The ENRGISE (ENabling Reduction of Low-Grade Inflammation in SEniors) Pilot Study
Phase: PHASE2
Conditions: Inflammation
Sponsor: University of Florida

 
  ENRGISE Pilot Study Protocol – Version 5.0 – 10/18/2016 
 
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The ENRGISE Pilot Study 
Protocol 
 
 
1. Project Title: 
 
The ENRGISE Pilot Study 
 
 
2. Investigators 
 
 
Dual PI grant:   
 
 
Marco Pahor PI (ACC) 
Walter Ambrosius PI (DMAQC)  
 
DMAQC: 
 
 
 
Michael Miller, Mark Espeland, Daniel Beavers, Kristina Lewis 
Biorepository:   
 
 
Russell Tracy 
ACC:  
 
 
 
Christiaan Leeuwenburgh, Samuel Wu 
 
Field Centers 
Wake Forest School of Medicine:  
Stephen Kritchevsky  
University of Florida:   
 
Todd Manini, Stephen Anton 
Northwestern University:  
 
Mary McDermott 
Tufts University:  
 
 
Roger Fielding, Christine Liu 
University of Pittsburgh:  
 
Anne Newman, Jane Cauley 
 
 
3. Abstract 
 
Growing evidence shows that low-grade chronic inflammation, characterized by elevations in plasma C-
reactive protein, tumor necrosis factor alpha, and particularly Interleukin-6 (IL-6), is an independent risk factor 
of disability, impaired mobility, and lower walking speed. Low-grade chronic inflammation is a modifiable risk 
factor. However, it is unknown whether interventions that reduce the levels of inflammatory markers per se 
improve mobility, or avert decline in mobility in older persons. 
To address this gap in evidence we conduct the randomized clinical trial ENRGISE (ENabling 
Reduction of low-Grade Inflammation in SEniors) Pilot Study to test the ability of anti-inflammatory 
interventions for preventing major mobility disability by improving or preserving walking ability. We have 
maximized the public health impact by selecting interventions that are safe, tolerable, acceptable, and 
affordable for vulnerable older persons. Specifically, in this trial we test the efficacy vs. placebo of the 
angiotensin receptor blocker losartan and omega-3 polyunsaturated fatty acids in the form of fish oil, alone and 
in combination. Both angiotensin receptor blockers and omega-3 polyunsaturated fatty acids have shown to 
reduce IL-6 in clinical trials and preliminary data suggest that they may improve physical function.  
We recruit older persons who are at risk for, or with, mobility impairment, as measured by slow gait 
speed and self-reported mobility difficulty, and who have elevated levels of IL-6, the marker most consistently 
associated with mobility limitations. Preliminary data regarding feasibility need to be gathered before such a 
trial can be effectively designed and implemented. We conduct The ENRGISE Pilot Study to assess the effects 
of the interventions on several inflammatory markers, walking speed, physical function and strength. This 
allows us to refine the design, recruitment yields, target population, adherence, retention, tolerability, sample-
size, and cost for the main ENRGISE trial.  
 
 

 
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4. Specific aims 
Growing evidence from our group and others shows that low-grade chronic inflammation, characterized by 
elevations in plasma C-reactive protein (CRP), tumor necrosis factor Alpha (TNF-α), and particularly 
interleukin-6 (IL-6),1-7 is an independent risk factor for disability, impaired mobility, and slow walking speed.1, 6-11 
Low-grade chronic inflammation is a modifiable risk factor. However, it is unknown whether interventions that 
reduce the levels of inflammatory markers per se improve mobility, or avert decline in mobility in older persons. 
To address this gap in evidence, we will conduct the main randomized clinical trial (RCT) ENRGISE 
(ENabling Reduction of low-Grade Inflammation in SEniors) to test the ability of anti-inflammatory 
interventions for improving or preserving walking ability. We have maximized the public health impact of the 
interventions by selecting interventions that are safe, tolerable, acceptable, and affordable for vulnerable older 
persons. Based on an extensive literature review, we test the efficacy of the angiotensin receptor blocker 
(ARB) losartan (LO) and omega-3 polyunsaturated fatty acids (ω-3) as fish oil. To maximize the anti-
inflammatory effect and potential benefit on mobility, we explore both individual and combination interventions 
that target different, but complementary and potentially synergistic anti-inflammatory mechanisms.12-15 We plan 
to recruit older persons at risk for, or with, mobility impairment, as measured by slow gait speed and self-
reported mobility difficulty, and who have elevated blood levels of IL-6 (>2.5 pg/ml), the marker most 
consistently associated with mobility limitations.1-7 Preliminary data on feasibility and information required for 
obtaining sample size estimates are needed to effectively design and implement the main trial. Thus, we 
implement the following specific aims for the ENRGISE Pilot Study: 
 
a. Conduct a pilot RCT in 300 older persons at risk of mobility decline to assess: (i) compared with placebo, 
the effects of LO, ω-3, and LO+ω-3 on IL-6 and walking speed; (ii) the recruitment yields, the target 
population, adherence, retention, tolerability, sample-size, design, and cost for the main RCT; and (iii) the 
intra-subject variability of IL-6, and the dosage and safety of the interventions.  
b. Measure novel and established inflammatory markers to:  
i. 
Characterize the effect of LO and ω-3 on these biomarkers and determine: (a) Are these 
cytokines/pathways impacted by the interventions? (b) Are the changes in these markers 
pre- vs. post-intervention less than, equivalent to, or greater than that of IL-6? and  
ii. 
Assess if these biomarkers yield independent information from IL-6 with respect to change 
in walking speed and/or provide benefit for screening participants for the main trial. 
 
ENRGISE addresses critical public health issues regarding mobility disability prevention. We test the anti-
inflammatory effects of widely available inexpensive interventions and their impact on mobility in a highly 
vulnerable population of older adults at risk of mobility disability.  
 
 
5. Background 
 
Mobility outcomes. Preserving mobility is central to maintaining a high quality of life and participation in 
activities to be fully independent in the community.16, 17 Mobility limitation is associated with subsequent 
hospitalization, nursing home placement, increased healthcare costs, and death.18-22 
Low-grade chronic inflammation and mobility outcomes. Acute inflammation is tightly regulated to promote 
healing and response to infection. In young, healthy persons, levels of inflammatory markers are low, while 
many older adults, even if apparently disease-free, have persistent, chronic, mild elevations of CRP, IL-6, TNF-
α, and other markers. The degree of these elevations predicts disability, mobility limitations1, 3 and mortality23-25 
independently of other risk factors. The basis for these elevations in older adults is not well understood. 
Smoking history and obesity,26 especially visceral adiposity,26 play a role, yet age itself remains the strongest 
predictor of these elevations in inflammatory markers.1 Studies of the aging process suggest that senescent 
cells secrete IL-6, initiated by IL-1α and regulated by TGFβ.27 Due to cell cycle arrest, senescent cells are less 
prone to malignant transformation, but may disrupt normal tissue function, promoting central adiposity, 
atherosclerotic plaque, and osteoporosis.27, 28 Reduced renal function29 and atherosclerosis30, 31 may contribute 

