes will be done on whole blood 
samples collected immediately before the first eritoran dose (Day 1) and just before the insulin clamp 
(Day 4). To confirm the inhibitory effect of eritoran on TLR4, an ex vivo LPS stimulation test will be 
performed on whole blood samples (59). 
 
6.4. 
Visit 4 
5-7 days after Visit 3 all subjects come to the BRU for inspection of the biopsy sites.  Subjects 
will maintain an isocaloric diet and will refrain from regular exercise until the next infusion 
protocol. If the lipid levels increased >10% during the Visit 3 hospital stay (from Day 1 to Day 
3 measurements), we may repeat lipids (LDL cholesterol) at Visit 4.  
 
6.5. 
Visit 5 
Approximately 4 - 10 weeks after Visit 4 all subjects come to the BRU to undergo the second infusion 
protocol (same as Visit 3). 
6.6. 
Visit 6 
5-7 days after Visit 5 all subjects come to the BRU for inspection of the biopsy sites.  If a 10% 
increase in lipid levels was observed during the Visit 5 hospital stay (from Day 1 to Day 3), we 
may repeat the lipid levels (LDL cholesterol) on Visit  6. 
 
7. Statistical Plan 
7.1. 
Sample Size Determination 
The primary hypothesis is that the TLR4 antagonist will increase insulin sensitivity, especially in the 
insulin resistant subjects (obese and T2DM). The effect of a TLR4 inhibitor on insulin sensitivity has 
never been tested in humans. Therefore, we conducted the sample size calculation based on data 
using acipimox, a drug which rapidly improves insulin sensitivity by reducing the plasma level of FFA (a 
TLR4 ligand). In prior studies, the effect of acipimox on insulin sensitivity in T2DM subjects was a mean 
increase in Rd of 2.2 mg/kg.min with a SD of 3.46 (67). Based on this, the effect size would be 
approximately 0.64 assuming a within subject correlation of 0.5. 
 
 


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Hence with n=20 completers per each group (lean, obese nondiabetic, obese T2DM) and an effect size 
of 0.64, a paired t-test of the within subject difference in insulin sensitivity (between the vehicle and the 
TLR4 inhibitor) would have a power of 77% with a two-sided α of 0.05. We will enroll ~90 subjects to 
account for a ~30% dropout rate. 
7.2. 
Statistical Methods 
Analytical Approach. The goal of the analysis is to identify treatment-related changes in insulin 
sensitivity. With this crossover design, we will compare means between treatment groups using a 
repeated-measures General Linear Model (GML) analysis. This is expected to yield better statistical 
power than the use of the paired t-test used for the power calculation. We will determine the relationship 
between measurements 
of inflammation (TLR4 signaling, inflammatory genes) vs. insulin 
signaling/sensitivity using this regression approach as well as model the effect of selected covariates 
(e.g., sex and race/ethnicity). Scatter plots will be generated to identify outliers and to verify statistical 
assumptions. 
 
 
 
8. Safety and Adverse Events 
8.1. 
Definitions 
Unanticipated Problems Involving Risk to Subjects or Others (UPIRSO) 
Any incident, experience, or outcome that meets all of the following criteria:  
• Unexpected in nature, severity, or frequency (i.e. not described in study-related documents such 
as the IRB-approved protocol or consent form, the investigators brochure, etc) 
• Related or possibly related to participation in the research (i.e. possibly related means there is a 
reasonable possibility that the incident experience, or outcome may have been caused by the 
procedures involved in the research) 
• Suggests that the research places subjects or others at greater risk of harm (including physical, 
psychological, economic, or social harm). 
 
Adverse Event (AE) 
In general, AE is used very broadly and encompasses physical and psychological harms and includes:   
 
Any untoward or unfavorable medical occurrence in a human subject, including any abnormal sign 
(for example, abnormal physical exam or laboratory finding), symptom, or disease, temporally 
associated with the subject’s participation in the research, whether or not it is considered related to 
the subject’s participation in the research. 
Intercurrent illnesses or injuries should be regarded as adverse events.  Abnormal results of diagnostic 
procedures are considered to be adverse events if the abnormality: 
• results in study withdrawal 
• is associated with a serious adverse event 
• is associated with clinical signs or symptoms 
• leads to additional treatment or to further diagnostic tests 
• is considered by the investigator to be of clinical significance 
 
Serious Adverse Event (SAE) 
Adverse events are classified as serious or non-serious.  A serious adverse event is any AE that:  
• results in death; 
• is life-threatening (places the subject at immediate risk of death from the event as it occurred); 
• results in inpatient hospitalization or prolongation of existing hospitalization; 
• results in a persistent or significant disability/incapacity; 


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• results in a congenital anomaly/birth defect; or 
• based upon appropriate medical judgment, may jeopardize the subject’s health and may require 
medical or surgical intervention to prevent one of the other outcomes listed in this definition 
(examples of such events include allergic bronchospasm requiring intensive treatment in the 
emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient 
hospitalization, or the development of drug dependency or drug abuse). 
 
Adverse Event Reporting Period 
The study period during which adverse events must be reported is normally defined as the period from 
the initiation of any study procedures to the end of the study treatment follow-up.  For this study, the 
study treatment follow-up is defined as 30 days following the last administration of study treatment.  
 
Pre-existing Condition 
A preexisting condition is one that is present at the start of the study.  A pre-existing condition should be 
recorded as an adverse event if the frequency, intensity, or the character of the condition worsens 
during the study period. 
 
General Physical Examination Findings 
At screening, any clinically significant abnormality should be recorded as a preexisting condition.  At the 
end of the study, any new clinically significant findings/abnormalities that meet the definition of an 
adverse event must also be recorded and documented as an adverse event.  
 
