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: 10
Healthy volunteers: yes

Brief summary:
The purpose of this study is to determine whether pharmacologic inhibition of Toll-like receptor 4 (TLR4) with eritoran for injection (E5564) prevents lipid-induced insulin resistance in lean, normal glucose tolerant (NGT) subjects.

Detailed description:
E5564 = Eritoran

Arms:
arm 1: IV administration of 5% Dextrose in water/D5W (vehicle) 12 mg every 12 hours plus Saline infusion at a rate of 30 mL/hour arm 2: IV Administration of 5% Dextrose in water /D5W (vehicle) 12 mg plus Intralipid infusion at a rate of 30 ml/hour arm 3: IV administration of Eritoran 12 mg every 12 hours plus Intralipid infusion at a rate of 30 ml/hour

Interventions: intervention 1: Eritoran intervention 2: D5W (5% Dextrose in water)
Intervention descriptions: intervention 1: Eritoran = E5564 Eritoran is a pharmacologic inhibitor of TLR4. intervention 2: D5W = 5% Dextrose Water Vehicle

Protocol document:
Prot_SAP_000.pdf: 
 
14-497H, Musi, Form BB, 02-10-16, AMD 
CONFIDENTIAL 
CLINICAL RESEARCH PROTOCOL 
NIH 
 
 
COVER PAGE FOR PROTOCOL AND STATISTICAL ANALYSIS 
PLAN 
 
 
 
 
 
Official Study Title: THE ROLE OF TLR4 ON LIPID-INDUCED  
INSULIN RESISTANCE (NIH_ERIT-001) 
 
 
NCT number: NCT02321111 
 
IRB Approval Date: 02-10-2016 


NIH_ERIT-001 
 
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CONFIDENTIAL 
_ERIT-001 
 
 
 
THE ROLE OF TLR4 ON LIPID-INDUCED  
INSULIN RESISTANCE (NIH_ERIT-001) 
 
 
 
 
 
 
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-001  
IND Number: 
123955-0007 
IRB Tracking Number: 
20140497H 
NCT#: 
02321111 
 
 
 
 
 
 
 
Initial version: 
v1.0_08.27.2014 
Amended: 
v1.7_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 .......................................................................................... 12 
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 ...................................................................................................... 1617 
6.4. 
VISIT 4 ............................................................................................................................................. 18 
6.5. 
VISIT 5 ............................................................................................................................................. 18 
6.6. 
VISIT 6 ............................................................................................................................................. 18 
6.7. 
VISIT 7 ............................................................................................................................................. 18 
6.8. 
VISIT 8 ............................................................................................................................................. 18 
7. 
STATISTICAL PLAN .............................................................................................................................. 18 
7.1. 
SAMPLE SIZE DETERMINATION ........................................................................................................... 18 
7.2. 
STATISTICAL METHODS ..................................................................................................................... 19 
8. 
SAFETY AND ADVERSE EVENTS ....................................................................................................... 19 


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8.1. 
DEFINITIONS ..................................................................................................................................... 19 
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 .............................................................. 22 
8.3.3. 
Sponsor reporting: Notifying the FDA .................................................................................... 23 
8.4. 
MEDICAL MONITORING ...................................................................................................................... 23 
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  ............................................................................................................................. 24 
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 ......................................................................................................................... 25 
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 ............................................................................................................................ 27 
12.1. 
FUNDING SOURCE ............................................................................................................................. 27 
12.2. 
CONFLICT OF INTEREST ..................................................................................................................... 27 
12.3. 
SUBJECT STIPENDS OR PAYMENTS .................................................................................................... 28 
13. 
PUBLICATION PLAN ......................................................................................................................... 28 
14. 
REFERENCES ................................................................................................................................... 28 
15. 
ATTACHMENTS ................................................................................................................................. 28 
ATTACHMENT A. MATERIAL TRANSFER AGREEMENT ........................................................................... 29 
ATTACHMENT B. EISAI, INC., SERIOUS ADVERSE EVENT REPORTING FORM .................................... 30 
ATTACHMENT C. STUDY EVENT SCHEDULE ............................................................................................ 31 
ATTACHMENT D. SAMPLE CONSENT FORM ............................................................................................. 32 
ATTACHMENT E. REFERENCES .................................................................................................................. 33 
ATTACHMENT F. INVESTIGATOR BROCHURE .......................................................................................... 38 
ATTACHMENT G. GRADING SCALE FOR PHLEBITIS ............................................................................... 39 
ATTACHMENT H. SUMMARY OF PROTOCOL CHANGES ......................................................................... 40 
 