 
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to the elevation of inflammatory markers, through decreased excretion and increased production of these 
markers. Thus, the “chronic inflammation” of aging may indicate the ongoing process of age-related damage 
and repair, and thus, contribute to the cumulative burden of disease-related damage, and cellular senescence.  
The age-related dysregulation of immune function may have direct adverse consequences on physical 
function and promote disability by causing fatigue and loss of muscle strength.7, 32, 33 CRP has been identified 
as an independent risk factor for cardiovascular disease (CVD) outcomes,34-38 and clinical trials targeting the 
up-regulation of inflammation to reduce CVD events are currently underway.39-41 In aging, IL-6 is thought to 
have a direct impact on fatigue and loss of muscle function. Injection of IL-6 produces acute and chronic 
atrophy of skeletal muscle in rats.42 IL-6 levels tracked best with functional decline,1, 4 though many 
inflammatory markers are involved and related to frailty.43, 44 While conditions, such as myocardial infarction, 
stroke and arthritis, may contribute to disability and inflammation, the inflammatory state appears to have a 
direct downstream causal effect on disability. It follows that treatments that moderate the inflammatory pathway 
and reduce IL-6 may ameliorate mobility. Inflammatory pathways are tightly regulated so that manipulation 
could have adverse effects such as infection or delay of healing. Though exercise and weight loss both reduce 
IL-6 levels and improve mobility function providing circumstantial support,45, 46 to our knowledge, the hypothesis 
that IL-6 reduction improves mobility function has not been directly tested. 
 
Detrimental mechanisms of action of inflammation in older persons. The sources and consequences of 
chronic inflammation are important for identifying promising interventions (Figure 5.1.). Inflammation in older 
persons results from: genes and their modifiers, the environment, and the in situ biochemical environment. 
Important exogenous environments include smoking, moderate (beneficial) and excessive alcohol 
consumption, and an anti-oxidative diet (beneficial). Pro-inflammatory endogenous factors include adiposity 
(especially visceral adiposity), subclinical disease burden, and oxidative stress, among others.  
 Regarding direct consequences, activation of NF-κB signaling increases muscle protein degradation and 
inhibits myogenesis,47, 48 except in very late differentiation where it promotes homeostasis.47 NLRP3-mediated 
IL-1β production plays a major role in mediating age-associated inflammation; ablation of this complex 
improves inflammation-associated detrimental changes with aging.49 This inflammosome is activated by 
several products of damage including certain free fatty acids, free cholesterol, and reactive oxygen species.49 
The cytokines TNF-α and IL-1β inhibit hormones critical for muscle homeostasis (e.g., growth hormone 
and IGF-1).47 Circulating IL-6 is negatively associated with myosin heavy chain synthesis in patients with heart 
failure.50 TNF-α activates muscle matrix metalloproteinase-9  (MMP-9), which degrades the extracellular 
matrix,51 and can cause muscle cell apoptosis, which is attenuated by caloric restriction.52 Cytokines also 
augment muscle infiltration by proinflammatory macrophage and lymphocytes,47 including proinflammatory 
senescence-associated secretory phenotype (SASP) cells.53 Aging muscle has accelerated mitochondria-
mediated apoptosis and mitophagy linked to increased oxidative stress.54  
ω-3 fatty acids are indicated to reduce triglyceride levels in adults with severe hypertriglyceridemia. ω-3 
fatty acids are also anti-inflammatory 55 via a specific ω-3 fatty acid receptor, GP120 56 which blocks NF-κB 
and JNK signaling, counter to saturated fatty acid-mediated proinflammatory TLR2 signaling.57 Saturated fats 
Genetic & Epigenetic Factors
Exogenous Factors
Smoking, Air pollution, Alcohol, 
Infectious burden, diet*
Endogenous Factors
Adiposity , Subclinical disease 
burden*, Oxidative stress status*, 
Innate and adaptive immune 
response capacity, hypertension
SOURCES
MOLECULAR AND BIOCHEMICAL 
CONSEQUENCES
PHYSIOLOGICAL 
CONSEQUENCES
Directly related :
Loss of muscle 
mass/function: sarcopenia*
MOBILITY 
LIMITATION 
and ultimately 
Major Mobility 
Disability
Directly related:
NF‐kB activation & cytokine production*&, loss 
of mitochondrial  function*, myocyte
apoptosis*&, loss of muscle regeneration*, 
proinflammatory lipid signaling*, etc.
Indirectly related:
Chronic diseases*& such as 
cardiovascular disease, 
diabetes, osteoporosis, etc
Indirectly related:
Monocyte & platelet activation Æ
atherosclerosis Loss of insulin signaling* Æ
diabetes  Increased osteoclast bone resorption 
Æ osteoporosis, etc.
* Potential effect of ω‐3
& Potential effect of ARB
Figure 5.1. The relation of inflammation to loss of physical function. The three main sources of inflammation in the elderly (genetic and epigenetic 
factors, exogenous factors and endogenous factors) combine to cause molecular and biochemical changes with important consequences, which in 
turn lead to physiological consequences, and ultimately to mobility limitation and mortality. These changes are complex with thousands of genes and 
other macromolecules involved; some examples of important pathways are listed. The potential impact of the chosen interventions are shown.