Abnormal Laboratory Values 
A clinical laboratory abnormality should be documented as an adverse event if any one of the following 
conditions is met:  
• The laboratory abnormality is not otherwise refuted by a repeat test to confirm the abnormality 
• The abnormality suggests a disease and/or organ toxicity 
• The abnormality is of a degree that requires active management; e.g. change of dose, 
discontinuation of the drug, more frequent follow-up assessments, further diagnostic 
investigation, etc. 
 
Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization should be documented 
and reported as a SAE unless specifically instructed otherwise in this protocol.  Any condition 
responsible for surgery should be documented as an AE if the condition meets the criteria for and 
adverse event.  
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported as an AE in 
the following circumstances: 
• Hospitalization or prolonged hospitalization for diagnostic or elective surgical procedures for a 
preexisting condition.  
• Surgery should not be reported as an outcome of an adverse event if the purpose of the surgery 
was elective or diagnostic and the outcome was uneventful. 
• Hospitalization or prolonged hospitalization required to allow efficacy measurement for the study. 
• Hospitalization or prolonged hospitalization for therapy of the target disease of the study, unless 
it is a worsening or increase in frequency of hospital admissions as judged by the clinical 
investigator. 
8.2. 
Recording of Adverse Events 
At each contact with the subject, the investigator or study staff will seek information about adverse 
events by specific questioning and, if appropriate, by examination.  Information on all AEs will be 
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form (CRF).  AEs will be tracked using the HSC IRB AE tracking form or data management tool (See 
Section 9.3) to be reviewed by Sponsor Investigator on a monthly and ad hoc basis, depending on 
severity and expected/unexpected nature of the event. A summary of AEs tracked will be reported in 
the quarterly report and cumulatively in the annual reports to Eisai and FDA. 
 
 
All AEs occurring during the study period will be recorded. The clinical course of each event will be 
followed until resolution, stabilization, or until it has been determined that the study treatment or 
participation is not the cause.  SAEs that are still ongoing at the end of the study period will be followed 
up to determine the final outcome.  Any SAE that occurs after the study period and is considered to be 
possibly related to the study treatment or study participation will be recorded and reported per 
Institutional policy and according to FDA requirements. 
 
8.3. 
Reporting of Serious Adverse Events and Unanticipated Problems 
 
SAEs and UPIRSOs will be reported per IRB policy and procedure and in accordance with Eisai Inc., 
Material Transfer Agreement (See Section 15, Attachment A, Material Transfer Agreement, pages 18-
21).  
 
Each subject is evaluated for any adverse events. Any event that is reported to either the principal 
investigator or designated research associates by the subject or medical staff caring for the subject and 
which meets the criteria will be documented as such.  Any serious event that is reported will then 
generate an adverse event report, which will be submitted to the IRB, R&D Committee, and the 
designated safety officer.   
 
Unanticipated risks to subjects or others (UPIRSO) that are a result of study participation are promptly 
reported to the IRB, the R&D Committee, and the designated safety officer. The report will include a 
description of the event, when and how it was reported, as well as any official chart records or 
documentation to corroborate the event or the reporting of the event. All adverse events will be graded 
as mild, moderate, or severe.  All adverse events will be summarized annually and submitted to the IRB 
and R&D Committees.  Any action resulting in a temporary or permanent suspension of this study (e.g. 
IRB actions, R&D actions or actions by Eisai Inc.) will be reported to the appropriate VA and NIH 
program official. 
8.3.1. Investigator reporting: notifying the study sponsor 
 
The Sponsor Investigator will comply with all safety reporting regulations as set forth in the Code of 
Federal Regulations. Any correspondence to the FDA regarding adverse events or other safety issues 
will be simultaneously copied via facsimile or email to Eisai. 
 
Institution and Sponsor Investigator shall cooperate with Eisai in all of their efforts to capture 
information on the safety profile of the Study Drug, including, but not limited to the following:  
 
Notifying Eisai of all serious suspected adverse drug reactions occurring in an individual who 
has been exposed to the Study Drug, and where the Study Drug is the suspected product. 
 
Where adverse drug reaction is defined as: a noxious and unintended response to a medicinal 
product related to any dose. A causal relationship between the medicinal product and the 
adverse response is at least a reasonable possibility. Serious is defined in Section 8.1, 
Definitions, see SAE.  
 


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A study-related UPIRSO, involving any type of SAE, must be reported to Eisai Inc. by telephone within 
24 hours of the event.  To report such events, a Serious Adverse Event (SAE) form must be completed 
by the investigator and faxed to the study sponsor within 24 hours, using the SAE form provided in the 
Material Transfer Agreement, Attachment B, and also referenced in Attachment B Section 15.  The 
investigator will keep a copy of this SAE form on file at the study site.   
 
Report SAEs by email and facsimile to: 
 
 
Sally T. Ishizaka, Ph.D. 
Sally_Ishizaka@eisai.com  
Director of Biology 
CINO Group, EISAI Inc. 
Andover, MA 01810 
978-837-4642 
 
Within the following 48 hours, the investigator must provide further information on the SAE or the 
UPIRSO in the form of a written narrative.  This should include a copy of the completed SAE form, and 
any other diagnostic information that will assist the understanding of the event.  Significant new 
information on ongoing serious adverse events should be provided promptly to Eisai, Inc. A summary of 
non-serious AEs will be reported in the quarterly and annual reports to Eisai, Inc., and in the annual 
report to FDA. 
 
 
 


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8.3.2. Investigator reporting: notifying the UTHSCSA IRB 
 
Reporting Process 
All adverse events will be summarized annually and submitted to the IRB and R&D Committees.  Any 
action resulting in a temporary or permanent suspension of this study (e.g. IRB actions, R&D actions or 
actions by the sponsor) will be reported to the appropriate VA program official. 
 
Unanticipated problems posing risks to subjects or others as noted above will be reported to the UTHSCSA 
IRB using the form: UPIRSO Reporting Form or as a written report of the event (including a description of 
the event with information regarding its fulfillment of the above criteria, follow-up/resolution and need for 
revision to consent form and/or other study documentation). 
 