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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 
 
 
 


NIH_ERIT-001 
 
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14-497H, Musi, Form BB, 02-10-16, AMD 
CONFIDENTIAL 
       Study Summary 
Title 
The Role of TLR4 on Lipid-Induced Insulin Resistance 
Protocol Number 
NIH_ERIT-001 
Phase 
Phase II 
Methodology 
Lean, normal glucose tolerant subjects will receive I.V. Intralipid (or saline) for 
72 h to induce insulin resistance.  The study will examine whether co-
administration of the TLR4 inhibitor eritoran (E5564/eritoran for injection) 
prevents/ameliorates lipid-induced insulin resistance.  Subjects will be 
randomized to receive I.V. eritoran or vehicle (D5W) in a double blind design. 
Study Duration 
This study is funded by a 4 year NIH grant.  Each subject will undergo 8 study 
visits over a period ranging between 4 and 9 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) 
prevents lipid-induced inflammation and insulin resistance in lean, normal 
glucose tolerant subjects. 
Number of Subjects 
20 completers (30 consented assuming ~30% screen failure or withdrawals) 
Inclusion Criteria 
Lean, healthy, aged 18-65 years, normal glucose-tolerant, without a family 
history of T2DM; all ethnicities; both genders; premenopausal women must be 
non-lactating and non-pregnant; postmenopausal women on stable dose of or 
not exposed to hormone replacement for ≥6 months; with labs: HCT ≥34%, 
serum Cr ≤1.4 mg/dL, normal electrolytes, normal urinalysis, and normal 
coagulation tests. Liver function tests up to 2x normal range, stable body 
weight (±1.5%) for ≥3 months and ≤1 exercise session/week for last 6 months.  
 
Exclusion Criteria 
Presence of diabetes or impaired glucose tolerance based on American 
Diabetes Association (ADA) criteria; family history of diabetes; Subjects with 
fasting triglycerides >200mg/dL at screening; Drugs that affect glucose and 
lipid homeostasis (subjects on a stable dose of statin >3 months are eligible); 
family history of hyperlipidemias; Non-steroidal anti-inflammatory drugs or 
systemic steroid use for >1 wk within 3 months; History of coagulopathy; 
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 heart disease (NY Classification > class II), peripheral 
vascular disease, pulmonary disease, smokers; Poorly controlled blood 
pressure (systolic BP>160, diastolic BP>90 mmHg); Active inflammatory, 
autoimmune, infectious, hepatic, gastrointestinal, malignant, and 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 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.    


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     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).   
       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.   
 


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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-001) will test whether a selective TLR4 
antagonist will protect against lipid-induced 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. 4A  
Fig. 4B  
Fig. 5A 
Fig. 5B 
Fig. 5C 


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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 increasesTLR4 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. 
 
 
g) Plasma LPS concentration is 
increased in obese and T2DM individuals. We measured LPS concentration in 
Fig. 6A 
Fig. 6C 
7B 
Fig. 8 
Fig. 9 
Fig. 6B 


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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 (10B) (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). Male Sprague-
Dawley rats (~200 g) were pre-treated with eritoran (E5564) (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 
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 
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, in this study (NIH_ERIT-001 we will test 
the hypothesis that blocking TLR4 will prevent the insulin resistance caused by lipids in lean normal glucose-
tolerant human subjects. In project NIH_ERIT-002, 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 
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 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 (E5564) prevents lipid-
induced insulin resistance in lean normal glucose-tolerant subjects. Rationale. Most obese and 
T2DM subjects have elevated concentrations of nutritional FFA [17]. In insulin resistant subjects, an acute 
reduction in plasma FFA improves insulin sensitivity [18, 19], and an experimental elevation of plasma FFA in 
normal glucose-tolerant subjects rapidly induces insulin resistance [20, 21]. Despite the considerable evidence 
implicating lipids (i.e. FFA) in the pathogenesis of insulin resistance, how lipids impair insulin action, 
particularly in humans, is not well understood. Two prevailing hypotheses have been proposed to explain how 
lipids impair insulin action. One hypothesis suggests that lipid metabolites (diacylglycerol, ceramides) activate 
Fig.16A 
Fig.16B 


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signaling intermediaries such as MAPKs, IKK-NFKB, and PKCs that interfere with the insulin-signaling pathway 
[53]. Another hypothesis proposed more recently considers FFA as ligands of TLR4, which also impair insulin 
action by stimulating pro-inflammatory pathways (NFKB, MAPK) [25, 26]. To date, this hypothesis has not 
been directly tested in human subjects. To test this hypothesis, here we will use a selective TLR4 
antagonist. 
 