 
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are generally pro-inflammatory,58 and can cause a decline in muscle protein synthesis via ER stress.59 ω-3 
supplementation down-regulates inflammation60 in part through decreased TNF-α expression and ω-3 fatty 
acids are likely to improve mTOR signaling, thereby stimulating muscle protein synthesis in humans.61 A meta-
analysis of several RCTs has shown that ω-3 reduce both IL-6 and CRP in chronic non-autoimmune disease.62 
ARBs are primarily indicated for the treatment of hypertension. The ARB LO is also a partial PPAR-γ 
agonist,63 yielding potent anti-inflammatory effects.64 LO reduces IL-6 in human65 and animal studies,66 
possibly through PPAR-γ/AMPK agonism, blockage of proinflammatory angiotensin II AT1 signaling, or both.64, 
67 ARBs mitigate lipopolysaccharide -mediated inflammation, and inhibit TNF-α-mediated endothelial Receptor 
for Advanced Glycosylation Endproduct (RAGE) expression.68 LO has been shown to improve skeletal muscle 
related activity measures in older mice.66, 69 In summary, inflammation plays a major role in the loss of physical 
function through a wide variety of mechanisms, many of which offer interventional targets.  
All drugs have multiple effects. LO63, 67, 70 and ω-3,71, 72 in addition to averting inflammation, also affect 
vasculature, coagulation, metabolism, and skeletal muscle, all of which may benefit mobility (Figure 5.1.). 
Testing these two different interventions that avert inflammation may provide added support regarding the 
relevance of their common anti-inflammatory effect in possibly benefiting walking speed and ultimately mobility.  
Selection of anti-inflammatory interventions. A broad range of pharmacological, nutritional, and behavioral 
interventions have potential efficacy in 
reducing low grade chronic 
inflammation.39, 74 To maximize the 
public health impact of ENRGISE, we 
set the criteria outlined in Table 5.1. 
and 5.2. for prioritizing interventions. 
Based on the selection criteria and the 
evidence reviewed below, in the pilot 
phase we test LO and ω-3. Both have 
excellent safety records, are tolerable 
and acceptable, reduce elevated IL-6, 
have shown benefits in improving 
physical performance, have not been 
tested in similar trials, act with different 
but complementary biological 
mechanisms,12-15 do not negatively 
interfere with neuromuscular 
metabolism, and are broadly available 
at low cost. ω-3 reduced IL-6 by 
>50% in RCTs75, 76 and ARBs reduced IL-6 by >40%77, 78 and by 58%79 in RCTs. In Health ABC1 and 
EPESE,2 we have found that a 50% lower IL-6 plasma level was associated with approximately 50% 
lower risk of mobility disability over 2.5 and 4 years of follow-up, respectively. Change in IL-6 induced by LO 
and ω-3 may depend on the target population, baseline IL-6 level, dosage, compliance, and duration of 
treatment. Thus, the pilot study is of key importance to probe their anti-inflammatory effects in the ENRGISE 
population.  
Inclusion of potential candidate interventions (see Table 5.2.)  
Angiotensin Converting Enzyme Inhibitors (ACEIs) and ARBs have shown excellent safety in large 
hypertension and heart failure trials in older persons,80-84 Among ACEIs, perindopril85, 86 and enalapril65 have 
demonstrated reductions in IL-6. Perindopril has shown to prevent physical function and walking speed decline 
in older persons,87 to reduce CRP,88 and to have a favorable safety profile.89 Dual angiotensin blockade with 
ACEIs and ARB results in higher risk of adverse events,90 thus, we do not recommend to combine ACEIs and 
ARBs. Virtually all ARBs, except azilsartan, have been shown to reduce elevated IL-6.65, 77, 91-102 Telmisartan 
improved walking distance in patients with peripheral artery disease103 and running endurance in mice.15 In 
older mice LO reduced IL-6 and improved physical performance.66 We prioritize ARBs because of greater 
tolerability than ACEIs,83, 84, 104 and among ARBs, we prioritize LO due to its low cost (1/50 that of other ARBs). 
Among nutritionals, ω-362 and lipoic acid92, 105 reduce IL-662, 92, 105 and CRP62, 106 in RCTs. The evidence of the 
Table 5.1. Criteria for prioritizing the selection of the ENRGISE interventions 
1. Safety, tolerability and acceptability are key criteria. Vulnerable older persons 
use multiple drugs and have multiple comorbidities, and thus, are at high risk of 
adverse drug reactions. Newer drugs are often tested in younger or middle age 
adults for the treatment of a single condition and therefore, their safety and 
tolerability in older persons is not fully known.73 Furthermore, for the prevention of 
mobility limitations, vulnerable or frail older persons may not be willing to take, 
and their providers may not be willing to prescribe drugs that bear a risk of severe 
adverse events. Finally, we exclude interventions that may negatively affect 
skeletal muscle or neuromuscular metabolism.  
2. Reduction of IL-6 and possibly CRP in clinical trials is our primary criterion. 
3. Benefit on physical performance and/or skeletal muscle is a relevant, but not 
necessary criterion. 
4. Innovation. We have prioritized interventions that have not been, or are not 
being tested in RCTs for preventing mobility limitations. 
5. Biological mechanisms are considered to prioritize interventions that target 
different inflammatory mechanisms or may have synergistic effects. We exclude 
interventions that may negatively affect skeletal muscle metabolism. 
6. Practical and affordable for implementation in the US health care system. Cost 
is a major factor for this criterion to maximize the public health impact of the trial 

 
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anti-inflammatory effect is particularly strong for ω-3.62 In older women, ω-3 improved walking speed,107 and 
strength when supplemented to exercise.108 ω-3 are widely available at low cost primarily in the form of fish oil. 
Among behavioral interventions, compared with Western diet, the Mediterranean diet (MED) reduces IL-6109-113 
and CRP.109, 110, 112, 113 Olive oil contains oleocanthal acting as a natural anti-inflammatory compound that has a 
potency and profile similar to that of ibuprofen.55, 114 MED is associated with better physical performance, 
higher walking speed and lower risk of frailty.115-118 MED is promising, however, after consulting with diet 
experts, including Dr. Linda Van Horn at NWU, Dr. Mara Vitolins at WFSM, and Dr. Stephen Anton at UF, it 
was concluded that diet interventions are complex and too resource intensive to be considered in this context, 
primarily due to the need for dietary counseling. Additionally, the most successful MED trials were conducted in 
Spain and Italy,110, 113, 119 where MED is part of the local traditions. It is doubtful whether MED could be 
successfully imported in the ENRGISE older frail US population within the available resources of this trial. 
Thus, after careful consideration, we did not prioritize diet interventions. 
 
Exclusion of potential candidate interventions (see Table 5.2.) 
Criterion #1 excludes anti-TNF-α agents (etanercept, infliximab, adalimumab),120 anti-IL-6 agents 
(siltuximab),121 anti-IL1β (canakinumab – no long-term safety data available),122 and thiazolidinediones 
(rosiglitazone, pioglitazone)123 for risk of infections, liver toxicity, fluid overload, CVD, fractures, or possible 
cancer. Chloroquine and statins are excluded due to concerns of myotoxicity124-126 and lack of effect on walking 
speed for statins (Criteria #1, #5).127 Corticosteroids, aspirin, non-steroidal anti-inflammatory drugs (NSAIDs), 
and cyclooxygenase-2 (Cox-2) inhibitors are excluded for risk of bleeding,128-132 gastrointestinal toxicity, and 
CV events for NSAIDs and COX-2 inhibitors.133 Colchicine is excluded for risk of myotoxicity134, 135 and 
neuropathy (Criteria #1, #5).135 Low-dose methotrexate is potentially safe and effective in lowering IL-6. But, 
we found it is not acceptable by the older target population. A pilot study to assess effects on walking speed 
did not randomize a single participant, primarily because of refusal to take methotrexate expressed by 
participants or their health providers (section 3.d.3). Methotrexate bears the stigma of a “dangerous” anticancer 
drug in these older people. Many of the above excluded drugs would likely face similar non-acceptability 
problems; even if they were effective, they would have limited public health impact for prevention, due to high 
cost and risk of adverse effects. 
 
Criterion #2 excludes promising interventions, such as metformin, ghrelin, lactoferrin, oxytocin, salsalate, 
creatine, curcuma, probiotics, and resveratrol because of lack of clinical trial evidence of reducing IL-6. 
Metformin, while potentially promising, has shown in the DPP a small reduction in CRP (-12%),136 in part 
related to its modest weight-reducing effect, however other trials have shown no effect on IL-6137 or CRP.138-142  
Criterion #4. Physical activity and weight 
loss have shown in multiple RCTs both 
reduction of IL-6 and prevention of mobility 
limitations,45, 46, 143-147 and thus, are not 
prioritized as innovative approaches.  
Innovation and strengths. The following 
features of ENRGISE represent innovative 
approaches: 
• 
Targeting chronic low grade 
inflammation to achieve reduced 
inflammation and reduced mobility 
limitations 
• 
Targeting an older population at high 
risk of mobility disability, which is often 
excluded from large RCTs 
• 
Repurposing widely available 
inexpensive interventions (LO and ω-3)   
• 
Testing LO and ω-3 in combination to maximize their effects on inflammation and mobility 
• 
The use of several adaptive features in design and stratified screening for ENRGISE pilot study 
Table 5.2. Summary of selection criteria and candidate interventions (see details in the 
text) 
 Criteria
 