Copies of each report and documentation of IRB notification and receipt will be kept in the study file. 
 
8.3.3. Sponsor reporting: Notifying the FDA 
 
The Sponsor Investigator is required to report certain study events to Eisai, Inc. thereby enabling the 
company providing the Study Drug whose IND is cross-referenced in Dr. Musi’s IND application to report in 
an expedited fashion to the FDA in an IND safety report. The following describes the safety reporting 
requirements by timeline for reporting an associated type of event: 
 
• Within 7 calendar days 
Any study event that is: 
– associated with the use of the study drug 
– unexpected,  
– fatal or life-threatening, and  
 
• Within 15 calendar days 
Any study event that is: 
– associated with the use of the study drug, 
– unexpected, and 
– serious, but not fatal or life-threatening 
-or- 
– a previous adverse event that was not initially deemed reportable but is later found to fit the 
criteria for reporting (reporting within 15 calendar days from when event was deemed 
reportable). 
Any finding from tests in laboratory animals that:  
– suggests a significant risk for human subjects including reports of mutagenicity, teratogenicity, or 
carcinogenicity. 
 
Additional reporting requirements 
Eisai Inc. is also required to identify in IND safety reports all previous reports concerning similar adverse 
events and to analyze the significance of the current event in light of the previous reports. 
 
Reporting Process 
Adverse events may be submitted on FDA Form 3500A or in a narrative format. The contact information for 
submitting IND safety reports is noted below: 
 
Food and Drug Administration 


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Center for Drug Evaluation and Research 
Division of Metabolism and Endocrinology Products 
5901-B Ammendale Road 
Beltsville, MD 20705-1266 
Phone: (301) 796-2290 
Fax: (301) 796-9712  
8.4. 
Medical Monitoring 
The Sponsor Investigator will review the safety and progress of this study on a monthly basis or when 
needed if SAE or SAE-UPIRSO occurs.  The Designated Safety Officer is Sandra Sanchez Reilly, M.D.  
Dr. Sanchez Reilly is a Professor in the Department of Medicine, UTHSCSA, and Staff Physician at the 
Audie L Murphy VA Hospital.   
 
Twice a year the Sponsor Investigator (PI) will provide study safety information to the Designated Safety 
Officer (DSO) to conduct reviews of overall subject safety for the study. Within 30 days of receiving safety 
information for routine reviews, the Designated Safety Officer will provide the Sponsor Investigator a 
written synopsis of the review and identify safety concerns, if any, with a recommended plan of action. Ad 
hoc reviews in real-time may occur upon mutual agreement between Sponsor Investigator and 
Designated Safety Officer. 
 
8.5. 
Unblinding Procedures 
See Section 5.8 – If, in the case of SAE or SAE-UPIRSO, the Clinical Study Coordinator or appropriately 
designated study staff will contact the Designated Safety Officer (Section 8.6) to assess the need for 
initiation of unblinding procedures. Responsibilities of each individual are bulleted below. 
 
8.5.1. Designated Safety Officer 
 
• 
Assess SAE or SAE-UPIRSO to determine if unblinding is required and, if yes, notify pharmacist to 
request subject’s assignment 
• 
Compare event data to intervention/assignment to determine if SAE is related to study intervention 
• 
Assess study and subject safety to determine if stopping rules should be invoked 
• 
Create the Safety Officer record in a blinded report format and communicate to Sponsor 
Investigator and Clinical Study Coordinator with overall impression and recommended plan of 
action 
• 
Responsible for determining the need to “unblind” the investigator  
 
8.5.2. VA Research Pharmacist  
 
• 
Unblind subject assignment communicated to Designated Safety Officer upon request 
• 
Suspend dispensing of study drug until SAE reporting is complete 
 
8.5.3. Sponsor Investigator 
 
• 
Receive and review Designated Safety Officer report with Clinical Study Coordinator, Pharmacist 
and Study Statistician 
• 
Determine and implement stopping rules, if required 
• 
Ensure that IRB, FDA, Eisai and NIH are notified as appropriate to local or agency policy 
 
8.5.4. Clinical Study Coordinator  
 


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• 
Obtain a copy of Designated Safety Officer report/record to file in study binder/file and forward a 
copy to the Regulatory Coordinator 
• 
Ensure appropriate medical care and follow up is scheduled and implemented until SAE resolved 
• 
Create FDA report (MedWatch), if applicable 
8.6. 
Stopping Rules  
 
Clinical or laboratory excursions that a subject may experience that may warrant stopping study 
participation or drug administration, either temporarily or permanently, include but are not limited to: 
 
Clinical criteria:  
• 
Fever with a temperature >101°F 
• 
Allergic reaction to drug 
• 
Blood pressure >160/95 or <80/50 
• 
Severe vomiting 
• 
Phlebitis grade 4 or 5 (see Attachment G.) 
 
Laboratory criteria: 
• 
Liver function test results >2x normal range 
• 
WBC ≥14,000 or ≤3,000 
• 
HCT ≥55 or ≤33 
• 
Platelets ≤100,000 or ≥700,000 
 