3. Study Design 
 
3.1. 
Subjects 
20 healthy, lean (BMI <26 kg/m2) normal glucose-tolerant subjects (completers) without a family history of 
T2DM will be enrolled (~50% males). Subjects will be studied in the Bartter Research Unit (BRU), which is the 
Clinical Research Center located in the Audie L. Murphy VA Medical Center in San Antonio, TX.  Subjects will 
be recruited through local advertisement. ~30 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 8 study visits over a period of 4-5 months.    
 
Intralipid 20% (Baxter, Deerfield, IL)] or Saline (control) will be infused at a rate of 30 ml/h for 72 h to raise 
plasma FFA from ~300-350 to 450-600 M, which are the concentrations typically seen in obesity and T2DM. 
Eritoran or vehicle (D5W), randomly assigned, will be administered at a dose of 12 mg every 12 h to block 
TLR4.  Each eritoran dose is diluted in 100 ml of D5W and infused over 4 h.   
 
Following a screening period, which can be up to  three months, each subject will be admitted  to the BRU on 3 
different occasions (separated by 4-10 weeks each) to receive the following 3 infusion protocols (in random 
order): 1) Vehicle + 0.9% Saline; 2) Vehicle + Intralipid 20%; 3) eritoran + Intralipid 20%. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fig. 17.  Study Design.  Subjects will be admitted to the clinical research center (Bartter Research Unit) on 3 
different occasions (randomly assigned), separated by  4-10 weeks, to receive the 3 protocols shown above. 
At the end of the infusions, insulin sensitivity will be measure with a 2-stepped insulin clamp.  Mus 
biop=muscle biopsy.   


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3.3. 
Study Endpoints 
Primary outcome:  Protective effect of eritoran on muscle insulin action (insulin signaling and glucose 
disposal) and hepatic insulin sensitivity (insulin suppression of EGP) in response to a lipid infusion. 
 
Secondary outcomes:  Effect of eritoran on: 
- Inflammatory signaling in muscle: TLR4 signaling and inflammatory gene expression. 
- 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 792 mL.  Hematology, metabolic panel and lipids will be repeated during the Infusion 
Visit to assess effects on blood cell count, liver function and lipid metabolism. 
 
c) IV lines.  Catheters will be placed in an antecubital vein and a hand vein for the euglycemic clamps.  
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.  
 
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) Subjects will be exposed to radioactivity during these studies.  The radiation exposure from tritiated 


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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) No family history of diabetes. 
3) Normal glucose tolerance determined by OGTT. 
4) Both genders. 
5) Age=18-65 years. Older subjects are excluded because aging is a pro-inflammatory state. 
6) All ethnic groups. 
7) 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. 
8) Lab: Hematocrit ≥34%, serum creatinine ≤1.4 mg/dl, normal electrolytes, urinalysis, and 
coagulation tests. Liver function tests up to 2X normal range. 
9) Stable body weight (±1.5%) for ≥3 months. 
10) One or less sessions of strenuous exercise/wk for last 6 months. 
4.2.      Exclusion Criteria 
1) Presence of diabetes or impaired glucose tolerance based on American Diabetes Association 
(ADA) criteria; family history of diabetes. 
2) Subjects with fasting triglycerides >200mg/dL at screening will be excluded. Current treatment 
with drugs known to affect glucose and lipid homeostasis. Subjects on a stable dose of statin 
(>3 months) are eligible. Subjects with a family history of hyperlipidemias will be excluded. 
3) Non-steroidal anti-inflammatory drugs or systemic steroid use for more than 1 week within 3 
months 
4) History of coagulopathy will be excluded. 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.  
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 
psychiatric disease. 
4.3. 
Subject Recruitment and Screening 
Subjects will be recruited by advertisement and through the outpatient clinics of the South Texas 


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Veterans Health Care System and the Texas Diabetes Institute (TDI) in San Antonio, TX.  
 