Interventions 
1. Safe, 
tolerable, 
acceptable 
2. IL-6 
redu-
ction  
3. Physical 
perfor-
mance 
4. 
Inno-
vation 
5. 
Mecha
-nism 
6. 
Practical, 
affordable
ACEIs, ARBs 
+ 
+ 
+ 
+ 
+ 
+ 
ω-3 
+ 
+ 
+ 
+ 
+ 
+ 
Mediterranean diet 
+ 
+ 
+ 
+ 
+ 
- 
Physical activity, weight loss 
+ 
+ 
+ 
- 
+ 
+? 
Vitamin D 
+ 
+ 
+ 
- 
+ 
+ 
Anti-TNF-α, -IL6,-IL1; 
methotrexate thiazolidinediones 
- 
+ 
? 
+ 
+ 
? 
Statins, chloroquine, colchicine 
- 
+ 
- ? 
+ 
- 
+ 
Corticosteroids, aspirin, 
NSAIDs, cox-2 inhibitors 
- 
+ 
? 
+ 
+ 
+ 
Metformin, fosinopril, ghrelin, 
lactoferrin, oxytocin, salsalate, 
curcuma, creatine, probiotics, 
resveratrol 
+ 
- ? 
- ? 
+ 
+ ? 
+ 
+ positive evidence, - negative evidence, ? evidence lacking 

 
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• 
The assessment of novel inflammatory markers in ENRGISE pilot study 
 
 
6. Research Plan 
6.1. Design. We test LO, ω-3 and their combination in a double-blind, placebo-controlled RCT (The ENRGISE 
Pilot Study). Randomization is concealed via the secure web-based data management system. We use a 
permuted block algorithm (with random block lengths).   
LO and ω-3 are considered safe in elders.80-84, 148, 149 We conduct this pilot RCT to (a) decide whether 
we should proceed to the main ENRIGISE trial with 1 or 2 interventions, and (b) if two interventions are 
warranted whether we should proceed as a three-arm (without the combination group) or a 2x2 factorial study. 
The pilot is designed to achieve the following goals: (1) estimate recruitment yields including percent 
consenting; (2) confirm safety and tolerability; (3) assess the impact of interventions on IL-6 and walking speed 
as well as the secondary outcomes of Short Physical Performance Battery (SPPB), frailty, and muscle 
strength; (4) evaluate the strength of relationship between the change in IL-6 with the change in walking speed; 
(5) allow selection of the interventions that demonstrate high participant compliance and retention; (6) collect 
data on outcomes of major mobility disability and other mobility measures; (7) support the decision to proceed 
with a factorial or parallel design; (8) examine intra-subject variability of IL-6; (9) explore the impact of the 
interventions on 
additional biomarkers, 
their relationship with 
walking speed and their 
impact  
on recruitment and 
effect size estimates; 
and (10) estimate the 
cost of the main trial. 
Recruitment is planned to last 1 year and each participant is being followed for 1 year. To assess 
recruitment yields according to different targets, we stratify on ACEI/ARB and baseline ω-3 use. The first 
stratum (people not using ω-3) is randomized to placebo or ω-3. The second stratum (people not using ACEI 
or ARB) is randomized to placebo or LO. The third stratum (people not using ACEI, ARB, or ω-3) is 
randomized to placebo, LO, ω-3, or the combination. The original ENRGISE targets, based on the estimated 
proportions in LIFE are presented in Table 6.1.  (We note that during the ENRGISE DSMB’s August 23, 2016 
phone call, the DSMB approved the investigators’ request to lift the upper limit on the number recruited into 
stratum 1.  In the text which follows, the original sample sizes remain as we do not know how many we will 
recruit into each stratum.  The randomization weights will remain unchanged.)  We expect to have a slight 
imbalance in the overall numbers within each treatment group of 90, 75, 75, and 60. Oversampling of the 
control group allows us to better estimate parameters associated with IL-6, walking speed, and major mobility 
disability in that group; this is important for planning the sample-size of the main trial. Note that anybody 
eligible for stratum 3 is also eligible for strata 1 and 2. To achieve overall balance between the two single 
interventions, we focus attention on having equal numbers in strata 1 and 2 but do not worry if either is less 
than 25% of the total as randomizing additional people in strata 3 provides similar information. If 20% were in 
each of strata 1 and 2, the overall weights would be 0.28, 0.24, 0.24, and 0.24; if 15%, then 0.26, 0.23, 0.23, 
and 0.28. Both are close to Table 6.1. and do not markedly reduce power for any comparison. 
6.2. Interventions and compliance. . 
6.2.a. ω-3 fish oil and placebo (corn oil) are obtained by Epax in 0.7 g gel caps and they have identical 
shape, color, taste and weight. Each 0.7 g of fish oil ω-3 contains 400 mg eicosapentaenoic acid (EPA) and 
200 mg docosahexaenoic acid (DHA). We start with 1.4 g/day of fish oil and continue until the 6 month visit. If 
the average of IL-6 measured at 3- and 6-month visits does not decrease by >40% vs. baseline (average of 
screening visits 1 and 2), we increase the dose to 2.8 g/day. To limit the effect of within subject variation, we 
use the average of 2 measures. If there is an acute illness within one month prior to the visit, the 3-month or 
the 6-month IL-6 measure is postponed or excluded from titration algorithms. In previous RCTs, ω-3 reduced 
IL-6 by >50% using 1 to 2 g/day of fish oil.75, 76 In other trials in elders, fish oil was used in comparable doses 
Table 6.1. Randomization strata according to medication use at baseline – Total N=300 
Strata according to 
medication use at baseline 
LIFE 
N (%) 
Observed 
ENRGISE pilot
N (%) 
Proposed 
Randomization weights 
Placebo LO 
Placebo ω-3 
LO 
Placebo ω-3 
Placebo LO
ω-3 
LO 
ω−3 
1 No ω-3 (ACEI/ARB ok) 
637 (44.1) 
75 (25%) 
0.4* 
0 
0.6* 
0 
2 No ACEI/ARB (ω-3 ok) 
154 (10.7) 
75 (25%) 
0.4** 
0.6** 
0 
0 
3 No ACEI/ARB, No ω-3 
655 (45.3) 
150 (50%) 
0.2 
0.2 
0.2 
0.4 
Overall 
 
300 (100%) 
0.3 (n=90) 
0.25 (75) 
0.25 (75) 
0.2 (60)
* Does not receive placebo LO, and  ** does no not receive placebo  ω-3 as the use of the corresponding drugs is permitted at 
baseline  

 
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ranging from 0.6 to 2.3 g/day.62 
 