In the unlikely event that a study-related death or SAE occurs, the decision to stop the trial, either 
temporarily or permanently, will be the responsibility of the Designated Safety Officer in collaboration with 
the Sponsor Investigator. 
9. Data Handling and Record Keeping 
9.1. 
Confidentiality 
Information learned about all subjects will be kept confidential.  All data and protected health information 
in paper form will be kept confidential by assigned anonymous identifier and kept secured (password 
protected and/or double locked). Subjects will not be identified in any way in any publication.   
9.2. 
Source Documents 
Source data are contained in source documents found in paper subject files at the research site and in 
VA CPRS medical records.  Print all entries legibly in blue or black ink.  Erasures and white-out material 
are prohibited. If any entry error has been made on paper, to correct such an error, draw a single straight 
line through the incorrect entry and enter the correct data above it.  All such changes must be initialed 
and dated.  For clarification of illegible or uncertain entries, print the clarification above the item, then 
initial and date it. 
9.3. 
Case Report Forms 
The study case report forms (CRF) are the primary data collection instruments for the study.  Data 
requested on the CRF will be collected from the subject encounter and from the subject diaries, 
pharmacy logs, and medical records (VA CPRS), then entered into the IDEAS database managed by the 
Department of Epidemiology and Biostatistics.  All missing data will be routinely queried, corrected, and 
or explained.  If a space on the CRF is left blank because the procedure was not done or the question 
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9.4. 
Data Management 
Database Management Software: All data management for this project will be maintained using the 
Informatics Data Exchange and Acquisition System (IDEAS) which was developed by the Department of 
Epidemiology and Biostatistics. The database engine consists of Oracle Enterprise which is maintained on 
a cluster of 22 Sun Microsystems servers. Database administration is performed via UNIX-based scripts, 
Oracle Enterprise Manager (OEM) and Tool for Oracle Application Developers (TOAD). The data system 
model is described below. 
Data System: IDEAS is a computing environment consisting of a collection of applications, systems, 
policies, and procedures that govern its informatics operations. While the IDEAS integrates a variety of 
data from different sources, it was developed around support for clinical/translational research 
investigations. Starting with security and acting as an information broker facilitating data collection, 
analysis, and reporting, the system was designed to integrate disparate data from multiple and 
geographically separate collection and treatment facilities. Data applications are designed to be end-user 
oriented and constructed to optimize workflow and minimize errors. The IDEAS system capitalizes on its 
tiered architecture to implement numerous business rules and business objects to enforce a number of 
Quality Control (QC) procedures, IDEAS incorporates and utilizes pre-defined standardized Common Data 
Elements (CDEs), with the ability to incorporate new CDEs as they become available. It has been designed 
to follow the necessary standards and if needed, set the standards. IDEAS server resources are divided 
into four primary categories: 1) informatics and statistical computing; 2) data collection and reporting; 3) 
software development; and 4) infrastructure: file and naming services. This system has been in use for 5 
years and has proven to be flexible, scalable and easily extendible to meet research, production, 
regulatory, reporting, and analytical needs. IDEAS modules support clinical, specimen, pathological, 
personnel, and project tracking applications. In addition to data collection and analysis, IDEAS facilitates 
performance monitoring and the enforcement of all human subject requirements (both local as well as 
Federal). IDEAS is the system used by our larger CTSA projects as well as by our National Cancer Institute 
P30-funded cancer center. We will utilize IDEAS to support all data management. 
All data will be input using a web front-end interface. All users are individually assigned authorization for 
access to specific components of the database application. Information that is input is checked for logical 
and range consistency and mandatory data fields must be entered in order to input a record. We use a 
formal revision tracking procedure where all SAS code is documented by the analyst, giving the date, 
version and purpose. All database tables are fully labeled. Common field formats, such as those for sex 
and ethnicity, are used for all data applications. For analysis, al project data are directly accessible via 
direct SQL calls within SAS to Oracle (using PROC SQL). Reports are available using both Microsoft Word 
and Adobe pdf documents and automated tables and graphics generated using the SAS Output Delivery 
System (ODS).  
9.5. 
Records Retention 
The NIH Principal Investigator is responsible for maintaining study essential documents for at least 3 
years after the NIH grant period ends or at least 2 years have elapsed since the formal discontinuation of 
clinical development of the investigational product, whichever is longer.  These documents should be 
retained for a longer period if required by an agreement with the sponsor.  In such an instance, it is the 
responsibility of the sponsor to inform the investigator/institution as to when these documents no longer 
need to be retained.  
 
10.   Study Monitoring, Auditing, and Inspecting 
10.1. Study Monitoring Plan 
The Sponsor Investigator will ensure that the designated study monitor, compliance specialist or other 
quality assurance reviewer is given access to all the above noted study-related documents and study 


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related facilities (e.g. pharmacy, diagnostic laboratory, etc.), and has adequate space to conduct the 
study monitoring visits. 
 
Monitoring elements are described below: 
1. The quality and content of the informed consent, and the consent process will be monitored 
throughout the study period.  
2. The accuracy of the collection and processing of laboratory specimens will be monitored throughout 
the study period.  
3. The timeliness and conformity with established clinical monitoring of the subjects will be followed on 
a quarterly basis. 
4. The forms and source documents in the subjects’ research records will be current and maintained 
in a timely manner. 
5. The physical and mental condition of the subject and any type of AE will be assessed and 
monitored prior to beginning the study by the investigator(s) and staff, throughout the study period. 
 
Areas of focus for study monitoring by the designated study monitor: 
1. Regulatory documentation and compliance with local policy will be monitored quarterly and 
throughout the study period. 
2. The safe handling and accountability of drugs, which includes the use of protective equipment and 
disposal of waste, will be maintained prior to the start of the study and semi-annually throughout the 
study period. 
3. Compliance with the protocol and conduct of the study, including the consenting process, along with 
source data compared to collected data (SDV), will be monitored on a quarterly basis throughout 
the study period.  
a. 50% of subject records and 20% of data for each subject will be monitored.  
b. The first and the last subject enrolled will be monitored for consent practices and 100% of 
data collected. 
4. Adverse event logs will be monitored quarterly and research records analyzed for AEs that were 
overlooked or otherwise not documented properly in the study files. 
5. The condition of the physical space used by the investigator and subject for study purposes will be 
monitored prior to the start of the study and annually throughout the study period.  
6. The safety assurance and condition of any electrical, radiological, magnetic, gaseous, and 
hazardous equipment, tools or apparatus that will be used by the investigators and subjects will be 
monitored prior to the start of the study and annually throughout the study period.  
7. The physical safety and security of the staff prior to the start of the study and throughout the study 
period will be maintained and monitored annually prior to continuing review. 
10.2. Auditing and Inspecting 
The Sponsor Investigator will permit study-related monitoring, audits, and inspections by the IRB, the 
funding sponsor, government regulatory bodies, and University compliance groups of all study related 
documents (e.g. source documents, regulatory documents, data collection instruments, study data etc.).  
The investigator will ensure the capability for inspections of applicable study-related facilities (e.g. 
pharmacy, diagnostic laboratory, etc.). 
 