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 CRC 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.  Only subjects with normal glucose 
tolerance based on ADA criteria will be enrolled.  
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 (E5564, eritoran for injection) is a drug originally developed by Eisai Pharmaceuticals 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). 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 


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tetrasodium (as the tetrasodium salt) or 6.5 mg of eritoran (as the free acid) active packaged in a 10-mL 
glass vial.  
 
 
 
 
5.2. 
Treatment Regimen 
All subjects will receive the following infusions separated by  4-10 weeks, at Visits 3, 5 and 7 (in 
random order): 
1) Vehicle (D5W) + Normal Saline (0.9% sodium chloride)  or 
2) Vehicle + Intralipid or 
3) Eritoran + Intralipid  
Infusion Protocol. Intralipid (Baxter, Deerfield, IL)] or normal saline (control) will be infused at a rate 
of 30 ml/h for 72 h to raise plasma FFA from ~300-350 to 450-600 µM, which are the concentrations 
typically seen in obesity and T2DM. Eritoran or vehicle (D5W), randomly assigned and blinded, will be 
administered every 12 h to block TLR4.  Each subject will receive a total of 6 (12 mg) I.V. doses (72 
mg total).  Each dose is diluted in 100 ml of D5W and infused over 4 hours. 
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 source 
documents, data records and specimens in a de-identified method using only the unique Subject 
ID/Randomization number. 
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 
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. 


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5.7. 
Packaging    
Per MTA, Eisai will supply a total of 1,220 7mg vials of Eritoran 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 of diluent (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 
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. 


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After reconstitution and admixture, storing solutions of Eritoran will be limited to 4 hours at 15-30°C or 
up to 24 hours refrigerated at 2-8°C. 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 maintains 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. 
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. 
 
After eligibility requirements for the study are completed, and if there are abnormal lab results during 
the course of the study, it may be necessary to repeat a lab test to ensure subject safety during 
participation in the study. 
 
6.3. 
Visit 3 – Infusion Protocol 
Visit 3: Within 3-45 days after Visit 2, subjects will return to the BRU for Visit 3.  During this Visit, 
subjects will undergo one of the 3 infusion protocols shown below (each subject will be admitted 
three times to receive the 3 different protocols, separated by 4-10 weeks). Hematology, 


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comprehensive metabolic panel, including liver function tests and lipid panel will be performed prior 
to the infusion at Visits 3, 5 and 7 (on Days 1, 2 and 3). If LDL cholesterol increases more than 10% 
from Day 1 to Day 3 (on Visit 3, 5 or 7), lipids will be re-assessed on the following visit. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
For Visit 3, subjects will be admitted to the BRU at approximately 7am. At ~10 AM (Time=0), 
eritoran or vehicle will be administered at a dose of 12 mg IV every 12 h. At ~12 PM (T= +2 h), 
subjects receive Intralipid 20% or saline (Baxter, Deerfield, IL) via an antecubital vein at a 30 ml/h 
rate for 72 h. While in the BRU, subjects remain ambulatory and are fed 3 times a day based on 
their habitual diet and food preferences, except that total oral caloric intake is decreased 
appropriately to maintain a neutral energy balance. 68 h after starting Intralipid/saline (T= +70 h; 8 
AM), a 2-step euglycemic clamp with tritiated glucose is done. During Step 1 (0-120 min) insulin is 
infused at 20 mU/m2.min rate to measure hepatic insulin sensitivity. During Step 2 (120-240 min) 
insulin is infused at a 80 mU/m2.min rate to measure peripheral (muscle) insulin sensitivity (61). 
Indirect calorimetry is performed three times for 30 minutes: immediately before the clamp and at 
the end of each clamp step. Vastus lateralis muscle needle biopsies are performed just before and 
at the end of the clamp (62). 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).   
 
Analyses.  
Muscle: On the basal (pre-clamp) muscle samples we will perform assays of TLR4 signaling [TLR4 
content, IKK/NFB, MAPK (JNK, p38, ERK) phosphorylation/activation; ~50 mg tissue], inflammatory 
Fig. 18. Schema of Visit 3.  On Visit 3, each subject will undergo one of these 3 infusion protocols 
(randomly assigned).  Each subject will be admitted 2 more times (on Visits 5 and 7) to receive the other 
2 infusions, determined randomly.   


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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, 62, 63). Insulin signaling assays will 
be done on pre-clamp and insulin-stimulated muscle samples (~30 mg), as described (64).   
  