6.2.a.1. Risks and risk protection and dose adjustments related to administration of ω-3  
There are no contraindications to the use of ω-3 fish oil, except intolerance or allergy to fish oil. ω-3 are 
considered very safe and produce only mild adverse effects involving gastrointestinal upset and fishy 
aftertaste.148, 149 FDA has affirming menhaden fish oil as Generally Recognized As Safe (GRAS) under 21 
CFR 4 184.1472 provided that the daily intake of EPA and DHA combined do not exceed 3 g per person per 
day. http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=184.1472 and 
http://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/SCOGS/default.htm accessed on 9/11/2014. 
ω-3 may rarely cause clinical or subclinical bleeding, worsening of glycemic control in people with 
impaired glucose tolerance, rise in LDL cholesterol, and possibly hypotension when combined with 
antihypertensive treatment.  
We start with a dose of 1.4 g/day of ω-3 fish oil. Adverse effects, such as gastrointestinal discomfort are 
monitored. Dose will be adjusted as below based on serum glucose, LDL cholesterol and hemoglobin 
measured at baseline and at month 3, 6, and 9. ω-3 fish oil dose will be decreased or discontinued if any of the 
following criteria are met at follow-up:  
• 
New onset paroxysmal or persistent atrial fibrillation 
• 
Hemoglobin decreases by ≥20% from baseline (dose will be discontinued),  
• 
The participant is not diabetic and fasting glucose is > 126 AND has increased by >30% from baseline,   
• 
The participant is diabetic and fasting glucose > 300, 
• 
Baseline LDL > 130 AND increases by ≥ 20% from baseline,  
• 
The participant is taking a blood pressure lowering agent OR is also participating in the Losartan arm of 
the study AND blood pressure is <90/50, or  
• 
Participant does not tolerate the fish oil.  
The principal investigator/study physician at each site has the discretion to opt out of ω-3 fish oil dose 
adjustments due to glucose and/or LDL levels.     
We increase the dose of ω-3 fish oil to 2.8 g/day if the average of IL-6 measured at 3- and 6-month 
visits has not decreased by at least 40% vs. baseline (average of screening visits 1 and 2). The 40% 
threshold is selected based on previous trials with ω-3 fatty acids. ω-3 fatty acids have been shown to reduce 
IL-6 by >50% in RCTs75, 76 and to be consistent with the threshold used below for losartan. To minimize the 
impact of outliers due to acute illness, the 3-month or the 6-month IL-6 measure are excluded or postponed if 
there is an acute illness within one month prior to the visit. In the rationale for dose titration we use the 
following assumptions: 
1. IL-6 biovariation as a CV is ~30%. This is a reasonable estimate based on our own estimates 
(unpublished data) and those of others.150, 151 This means that a SD of each person’s day to day 
variability is about 30% of the mean value. 
2. We have two measures at baseline that can be averaged to yield a reasonable estimate of each 
person’s average value. 
3. For dose adjustment we use IL-6 measured at 3 months and 6 months. 
4. The half-life of IL-6 is extremely short (<30 minutes), so response to interventions is essentially 
coincident to the effect of the interventions. 
5. We screen for outliers by excluding participants who experienced an acute illness within a month of the 
visit 
 
6.2.b. LO and placebo (cellulose based) are obtained in 25 mg and 50 mg capsules. The shell capsules are 
cellulose based. Placebo and LO have identical shape, color, taste and weight. We start with 25 mg/day, if 
tolerated, we step up to 50 mg/day. If there are no safety concerns, we continue with 50 mg/day until the 6 
month visit. If the average of IL-6 measured at 3- and 6-month visits does not decrease by >40% vs. baseline 
(average of screening visits 1 and 2), we increase the dose to 100 mg/day. Equivalent doses of ARBs have 
shown to reduce IL-6 by >40%77, 78 and by 58%79 in RCTs.  
 
 

 
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8 
 
6.2.b.1. Risks, risk protection and dose adjustment related to administration of the ARB Losartan. 
ARBs are reported to have a side effect profile indistinguishable from that of placebo but adverse reactions can 
occur rarely. Losartan has been safely used in large long-term clinical trials in older persons with congestive 
heart failure.83, 84 ARBs are particularly well tolerated with very few side effects. Because ARBs do not 
influence kinin metabolism, dry cough is not seen and angioedema is very rare. Impairment in renal function 
may arise in patients with bilateral renal artery stenosis. Renal failure, reversible on discontinuation of ARB, 
may be precipitated. 
 
• 
Hyperkalemia due to potassium retention mediated by reduction of aldosterone. Rare except in renal 
impairment. 
• 
Impairment of renal function. Caution if bilateral renal artery stenosis suspected. 
• 
Dizziness and syncope. Rare but may be precipitated by volume depletion. 
• 
Angioedema. Very rare. 
• 
Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) Including Selective Cyclooxygenase-2 Inhibitors 
(COX-2 Inhibitors): In patients who are elderly, volume-depleted (including those on diuretic therapy), or 
with compromised renal function, co-administration of NSAIDs, including selective COX-2 inhibitors, 
with angiotensin II receptor antagonists (including losartan) may result in deterioration of renal function, 
including possible acute renal failure. These effects are usually reversible. We monitor renal function 
periodically in patients receiving losartan and NSAID therapy as outlined below. 
A full list of potential side effects of losartan can be found in the current package insert.   
 
6.3. Safety dose titration. ARBs produce blood pressure reductions similar to those seen with ACE inhibitors 
and other antihypertensive classes. These drugs vary in efficacy and duration of action but all are 
recommended for once daily dosing. Like ACE inhibitors, duration of antihypertensive effects is dose-
dependent; therefore, smooth blood pressure control over 24 hours is most likely at the maximum 
recommended dose. To avoid precipitous initial fall in blood pressure or decline in renal function, we start 
therapy with the lower dose of losartan (25 mg/day). If there are no safety concerns, the dose is increased to 
50 mg/day and safety is again checked in about 1 week. To identify abrupt reduction in renal function that 
warrants drug dose reduction and to avoid dangerous hyperkalemia, we measure eGFR, potassium, and blood 
pressure before and soon (approximately 1 week) after starting losartan and after any dose adjustment. 
However, dose adjustments downward that are not related to renal function or hyperkalemia will not require a 
short-term follow-up blood draw.  
In general, losartan dose will be maintained if ALL of the following criteria are met: 
• 
Blood pressure is ≥ 100/50 and < 110/50,  
• 
Potassium < 5.5,  
• 
eGFR has NOT declined by ≥ 20% from baseline, and 
• 
No symptoms of lightheadedness or dizziness. 
Losartan dose will be decreased if ALL of the following criteria are met: 
• 
Blood pressure is ≥ 90/50 and < 100/50,  
• 
Potassium < 5.5,  
• 
eGFR has NOT declined by ≥ 20% from baseline, and 
• 
No symptoms of lightheadedness or dizziness. 
Losartan dose will be stopped if ANY of the following criteria are met: 
• 
Blood pressure < 90/50,  
• 
Potassium ≥ 5.5, 
• 
eGFR decline ≥ 20% from baseline, or 
• 
Symptoms of dizziness, lightheadedness, or syncope.  
The principal investigator/study physician at each site has discretion to alter the losartan titration 
schedule on a case by case basis.   
We increase the dose of losartan to 100 mg/day if the average of IL-6 measured at 3- and 6-month 
visits is not decreased by at least 40% vs. baseline (average of screening visits 1 and 2). The 40% threshold 

 
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9 
 
was selected based on previous trials with ARBs. ARBs have been shown to reduce IL-6 by >40%77, 78 and by 
58%79 in RCTs, and to be consistent with the threshold used above for ω-3 fish oil. To minimize the impact of 
outliers due to acute illness, the 3-month or the 6-month IL-6 measure are excluded or postponed if there is 
acute illness within one month prior to the visit. 
As part of the safety monitoring of the ENRGISE interventions, we measure blood pressure, blood 
hemoglobin, serum glucose, eGFR, LDL cholesterol, and potassium at baseline and at 3, 6, and 9 months of 
follow-up. In addition for those in the losartan arm of the trial, blood pressure, serum potassium, and eGFR, are 
measured about 1 week after randomization and about 1 week after any losartan dosage adjustment.  
However, serum potassium and eGFR are not measured one week after a downward dose adjustment that 
was not based on potassium or eGFR values. 
 