Participation as an investigator in this study implies acceptance of potential inspection by government 
regulatory authorities and applicable University compliance and quality assurance offices. 
11.    Ethical Considerations 
This study is to be conducted according to US and international standards of Good Clinical Practice (FDA 
Title 21 part 312) applicable government regulations and Institutional research policies and procedures. 
 
This protocol and any amendments will be submitted to the Institutional Review Board (IRB), in 


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agreement with local legal prescriptions, for formal approval of the study conduct.  The decision of the 
IRB concerning the conduct of the study will be made in writing to the investigator and a copy of this 
decision will be provided to the funding sponsor before commencement of this study.   
 
All subjects for this study will be provided a consent form describing this study and providing sufficient 
information for subjects to make an informed decision about their participation in this study.  See 
Attachments, Section 15, Attachment D., Sample Informed Consent Form.  This consent form will be 
submitted with the protocol for review and approval by the IRB for the study.  The formal consent of a 
subject, using the IRB-approved consent form, must be obtained before that subject undergoes any study 
procedure.  The consent form must be signed by the subject or legally authorized representative, and the 
investigator-designated research professional obtaining the consent.  
12.  Study Finances 
12.1. Funding Source 
This study is financed through a grant from the US National Institutes of Health/National Institute of 
Diabetes and Digestive and Kidney Diseases (NIH-NIDDK), R01DK080157.  
12.2. Conflict of Interest 
None reported. 
12.3. Subject Stipends or Payments 
This study will reimburse subjects. A schedule of payments is shown below. The total potential 
reimbursement to a subject is $1,680 for the study for all visits, or payments may be prorated to include 
the last visit completed if study participation is terminated early. Manual payments for additional visits, if 
necessary, will be handled on an ad-hoc basis with prior approval from the Sponsor Investigator. 
 
Visit 1  
$10 
Visit 2  
$50 
Visit 3  
$800 
Visit 4  
$10 
Visit 5  
$800 
Visit 6  
$10 
Unscheduled Visit 
Up to $100 
 
13.    Publication Plan 
The Institution, Sponsor Investigator or respective designees may present or publish the results of a 
scientific investigation involving this Study in accordance with Attachment A., Material Transfer 
Agreement, Article 7., Publication/Release of Information. 
14.   References 
See also References in Attachments, Section 15. 
 
15.   Attachments 
A. EISAI Inc - Material Transfer Agreement (signed May 20, 2014) 
B. EISAI Inc - Serious Adverse Event Reporting Form (Attachment to MTA) 
C. Study Event Schedule  
D. Sample Consent Form 


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E. Reference List 
F. EISAI Inc - Investigator Brochure 
G. Grading Scale for Phlebitis 
H. Summary of Protocol Changes 
 
 
 


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ATTACHMENT A. MATERIAL TRANSFER AGREEMENT 


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ATTACHMENT B. EISAI, INC., SERIOUS ADVERSE EVENT REPORTING FORM  


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ATTACHMENT C. STUDY EVENT SCHEDULE 


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ATTACHMENT D. SAMPLE INFORMED CONSENT 


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ATTACHMENT  E. REFERENCES 
 
 
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Sun L, Yu Z, Ye X, Zou S, Li H, Yu D, et al. A marker of endotoxemia is associated with obesity and 
related metabolic disorders in apparently healthy Chinese. Diabetes Care. 2010;33(9):1925-32. Epub 
2010/06/10. doi: dc10-0340 [pii] 
10.2337/dc10-0340. PubMed PMID: 20530747; PubMed Central PMCID: PMC2928335. 
 
 


NIH_ERIT-002 
 
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ATTACHMENT F. INVESTIGATOR BROCHURE 
 
 
 
 


NIH_ERIT-002 
 
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Version: 2.0 
CONFIDENTIAL 
ATTACHMENT G. GRADING SCALE FOR PHLEBITIS 


NIH_ERIT-002 
 
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Version: 2.0 
CONFIDENTIAL 
ATTACHMENT H. SUMMARY OF PROTOCOL CHANGES 
 
Date of Change 
Version 
Number 
Section Modified 
Before Change 
After Change 
09-25-2014 
1.1 
Title Page 
IND Number:  Pending 
IND Number: 123955-0001 
09-25-2014 
1.1 
Table of Contents 
 
Added Attachment G., page 33 
09-25-2014 
1.1 
4.1, page 12 
Typographical error: Stable 
body weight “±1%” 
Corrected: Stable body weight “1.5%” 
09-25-2014 
1.1 
5.8.2, page 15  
After reconstitution and 
admixture, solutions of 
Eritoran may be stored for up 
to 24 hours at temperatures 
between 15-30°C (59 to 86 
°F) or under refrigeration. 
After reconstitution and admixture, 
solutions of Eritoran may be stored for 
up to 24 hours under refrigeration at 
temperatures between 2-8 °C (36.5 to 
46.4°F) 
09-25-2014 
1.1 
ATTACHMENTS 
 
Added ATTACHMENT G. Summary of 
Protocol Changes 
12-03-2014 
1.2 
Header  
Incorrect version # on some 
pages 
Version “1.2” all pages 
12-03-2014 
1.2 
Title Page 
NCT# Pending 
NCT02267317 
12-03-2014 
1.2 
Section 
3.4 
Potential Risks to 
Subject 
Safety, 
page 11 
 