Blood/plasma: Plasma insulin, FFA and tritiated glucose are assayed before and every 10 min during 
the clamp. Plasma FFA also are measured at ~6 AM and 6 PM daily. Cytokines are assayed before 
administration of vehicle/eritoran and saline/intralipid, and before/after the clamp, using a multianalyte 
(45 assays) platform (Myriad-RBM, Austin, TX). Plasma LPS (Lonza, Walkersville, MD) and LBP (Cell 
Sciences, Canton, MA) are assayed at baseline because their concentration may affect basal TLR4 
signaling. Monocyte TLR4 expression and TLR4 signaling (MAPK and NFB phosphorylation) will be 
performed by fluorescence activated cell sorting (FACS) in samples collected before Intralipid/saline 
and before the clamp. To confirm the inhibitory effect of eritoran, an ex vivo LPS stimulation test is 
done in whole blood (59). 
 
6.4. 
Visit 4 
5-7 days after Visit 3 all subjects come to the BRU for inspection of the biopsy sites. 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. Subjects will maintain an isocaloric diet and will refrain from regular exercise 
until the next infusion protocol. . 
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 the lipid 
levels increased >10% during the Visit 5 hospital stay (from Day 1 to Day 3 measurements), we may 
repeat lipids (LDL cholesterol) at Visit 6. Subjects will maintain an isocaloric diet and will refrain 
from regular exercise until the next infusion protocol.  
6.7. 
Visit 7 
Approximately 4-10 weeks after Visit 6 all subjects come to the BRU to undergo the third infusion 
protocol (same as Visit 3).  
6.8. 
Visit 8 
5-7 days after Visit 7 all subjects come to the BRU for inspection of the biopsy sites.  A final 
lipid profile will be performed if LDL elevations of >10% over baseline were observed during 
the study. 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. 
 
7. Statistical Plan 
7.1. 
Sample Size Determination 
The primary hypothesis is that the TLR4 antagonist will attenuate the negative effect of Intralipid on 
insulin sensitivity. In prior studies conducted in lean subjects (64), the effect of Intralipid on insulin 
sensitivity was a mean decrease in glucose disposal (Rd) of 1.6 (=9.9-8.3) mg/kg.min with a SD of 2.4. 
If the TLR4 inhibitor preserves insulin sensitivity with a similar variability, the effect size would be 
approximately 0.7 (1.6/2.4) assuming a within-subject correlation of 0.5. In preclinical studies with rats 
(Figure 14), eritoran had a comparable effect size of 0.83 in preserving insulin sensitivity. Hence, with 
n=20 completers and an effect size of 0.7, a paired t-test of the within-subject difference would achieve 


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a power of 84% with a two-sided, Type I error of 0.05. We will enroll ~30 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; 
• 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 


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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 an 
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 
recorded immediately in the source document, and also in the appropriate AE section of the case report 
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 


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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.  
 
  
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 this Protocol in Section 15 as 
Attachment B.  The investigator will keep a copy of this SAE form on file at the study site.   
 
Sally T. Ishizaka, Ph.D. 
Sally_Ishizaka@eisai.com 


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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. 
 
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. 


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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 
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 


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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, if 
applicable, with the 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  
 
• 
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 5 (see Attachment G.) 


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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 was not asked, write 
“N/D”.  If the item is not applicable to the individual case, write “N/A”.   
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 


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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 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.  


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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 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. 


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12.3. Subject Stipends or Payments 
This study will reimburse subjects for time and transportation.  A schedule of payments is shown below. The 
total potential reimbursement to a subject is $2,490 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     
Visit 7  
$800   
Visit 8  
$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.E. 
 
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 
E. References 
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 CONSENT FORM 


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ATTACHMENT E. REFERENCES 
 
 
 
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training improves insulin sensitivity but does not inhibit inflammatory pathways in immune cells from insulin-
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activity in muscle from obese and type 2 diabetic subjects under basal and exercise-stimulated conditions. Am 
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63. 
Sriwijitkamol A, Christ-Roberts C, Berria R, Eagan P, Pratipanawatr T, DeFronzo RA, et al. Reduced 
skeletal muscle inhibitor of kappaB beta content is associated with insulin resistance in subjects with type 2 
diabetes: reversal by exercise training. Diabetes. 2006;55(3):760-7. PubMed PMID: 16505240. 
64. 
Liang H, Tantiwong P, Sriwijitkamol A, Shanmugasundaram K, Mohan S, Espinoza S, et al. Effect of a 
sustained reduction in plasma free fatty acid concentration on insulin signalling and inflammation in skeletal 
muscle 
from 
human 
subjects. 
J 
Physiol. 
2013;591(Pt 
11):2897-909. 
Epub 
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doi: 
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10.1113/jphysiol.2012.247510. PubMed PMID: 23529132; PubMed Central PMCID: PMC3690693. 
 