6.4. The Investigational New Drug (IND) Per requirement of the Institutional Review Board, #IND124103 was 
submitted and approved by the FDA with the mention “STUDY MAY PROCEED” 
6.5. Compliance 
To measure compliance with LO and ω-3, we use dual methods involving both pill count152 and participant 
recall.153 Pills are counted at each study visit.  
 
6.6. Recruitment – inclusion/ exclusion criteria - (details in Table 6.2.)  
We identify men and women aged >70 years who are able to complete the 400 m walk at baseline, and are 
at high risk of mobility disability assessed by  
• 
Self-reported difficulty walking ¼ mile or climbing a flight of stairs,  
• 
Usual walking speed <1 m/sec and >0.44 m/sec on the 4 m walk (in the pilot phase we explore the 
feasibility of recruiting at least 50% of participants who have a baseline walking speed of <0.80 m/sec and 
>0.44 m/sec),22, 154, 155 and  
• 
Chronic low-grade inflammation measured by IL-6 >2.5 pg/ml, the threshold we have found for increased 
risk of mobility limitation,1, 2 and <30 pg/ml to exclude high grade inflammation. To minimize within person 
variability,150, 156 IL-6 is the average of 2 measures taken at screening visits, with the first measure being 
>2.3 and <30 pg/ml, and the average of the two measures >2.5 and <30 pg/ml. 
We exclude subjects with acute, autoimmune and HIV illnesses and causes of low walking speed that 
may be related to neurological conditions, severe arthritis, or low vitamin D. Other exclusion criteria reflect 
conditions that may interfere with the conduct of the study, interventions or assessments. Those taking ACEI or 
ARB are excluded from the LO randomization and those eating>2 servings/week of fish in the past year or 
taking fish oil are excluded from the ω-3 randomization. We recruit from the community. We use recruitment 
strategies successfully applied in LIFE,157 LIFE-Pilot,158 and TTrial,159 which include primarily mass mailing, 
and newspaper, radio and TV ads and explore alternative recruitment strategies, such as electronic medical 
records to identify patients not taking study drugs and assess whether that strategy is effective in lieu of or in 
addition to mass mailing. 
 
Table 6.2. Inclusion and exclusion criteria  
Inclusion Criteria  
• 
Men and women age >70 years 
• 
Self-reported difficulty walking ¼ of a mile or climbing a flight of stairs  
• 
Walking speed <1 m/sec and >0.44 m/sec on the 4 m walk at usual pace. A walking speed of <0.44 m/sec would not be 
compatible with completing the 400 m walk in 15 min. (In the pilot phase we explore the feasibility of recruiting at least 50% 
of participants who have a baseline walking speed of <0.80 m/sec and >0.44 m/sec) 
• 
Able to complete the 400 m walk test within 15 minutes without sitting or the help of another person and without a walker, a 
cane is allowed 
• 
Blood level IL-6 >2.5 pg/ml and <30 pg/ml.  
• 
Willingness to be randomized to the intervention groups 
Exclusion criteria  
• 
Failure or inability to provide informed consent 
• 
Lives in a nursing home; persons living in assisted or independent housing are not excluded 
• 
Self-reported inability to walk one block 

 
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10 
 
• 
Significant cognitive impairment, defined as a known diagnosis of dementia, or a Mini-Mental State Exam (MMSE) score <24 
(<23 for racial/ethnic minorities or participants with less than 9 years of education) 
• 
Unable to communicate because of severe hearing loss or speech disorder 
• 
Neurological conditions that are causing impaired muscle function or mobility (may include stroke with residual paresis, 
paralysis, neuropathy, Parkinson disease, or multiple sclerosis) 
• 
Severe rheumatologic or orthopedic diseases, e.g., awaiting joint replacement, known active inflammatory or autoimmune 
disease (e.g. rheumatoid arthritis, lupus, Crohn’s disease, HIV) 
• 
Terminal illness with life expectancy less than 12 months 
• 
Severe pulmonary disease, requiring either steroid pills or injections  
• 
Other significant co-morbid disease that in the opinion of the field center PI would impair ability to participate in the trial, e.g. 
renal failure on hemodialysis, severe psychiatric disorder (e.g. bipolar, schizophrenia), excessive alcohol use (>14 drinks per 
week); drug addiction; treatment for cancer (radiation or chemotherapy) within the past 1 year; or other conditions 
• 
Lives outside of the study site or is planning to move out of the area in next 1 year or leave the area for >3 months during the 
next year 
• 
Exclusion criteria that apply only to those who receive losartan: 
o 
Intolerance or allergy to ARBs 
o 
Known bilateral renal artery stenosis or liver cirrhosis 
o 
Hypotension SBP<110 or DBP<60 mmHg 
o 
Serum potassium ≥5.0 mEq/L  
o 
Use of lithium salts 
o 
eGFR < 15  
o 
Congestive heart failure with ejection fraction < 40%  
• 
Exclusion criteria that apply only to those who receive ω-3: 
o 
Intolerance or allergy to ω-3 or fish/shellfish 
o 
Fatty fish intake >2 servings per week on average 
o 
History of paroxysmal or persistent atrial fibrillation 
• 
To maintain blinding, those who are not eligible to receive any active treatment (ω-3 or losartan) are excluded 
Temporary exclusion criteria  
• 
Myocardial infarction, CABG, or valve replacement within past 6 months; 
• 
Pulmonary embolism or deep venous thrombosis within past 6 months; 
• 
Uncontrolled diabetes with recent weight loss, diabetic coma, or frequent insulin reactions; 
• 
Stroke, hip fracture, hip or knee replacement, or spinal surgery within past 4 months; 
• 
Physical therapy for gait, balance, or other lower extremity training within the past 2 months; 
• 
Severe hypertension, e.g., SBP > 200, or DBP > 110 mmHg; 
• 
Hemoglobin <10 g/dL 
• 
Participation in another intervention trial within 3 months; participation in an observational study may be permitted; 
• 
Current smoking (within 6 months),  
• 
Acute infection (urinary, respiratory, other) or hospitalization within 1 month 
• 
Exclusion criteria that apply only to those who receive losartan: 
o 
Use of ACEI, ARB within 2 months  
o 
Use of aliskiren within 2 months in patients with type 2 diabetes or renal impairment with eGFR<60 161 
o 
Use of potassium sparing diuretics, other medications with potassium sparing properties (such as but not limited to 
spironolactone or eplerenone), potassium supplements, and salt substitutes containing potassium within 1 week 
o 
Transaminases >twice upper limit of normal to exclude participants with impaired liver function 
• 
Exclusion criteria that apply only to those who receive ω-3: 
o 
Use of ω-3 within 2 months  
To maintain blinding, those who are not eligible to receive any active treatment (ω-3 or losartan) are excluded 
 
 
6.7. Outcomes  
6.7.a. Primary Outcomes 
IL-6 and walking speed during the 400 m walk test are the main outcomes of the pilot study as outlined 
below.  
The 400 m walk test at usual pace is used.143 Major mobility disability (MMD),143, 162 defined as inability to 
walk ¼ mile or 400 m, is measured in the pilot study and is our preferred primary outcome for the main trial. 
MMD is of major public health significance. Ability to walk ¼ mile is measured in the US census163 and in most 
epidemiologic surveys.18 The MMD outcome based on the 400 m walk test is a feasible, objective, reliable,164 
well-validated and important clinical and public health outcome in older people,22, 143, 162 which has been 