A CBC and lipid panel will be obtained 
on Day 3 of Infusion Visit to assess 
effects on blood cell count and lipid 
metabolism. 
12-03-2014 
1.2 
6.3 
Visit 
3 
– 
Infusion 
Protocol, 
page 17 
Subjects will be given lunch 
at ~1PM, monitored for three 
hours, and discharged to 
home. 
Subjects will be given lunch at ~1 PM, 
monitored for three hours including a 
CBC and lipid panel obtained to assess 
effects on blood cell count and lipid 
metabolism,  then discharged to home 
12-03-2014 
1.2 
4.1 
Inclusion 
Criteria, item 6) 
Premenopausal women in 
the follicular phase, non-
lactating and with a negative 
pregnancy test. 
Premenopausal women, non-lactating 
and with a negative pregnancy test. 
12-03-2014 
1.2 
Title Page, Study 
Summary, Section 
1.4, 5.1, and 5.7 
Eritoran injection 
Eritoran for injection (per Eisai request 
to be consistent) 
12-03-2014 
1.2 
Table of Contents 
and 
ATTACHMENTS 
ATTACHMENT E & G 
Added placeholders for 
ATTACHMENTS A, B, C, D, and F for 
use when attachments are updated 
12-03-2014 
1.2 
Section 5.1, Page 
14 
“Since these will be a 
double-blind studies, 
matching placebo vials will 
be used.” 
Deleted typo and entire sentence: 
“Since these will be a double-blind 
studies, matching placebo vials will be 
used.” 
12-03-2014
 
 
 
 
1.2 
Title Page 
 
 
Added  IRB Tracking Number 
20140498H 
12-03-2014            
 
1.2           Section 
5.3, 
page 14     
Study staff maintains source 
documents, data records and 
specimens in a de-identified 
method using only the unique 
Subject ID/Randomization 
number. 
 
Study staff maintains specimens in a 
de-identified manner using only the 
unique Subject ID/Randomization 
number; source documents and data 
records are stored in electronic systems 
with restricted access and paper records 
in locked cabinets. 
 
04-14-2015 
1.3 
Study Summary, 
Page 1 (5 of 43), 
Inclusion Criteria 
Stable body weight (±1%) for 
≥ 3 months;  
Age 18-60 
Corrected typo: Stable body weight 
(±1.5%) for ≥ 3 months;  
Age 18-65 
 
04-14-2015 
1.3 
Study Summary, 
Exclusion 
Criteria  
 
1) 
Presence of glucose 
intolerance based on ADA 
criteria (lean/obese NGT) 


NIH_ERIT-002 
 
Page 42 
Version: 2.0 
CONFIDENTIAL 
2) 
Hemoglobin A1c ≥ 8.5% 
(diabetic subjects only). 
 
05-12-2015 
1.3 
3.1 Subjects, 
Page 11 
~80 subjects will be 
consented assuming ~30% 
screen failure or withdrawals. 
Corrected typo: ~90 subjects will be 
consented assuming ~30% screen 
failure or withdrawals. 
04-14-2015 and 
05-12-2015 
1.3 
3.4 Potential 
Risks to Subject 
Safety 
A CBC and lipid panel will be 
obtained on Day 3 of Infusion 
Visit to assess effects on 
blood cell count and lipid 
metabolism. 
 
Page 12 
 
 
Catheters will be placed in 
an antecubital vein and in a 
hand vein for the euglycemic 
clamps. 
The maximal amount of blood to be 
drawn during the entire study for any 
given subject will be approximately  
534 mL. Hematology comprehensive 
metabolic panel with lipids and liver 
function tests will be obtained at the 
Infusion Visit to assess effects on blood 
cell count, liver function and lipid 
metabolism. 
IV lines.  Intravenous catheters will be 
used for infusions and venipuncture.   
 
04-14-2015 
1.3 
Section 4.1 
Inclusion Criteria 
One of these 3 groups: … ; 
or (iii) lean (BMI <26 kg/m2) 
normal glucose tolerant. 
Added “Lean subjects have no family 
history of diabetes” 
04-14- 2015 
1.3 
Section 4.1, 
Inclusion 
Criteria, Page 12 
(16 of 43) 
Stable body weight (±1%) 
for ≥3 months. 
Age 18-60 
 
Corrected typo: Stable body weight 
(±1.5%) for ≥3 months. 
Age 18-65 
04-14-2015 
1.3 
Section 4.2 
Exclusion 
Criteria 
1) Presence of glucose 
intolerance based on 
American Diabetes 
Association (ADA) 
criteria; 
2) Non-steroidal anti-
inflammatory drugs or 
systemic steroid use 
for more than 1 wk 
within 3 months 
3) Current treatment with 
anticoagulants 
(warfarin), Aspirin (up 
to 325 mg) and 
clopidogrel will be 
permitted if these can 
be held for seven days 
prior to the biopsies 
 
1) 
For non-diabetic subjects, the 
presence of glucose intolerance on 
OGTT based on American 
Diabetes Association (ADA) criteria 
will be exclusionary. 
3) 
2)    Hemoglobin A1c ≥ 8.5% 
(diabetic subjects only) Removed 
abbreviation of “wk” to read more 
clearly. “Non-steroidal … 1 week 
within 3 months 
4) 
Added “History of, coagulopathy 
will be excluded.”  
04-14-2015 
1.3 
Section 5.8.2, 
page 15 
After reconstitution and 
admixture, solutions of 
Eritoran may be stored for 
up to 24 hours under 
refrigeration at 
temperatures between 2-8 
°C (36.5 to 46.4°F) 
Change storage instructions for 
reconstituted drug to allow time 
between admixture and infusion 
starting. “After reconstitution and 
admixture, storage of solutions of 
Eritoran will be limited to 4 hours at 15-
30°C or up to 24 hours at 2-8°C.” Do 
not freeze. 
04-14-2015 and 
05-12-2015 
1.3 
Section 5.2, 
Treatment 
Regimen, Page 
15 
Following a screening period, 
which can be up to one 
month, each subject will be 
admitted on 2 different 
occasions (separated by 3-4 
weeks each) to receive… 
Extend window to 3-6 weeks:  
Following a screening period, which can 
be up to one month, each subject will 
be admitted on 2 different occasions 
(separated by 3-6 weeks each) to 
receive… 
04-14-2015 and 
05-12-2015 
1.3 
 