 


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ATTACHMENT F. INVESTIGATOR BROCHURE 


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ATTACHMENT G. GRADING SCALE FOR PHLEBITIS  


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ATTACHMENT H. SUMMARY OF PROTOCOL CHANGES 
 
Date of Change 
Version 
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 
NCT#: Pending 
12-03-2014 
1.2 
Title Page 
Funding Sponsor:  
US National Institutes 
of Health/National 
Institute of Diabetes 
and Digestive and 
Kidney Diseases (NIH-
NIDDK), Grant number  
Deleted “Grant number” from 
Funding Sponsor:  
US National Institutes of Health/National 
Institute of Diabetes and Digestive and 
Kidney Diseases (NIH-NIDDK) 
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 
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 
Section 5.2, 
Treatment Regimen, 
page 13 
Hanging sub header at 
bottom of page   
Moved sub header to correct position at top 
of page 14 
12-03-2014 
1.2 
6.3 Visit 3 – Infusion 
Protocol, page 17 
(62). 
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 
Title Page, Study 
Summary, Sections 
1.4, 5.1, 5.7 
ERITORAN (eritoran 
injection)  
Changed to ERITORAN (eritoran for 
injection) per Eisai email request  
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 
Title Page 
 
Added  IRB Tracking Number 20140497H 
02-26-2015 
1.3 
Study Summary, 
Inclusion Criteria 
Age 18-60 
Age 18-65 
02-26-2015 
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. 
Hematology, comprehensive metabolic 
panel with lipids and liver function tests will 
be performed during the Infusion Visit to 
assess effects on blood cell count, liver 
function and lipid metabolism. 
02-26-2015 
1.3 
Title Page 
NCT# Pending 
NCT#: 02321111 
02-26-2015 
1.3 
4.1 Inclusion 
Criteria 
1) 
Subjects capable of 
giving informed 
consent. 
2) 
Normal glucose 
tolerance determined 
1) 
Subjects capable of giving informed 
consent. 
2) 
Added: No family history of diabetes. 
3) 
Renumbered: Normal glucose 
tolerance determined by OGTT. 


NIH_ERIT-001 
 
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Version: 1.7 
CONFIDENTIAL 
by OGTT. 
5) 
Age 18-60 
 
5) 
Age 18-65 
02-26-2015 
1.3 
4.2 Exclusion 
Criteria 
1) 
Presence of diabetes 
or impaired glucose 
tolerance based on 
American Diabetes 
Association (ADA) 
criteria. 
2) 
Current treatment 
with drugs known to 
affect glucose and 
lipid homeostasis. 
Subjects on a stable 
dose of statin (>3 
months) are eligible.  
3) 
Non-steroidal anti-
inflammatory drugs or 
systemic steroid use 
for more than 1 wk 
within 3 months 
4) 
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) 
Presence of … (ADA) criteria; Added 
family history of diabetes. 
2) 
Added “Subjects with fasting 
triglycerides >200mg/dL at screening 
will be excluded.” Current…” Subjects 
with a family history of hyperlipidemias 
will be excluded.” 
3) 
Removed abbreviation of “wk” to read 
more clearly. “Non-steroidal … 1 week 
within 3 months 
4) 
Added: “History of coagulopathy will be 
excluded.” Current treatment … 
biopsies 
02-26-2015 
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.  
02-26-2015 
1.3 
Section 6.3 and 6.8 
Visit 3… below (each 
subject will be admitted 
three times to receive the 
3 different protocols, 
separated by 3-4 weeks) 
Indirect calorimetry is 
performed for 30 
minutes: immediately 
before the clamp and at 
the end of each clamp 
step. 
 