 
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11 
 
successfully implemented in the LIFE-Pilot and LIFE.165, 166 We have shown it to be a more efficient outcome 
for clinical trials than self-reported disability or the Short Physical Performance Battery (SPPB).167 Public health 
agencies use ability to walk ¼ mile or 400 m to define need and policy impact of interventions.18 Finally, people 
reporting the inability to walk 400 m incur higher health care costs of $4,000 per person per year, compared 
with those not reporting inability to walk 400 m.18-22 MMD is operationalized as the inability to complete a 400 m 
walk test within 15 min without sitting or help of another person or walker.162 Completing the walk in >15 min 
would be in an extremely slow pace (<0.45 m/sec), which is of little utility in daily life.168 A higher cut point (30 
or 60 min), makes the assessment impractical and does not add to the clinical significance of the outcome. The 
time to walk 400 meters and the ability to complete the test provide data to test effects of the interventions 
resulting from both attenuation of decline and increase in walking speed. We hypothesize that the interventions 
reduce the risk of reaching the MMD outcome.  
Non-completion of this test can be addressed by adjudicating MMD based on objective inability to walk 
4 m in<10 sec, or self- or proxy-reported inability to walk across a room.  
 
6.7.b. Secondary and exploratory outcomes and measures 
SPPB. A low score on the SPPB based on 4 m walk, balance & chair stands tests is a risk factor for disability, 
institutionalization, morbidity and mortality in initially non-disabled older persons.1-3 The summary score and 
components of the SPPB have good reliability (ICCs range from 0.88 to 0.92).4,5  
Frailty is a state of increased vulnerability to endogenous and exogenous stressors.169, 170 Frailty involves 
fatigue, weight loss, infections, balance, strength and gait impairment leading to an increased risk of falls, 
delirium, and disability. In addition frailty is associated with inflammation43 and thus it may be averted by anti-
inflammatory interventions. We characterize frailty with Fried criteria developed by Fried et al. that employ self-
reported exhaustion, unintentional weight loss, low energy expenditure, slow gait speed, and weak grip 
strength.171 Those with >3 of the 5 factors are judged to be frail, those with 1 or 2 factors as pre-frail, and those 
with no factors as non-frail. 
Isometric hand grip strength is 
a commonly used measure of 
upper body skeletal muscle 
function and has been widely 
used as a general indicator of 
functional status.172, 173 Higher IL-
6 levels are associated with 
lower grip strength.3, 7, 174 Thus, 
reducing IL-6 with LO and ω-3 
may result is higher grip strength 
vs. placebo. Grip strength in 
both hands is measured as we 
have successfully applied in 
LIFE.162  
Isokinetic dynamometry of the 
knee extensors and flexors. 
Participants complete this 
measure at baseline and 12 
months. Observational studies 
have shown associations 
between knee extension 
isokinetic strength and physical 
functioning, disability and 
mortality.176 The reliability of this 
measure is generally excellent.177, 178 In addition, several randomized trials in older mobility limited populations 
have shown this measure to be responsive to therapeutic interventions.179, 180 
The Short Form (SF-36) Health Survey 181-184 is administered to assess the general health status as 
Table 6.3. Schedule of screening, assessments and follow-up procedures in the pilot trial 
Visit type 
Phone scr. 
Scr. visit 1 
Scr. visit 2 
Base. Visit  
Safety* 
3-Mo visit 
Safety * 
6-Mo visit 
Safety* 
9-Mo visit 
Safety* 
12-Mo visit 
Safety* 
Extra visit** 
Basic eligibility screening 
x 
 
 
 
 
 
 
 
 
 
Short consent and 4 m walk test 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
IL-6  
x 
x 
x& 
 
x& 
 
x&
 
x&
 
 
Medical history 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
MMSE 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
Safety blood tests  
 
 
x 
x 
x 
x 
x* 
x 
x* 
x 
x*
 
x*
x 
Additional biomarkers 
 
 
 
x 
 
x 
 
x 
 
 
 
x 
 
 
Vital signs 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Anthropometric measures 
 
 
 
x 
 
x 
 
x 
 
x 
 
x 
 
 
Main Informed consent  
 
 
x 
 
 
 
 
 
 
 
 
 
Physical performance measures 
 
 
x 
x 
 
x 
 
x 
 
x 
 
x 
 
 
Medical history update & adverse events
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Questionnaires  
 
 
 
x 
 
 
 
 
 
 
 
x 
 
 
Dispense study drugs 
x 
x 
 
x 
 
x 
 
 
 
 
Assess compliance 
 
 
 
 
 
x 
 
x 
 
x 
 
x 
 
 
Proxy interview (if needed) 
 
 
 
 
 
x 
 
x 
 
x 
 
x 
 
 
* BP, and serum potassium and eGFR are measured about 1 week after the LO dose adjustment 
(potassium and eGFR are not measured if LO dose is reduced only for BP values). ** Extra visits if there 
are safety concerns. & The IL-6 measure is excluded or postponed if there has been an acute illness 
within one month prior to the visit 

 
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12 
 
Participant Reported Outcomes (PRO).  
6.8. Screening, assessments and follow-up (Table 6.3.) 
Potential participants are screened by telephone interviews to assess the main inclusion/exclusion criteria. 
Those who qualify are invited for the first screening visit during which a brief informed consent is obtained and 
the 4 m walk at usual pace is administered. Blood level of IL-6 is tested in those with a walking speed <1 m/sec 
and >0.44 m/sec. Participants with qualifying IL-6 levels are invited for the second screening visit to review 
medical history and current medications, complete blood testing (vitamin D, CBC, chemical panel, and second 
IL-6 measure), vital signs (blood pressure, pulse and temperature), 400 m walk, and the Mini Mental State 
Examination (MMSE).185  The study medical safety officer will review the participant’s health history, 
medication use, vital signs, and laboratory test results, and clear for randomization according to the study 
inclusion and exclusion criteria. If there are no safety concerns, participants attend the baseline visit during 
which the following is completed: full informed consent, questions to assess frailty,171 blood collection for labs, 
vital signs and anthropometric measurements, the SPPB, isokinetic dynamometry of the knee extensors and 
flexors, grip strength, and questionnaires. Validated Minnesota physical activity186, depressive symptoms 
(CES-D)175, patient reported outcomes (SF-36), and fatigability198 questionnaires are completed at the baseline 
and 12 month visit. The physical activity results are used as potential modifiers in the data analyses. If there 
are no exclusion criteria identified, the participants are randomized, and the study drugs are dispensed. The 
baseline measures are repeated as outlined in Table 6.3. BP&P, blood tests and adverse events are measured 
about 1 week after randomization and after each visit when the LO dose is modified. Medication dose is 
adjusted as outlined in the above titration and safety algorithms.  
6.9. Measure of additional biomarkers Inflammation, characterized by higher blood cytokine levels related to 
the activation of innate immunity and other systems (coagulation), is part of the aging process, plays a role in 
virtually all diseases, and contributes to mobility limitations.1 While it is known that inflammation markers 
predict these outcomes and mortality, little is known about how anti-inflammatory interventions affect the 
plasma cytokine levels, or associated pathways. Thus, we measure novel and established markers of 
inflammation and aging at baseline, and follow-up, and relate these to both change in IL-6 and change in 
walking speed to:  
(1) Characterize the effect of the interventions on these cytokines: (1.a.) Are these cytokines/pathways 
impacted by the interventions? (1.b) Are the changes in these markers pre- vs. post-intervention less than, 
equivalent to, or greater than that of IL-6?  
(2) Assess if these biomarkers yield independent information from IL-6 with respect to the change in 
walking speed and/or provide benefit for screening participants in the main ENRGISE trial. 
6.10 Data analyses and power for the ENRGISE Pilot Study and biomarkers assays  
The data analyses and power section is presented in the order by which the aims are likely to be 
achieved rather than the order in which the aims are stated elsewhere in the protocol.  
 