 
 
 
Section 6.3, 
Page 17 
 
 
 
 
 
 
 
 
Adjusted visit window and added 
blood draws for safety to Visit 3 
and 5 per FDA recommendation. 
Visit 3:  Within 3-30 days after Visit 
2, subjects will return to the BRU 


NIH_ERIT-002 
 
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Version: 2.0 
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1.3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Section 6.3 
cont’d 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Page18 Analyses: 
Blood/plasma: Insulin, FFA 
and tritiated glucose are 
assayed every 10 minutes 
during the clamp. Plasma 
cytokines …monocytes will 
be done on whole blood 
samples collected 
immediately before the first 
eritoran dose and just before 
the insulin clamp. To confirm 
the inhibitory effect of 
eritoran on TLR4, an ex vivo 
LPS stimulation test will be 
performed on whole blood 
samples (59). 
 
for the first eritoran/vehicle infusion.   
 “. 
 
Hematology, 
comprehensive 
metabolic panel, including liver function 
tests and lipid panel will be performed 
prior to the infusion at Visits 3 and 5 (on 
Days 1, 2 and 3). If LDL cholesterol 
increases more than 10% from Day 1 to 
Day 3 (on Visit 3 or 5), lipids will be re-
assessed on the following visit. 
 
Changed time for admission from 9AM 
to 7AM. 
 
Changed start time for tritiated glucose 
to 5-6AM not 8AM. 
 
 
Blood/plasma: Insulin, FFA and tritiated 
glucose are assayed 18 times during 
the clamp. Plasma cytokines … 
monocytes will be done on whole blood 
samples collected immediately before 
the first eritoran dose (Day 1) and just 
before the insulin clamp (Day 4). To 
confirm the inhibitory effect of eritoran 
on TLR4, an ex vivo LPS stimulation 
test will be performed on whole blood 
samples (59). 
 
 
04-14-2015 and 
05-12-2015 
1.3 
Section 6.4, Visit 
4 
5-7 days after Visit 3 all 
subjects come to the BRU 
for inspection of the biopsy 
sites. 
 
Subjects 
will 
maintain an isocaloric diet 
and will refrain from regular 
exercise 
until 
the next 
infusion protocol.  
 
5-7 days after Visit 3 …regular exercise 
until the next infusion protocol.  
 
Added: “If the lipid levels increased 
>10% during the Visit 3 hospital stay 
(from Day 1 to Day 3 measurements), 
we may repeat lipids (LDL cholesterol) 
at Visit 4.”  
 
04-14-2015 and 
05-12-2015 
1.3 
Section 6.5 Visit 
5 
Approximately 3-4 weeks 
after Visit 4 all subjects 
come 
to 
the 
BRU 
to 
undergo 
the 
second 
infusion protocol (same as 
Visit 3). 
 
Extend visit window: 
Approximately 3-6 weeks after Visit 4 
all subjects come to the BRU to 
undergo the second infusion protocol 
(same as Visit 3). 
 
04-14-2015 and 
05-12-2015 
1.3 
Section 6.6 Visit 
6 
5-7 days after Visit 5 all 
subjects come to the BRU 
for inspection of the biopsy 
sites.   
5-7 days after Visit 5 … the biopsy sites.  
Added blood draws at final follow up per 
FDA recommendation: 
“.  If a 10% increase in lipid levels was 
observed during the Visit 7 hospital stay 
(from Day to Day 3), we may repeat the 
lipid levels (LDL cholesterol) on Visit 8. 
 
.” 
 
04-14-2015 
1.3 
Section 8.6 
Stopping Rules 
In the unlikely event that a 
study-related death or SAE 
occurs, the decision to stop 
the trial, either temporarily or 
permanently, will be the 
responsibility of the 
Added 
pre-specified 
clinical 
and 
laboratory 
parameters 
for 
stopping 
infusion or study participation (per FDA 
recommendation dated 12-23-2014). 
”Clinical or laboratory excursions that a 
subject 
may 
experience 
that may 


NIH_ERIT-002 
 
Page 44 
Version: 2.0 
CONFIDENTIAL 
Designated Safety Officer in 
collaboration with the 
Sponsor Investigator. 
warrant stopping study participation or 
drug administration, either temporarily 
or permanently, include but are not 
limited to: 
 
Clinical criteria:  
• Fever with a temperature >101°F 
• Allergic reaction to drug 
• Blood pressure >160/95 or <80/50 
• Severe vomiting 
• Phlebitis grade 4-5 (see Attachment 
G.) 
 
Laboratory criteria: 
• Liver function test results >2x normal 
range 
• WBC ≥14,000 or ≤3,000 
• HCT ≥55 or ≤33:” 
 
In the unlikely event… 
 
04-14-2015 
1.3 
Section 9.2, 
Source 
Documents 
Print all entries legibly in 
black ink. 
Print all entries legibly in blue or black 
ink. 
04-14-2015 and 
05-12-2015 
1.3 
Section 12.3, 
Subject Stipends 
or Payment 
 