Increased length of time between infusion 
visits from 3-4 weeks to 3-6 weeks after 
previous visit. 
Indirect calorimetry is performed three 
times for 30 minutes: immediately before 
the clamp and at the end of each clamp 
step. 
6.3 Added blood draws for safety to Visit 
3 and Visit 8 per FDA recommendation. 
“Hematology, comprehensive metabolic 
panel, including liver function tests and 
lipid panel will be performed prior to the 
infusion at Visits 3,5 and 7 and lipids 
assessed upon final follow up at Visit 8.” 
6.8 A final lipid profile will be performed if 
LDL elevations of >10% over baseline 
were observed during the study. 
02-26-2015 
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 
Designated Safety Officer 
in collaboration with the 
Sponsor Investigator. 
 
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 warrant 
stopping study participation or drug 
administration, either temporarily or 
permanently, include but are not limited 
to: 
 
Clinical criteria:  


NIH_ERIT-001 
 
Page 42 
Version: 1.7 
CONFIDENTIAL 
• Fever with a temperature >101°F 
• Allergic reaction to drug 
• Blood pressure >160/95 or <80/50 
• Severe vomiting 
• Phlebitis grade 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… 
 
02-26-2015 
1.3 
Participant payment 
 
Added a Manual payment up to $100 
02-26-2015 
1.3 
Table of Contents 
and 
ATTACHMENTS 
Attachment C – Infusion 
Visits 3, 5 and 7. 
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 
05-07-2015 
1.4 
Section 6, Visits 2 
and 3 
Within 3-14 days after 
Visit 1… 
Within 3-14 days after 
Visit 2,… 
Extend visit windows:  
 
Within 3-30 days after Visit 1,… 
Within 3-30 days after Visit 2,… 
05-07-2015 
1.4 
Section 6, Screening 
Visit 1 
 
Added: 
At the discretion of the PI, blood 
coagulation studies (platelets, PT, PTT) 
may be repeated every 4-6 months during 
the study.  
 
Added: 
After eligibility requirements for the study 
are completed, and if there are abnormal 
lab results during the course of the study, it 
may be necessary to repeat a lab test to 
ensure subject safety during participation in 
the study. 
 
05-07-2015 
1.4 
Section 6, Visit 4 
 
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. 
05-07-2015 
1.4 
Section 6, Visit 6 
 
Added: 
If the lipid levels increased >10% during 
the Visit 5 hospital stay (from Day 1 to Day 
3 measurements), we may repeat lipids 
(LDL cholesterol) at Visit 6.  
05-07-2015 
1.4 
Section 6, Visit 8 
 
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. 
 
05-07-2015 
1.4 
Section 3, 3.4.b - 
Blood withdrawal 
The maximal amount of 
blood to be drawn during 
the entire study for any 
given subject will be 
approximately 780 mL. 
Changed total amount to 792 mL. 
06-15-2015 
1.5 
Title Page 
Amended: v1.4_05-05-
2105 
Amended: v1.5_06-10-2015 
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, 
14-497H, Musi, Form BB, 06-10-2015, 


NIH_ERIT-001 
 
Page 43 
Version: 1.7 
CONFIDENTIAL 
05-07-2015, AMD 
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 study sponsor 
 
1) 
Add paragraph: “The Sponsor 
Investigator will comply…” 
2) 
Change sentence regarding study-
related UPIRSO: Replace “Any” with 
“A” and replace “and” with “involving” 
3) 
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 -6 
months 
Duration extended: a period ranging from 4 
to 9 months. 
08-05-2015 
1.6 
Section 3.4, 
Potential Risk 
(formatting change) 
3.4(a) ¶2 paragraph spacing corrected 
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 5.8.2, 
Storage 
(formatting change) 
¶3, page 15, word spacing corrected 
08-05-2015 
1.6 
Section 6, Study 
Visits, pages 16,18 
6.3, 6.5, 6.7…3-30 days 
Change: Extend window to 3-45 days 
between V1-V2 and V2-V3, and 4-10 
weeks between inpatient visits 3 and 5, and 
between visits 5 and 7. 
08-05-2015 
1.6 
Section 8.3.1 
(formatting change) 
¶3, page 21, word spacing corrected 
02-10-2016 
1.7 
Cover page and 
headers to update 
protocol version and 
IND version 
v1.6 08-06-2015 
 
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 sent 
to FDA on 11-12-15 extended drug expiry 
date (advance in IND due admin update of 
protocol to v1.6.2)  
This version 1.7 under IND 123955-0007 
(IND per FDA 1571-0007 for ERIT-002)