Added an Unscheduled Visit Payment 
of up to $100 for unusual circumstance 
04-14-2015 
1.3 
Table of 
Contents and 
ATTACHMENTS 
Attachment C – Event 
Schedule 
Changed Attachment C – Interval for 
infusion visits changed from 3-4 weeks 
to 3-6 weeks after previous visit.  
Added placeholder for ATTACHMENT 
G – Grading Scale for Phlebitis and 
moved Summary of Protocol Changes 
to Attachment H position 
05-12-2015 
1.3 
Title page 
version number 
Version 1.3 was modified 
twice, on 04-20-15 and 
05-12-2015 (NM) without 
changing the version 
number 
Should have been v 1.4 and dated 05-
12-2015 
06-15-2015 
1.5 
Title Page 
Amended: v1.4_05-05-
2105 
Amended: v1.5_06-15-2015 
06-15-2015 
1.5 
Header 
Version 1.3 
Version 1.5 (updated, was not changed 
with version 1.4) 
06-15-2015 
1.5 
Footer 
14-497H, Musi, Form BB, 
05-07-2015, AMD 
14-497H, Musi, Form BB, 06-15-2015, 
AMD (updated) 
06-15-2015 
1.5 
Section 3.4 
 
Added return to insert a line space 
between risk categories where none 
existed before 
06-15-2015 
1.5 
Page 17 
Extra white space before 
“For visit 3…” and after 
the figure “Schema of Visit 
3. 
Removed extra white space 
06-15-2015 
1.5 
Section 6.3, Visit 
3 – Infusion 
Protocol 
“Subjects will be given 
lunch at 1PM…” 
Remove phrase: “…including a CBC 
and lipid panel obtained to assess 
effects on blood cell count and lipid 
metabolism”… 
06-15-2015 
1.5 
Section 8.2, 
Recording 
Adverse Events 
 
Add sentence: “A summary of AEs 
tracked…” 
06-15-2015 
1.5 
Section 8.3.1, 
Investigator 
reporting: 
notifying the 
 
1) 
Add paragraph: “The Sponsor 
Investigator will comply…” 
2) 
Change sentence regarding study-
related UPIRSO: Replace “Any” 


NIH_ERIT-002 
 
Page 45 
Version: 2.0 
CONFIDENTIAL 
study sponsor 
with “A” and replace “and” with 
“involving” 
Add sentence: “A summary of non-
serious AEs will be reported…”  
06-15-2015 
1.5 
Section 8.4, 
Medical 
Monitoring 
 
Add paragraph: “Twice a year the 
Sponsor Investigator will provide study 
safety information to the Designated 
Safety Officer…” 
06-15-2015 
1.5 
Section 8.5.2-
8.5.4 
Medical Safety Monitor or 
Safety Officer 
Replace with “Designated Safety 
Officer” 
06-15-2015 
1.5 
Section 9.2, 
Source 
Documents 
Print all entries with black 
ink 
Change: Print all entries with “blue or 
black ink.” 
08-05-2015 
1.6 
Title page, 
headers and 
footers 
v1.5, <date> 
Updated in all places  
to v1.6, 08-05-2015 
08-05-2015 
1.6 
Study Summary 
Study Duration… 4 -4[sic] 
months 
Duration extended: 4-6 months 
08-05-2015 
1.6 
Study Design 
Study Design. The proposed 
study is a randomized, 
crossover, double blind, 
placebo-controlled study. 
Each subject will undergo 6 
study visits over a period of 3 
– 4 months.    
 
Deleted sentence, kept subheading 
Study Design. Redundant statement of 
previous paragraph. 
08-05-2015 
1.6 
Section 3.4, 
Potential Risk 
Muscle Biopsy 
Added: “There is the possibility that a 
future biopsy may not be done at the 
discretion of the PI in case the subject 
did not tolerate well a prior biopsy.” 
 
08-05-2015 
1.6 
Section 6, Study 
Visits, pages 16-
18 
6.2…3-14 days  
6.3, 6.5…3-30 days 
Change: Extend visit window between 
V1-V2 to 3-45 days and V2 –V3 to 3-45 
and 4-10 weeks between inpatient visits 
3 and 5. 
08-05-2015 
1.6 
Section 8.3.1 
(formatting change) 
¶3, page 21, word spacing and 
punctuation corrected 
02-10-2016 
2.0 
Cover page and 
headers to 
update protocol 
version and IND 
version 
v1.6 08-06-2015 
(proposed change to 
inclusion/exclusion criteria) 
Updated protocol versioning and serial 
IND numbers consistently: 
-Protocol v1.6 with 123955-0005 per 
FDA Annual Report 10-06-15 (advance 
in IND# due admin update of protocol to 
v1.6.1).  
-Protocol v1.6 with IND 123955-0006 to 
FDA on 11-12-15 extended drug expiry 
date (advance in IND due admin update 
of protocol to v1.6.2)  
-Protocol v2.0 will become IND 123955 
-0007 per changes listed below 
02-10-2016 
2.0 
Study Summary, 
Exclusion 
Criteria 
 
v1.6 08-06-2015 - Current 
statement reads: “The only 
anti-diabetic agents allowed 
for T2DM subjects will be 
sulfonylureas because of 
their negligible anti-
inflammatory properties.”  
Page 1, Exclusion Criteria - Revised 
statement to read: “The only allowed 
anti-diabetic 
agents 
will 
be 
sulfonylureas and metformin.” 
02-10-2016 
2.0 
Sections 4.2 and 
5.6, Exclusion 
Criteria 
v1.6 08-06-2015 – Current 
statement reads: 
“Subjects on sulfonylureas 
are eligible.” 
Pages 13 and 16, Exclusion Criteria - 
revised statement to read: “Subjects on 
sulfonylureas and/or metformin are 
eligible.” 
02-10-2016 
2.0 
Section 6.3 – 
Visit 3 Infusion 
Protocol 
v1.6 08-06-2015 
Page 18, adjusted spacing and lines to 
improve where page break occurs 
02-10-2016 
2.0 
Section 7.1, 
V1.6 08-06-2015 – 
Page 19, revised statement to read: 


NIH_ERIT-002 
 
Page 46 
Version: 2.0 
CONFIDENTIAL 
Statistical Plan 
Current statement reads: 
“…enroll ~88 subjects.” 
“…enroll ~90 subjects” to be consistent 
throughout document and consent form.