6.10.a. Feasibility and recruitment 
Feasibility of recruitment and identifying the study population are contained in Aim a (ii). In the 
ENRGISE Pilot Study we will use one-sided tests/confidence intervals because we wish to rule out conditions 
that would make the main study infeasible. Having a higher than hypothesized safe tolerable rate, higher yield 
for recruitment, or stronger relationship between the changes in IL-6 and walking speed would simply make the 
follow-up study easier to implement. For efficacy, we use tests at the 10% significance level;187, 188 this is 
consistent with the FDA’s aims for phase 2 versus phase 3 studies.189, 190 No formal adjustment for multiple 
comparisons is planned for the pilot study.  
 
We assess the recruitment yields in strata 1-3 presented in Table 6.1 and the proportions of people 
who tolerate LO and ω-3. One-sided exact confidence intervals of these proportions are calculated. If we need 
to screen 300 people to recruit 75 into strata 1 (People not using ω-3) or 2 (People not using ACEI or ARB) 
(25% yield), the 95% one-sided CI would set the lower bound of our recruitment yield at 20.9% using a 
Clopper-Pearson interval191 and 8.3% if we need to screen 750 (10% yield). For stratum 3 (People not using 
ACEI, ARB, or ω-3) (target 150), these lower limits would be 22.1% (25% yield) or 8.8% (10% yield). Note that 
those eligible for stratum 3 are also eligible for strata 1 and 2; these people are also used to estimate the 

 
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proportions eligible; thus, our CIs above are conservative. 
 
6.10.b. Intervention Effect 
We use the "intention to treat" approach assuming participants are grouped according to randomization 
for all analyses of the intervention effect. The stratified recruitment and randomization allow us to compare 
mean log(IL-6) between each of the three active groups to placebo using a linear mixed model with both main 
effects and their interaction, adjusted for baseline log(IL-6) and strata. Time is a repeated factor, and the 
primary comparison is based on a contrast at 12 months. The covariance structure will be assumed to be 
unstructured. The primary focus is on marginal comparisons (135 and 165/group) between each active 
intervention and placebo using one-sided hypothesis tests at the 10% level (we are not looking for definitive 
evidence, but to rule out small effects). We assume 5% loss to follow-up (LTFU); this is similar to the 5% we 
achieved in LIFE. Using the root mean squared error from LIFE-P (adjusted for baseline log(IL-6) and 
treatment) of 0.52, we have 91% power to detect a difference if the difference (on the log scale) is at least 
0.1625. This is equivalent to the difference between 4.20 and 3.57 pg/ml (or a 15% difference). We would have 
66% power for a 10% difference (4.20 vs. 3.78 pg/ml, 0.1054 on the log scale) and 99% power for a 20% 
difference (4.20 vs. 3.36 pg/ml, 0.2231 on the log scale). Individual treatment group comparisons (at least 60 
vs. 75/group) would have 44% power for a 10% difference, 68% power for a 15% difference, and 87% power 
for a 20% difference. This achieves the first part of Aim a (i). As sensitivity analyses, we also explore the 
impact of additional factors (e.g., diet, physical activity, medication use) by adding these factors to the models. 
Although the mixed model is robust against data missing at random (MAR), multiple imputation will be used in 
sensitivity analyses. 
 
 
From LIFE-Pilot, the SD of 400 m walk speed in IL-6 eligible participants is 0.21 m/s, with high 
correlation (r>0.80) between baseline and 12 month measures. With at least 60 and 75 participants per group 
(for pairwise comparisons), we project to have >99% power to detect a difference in means of >0.095 m/sec 
using a one-sided test at the 10% level (a substantial meaningful change).192 There is also 67% power to 
detect a >0.038 m/sec difference (a small meaningful change). Using groups of 135 and 165 (for main effects), 
we would have >99% power to detect >0.095 m/sec, and 86% power for a difference >0.038 m/sec in walking 
speed. This analysis uses linear mixed models, as for IL-6; a similar approach is used for the secondary 
outcomes of SPPB, frailty, and grip strength. This achieves the last part of Aim a (i). For people who are 
unable to complete the 400m walk, we will use their walking speed from the portion completed. 
 
 
6.10.c. Associations between inflammation and function 
We base our calculation of detectable SPPB effects on LIFE data. The correlations between baseline 
and 24 month follow-up SPPB outcomes were 0.49 (Successful Aging: SA) and 0.47 (Physical Activity: PA); 
we assume a correlation of 0.5 between baseline and 12 month in ENRGISE. Estimated standard deviations at 
24 months were 2.60 (SA) and 2.47 (PA); we assume 2.5. Under these assumptions, for the comparisons of 60 
to 65 people, we have 90% power to detect a difference of at least 0.99 SPPB units; for the comparisons of 
135 to 165 people, we have 90% power to detect a difference of at least 0.66 SPPB units. Both assume 1-
sided tests at the 10% level. 
 
 We will examine the relationship between the change in IL-6 and the change in walking speed using 
univariate regression. Assuming we assess ≥285 participants (of 300, ≥95%) at 12 months, we have 80% 
power to detect a correlation less than -0.126 (i.e. at least 1.59% of explained variability) using a one-sided test 
at the 10% level. We have 90% power if the correlation is less than -0.152 (Aim a (iii)).  
 
To assess intra-person variability in IL-6 (Aim a (iii)), we measure IL-6 at both screening visits. We use 
a Bland-Altman plot to assess agreement; this is summarized using a 95% CI of the difference at the two visits 
providing information on the expected differences over one week due to biological variability and measurement 
error. If agreement is high, one measure is likely sufficient for recruitment into the main study. If not, we may 
need to use two or more measures to determine eligibility and to assess participant-specific risk. 

 
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6.10.d. Additional inflammatory biomarkers:  
In addition to IL-6 levels, we examine additional markers of inflammation (Aim b (i) and (ii)). As with IL-
6, our primary focus is on the relationships between changes in cytokines with the change in walking speed 
using linear mixed models. We use stratified linear mixed models (as described above for IL-6) to examine the 
impact of interventions on these cytokines. These analyses assess whether changes in these cytokines are of 
similar magnitude to IL-6. As the absolute levels differ between cytokines, focus is on relative changes from 
baseline. We also examine these cytokines in models also including baseline and follow-up log(IL-6) to assess 
whether they provide additional predictive information. We also explore MMD event rates by baseline levels of 
IL-6 and additional cytokines to explore whether we can use an additional cytokine to select a population at 
greater risk to use for the main study, recognizing the potential tradeoff between ease of recruitment and 
selecting a population at higher risk. 
 
6.10.e. Informing the main study 
We assess compliance and retention rates (Aim a (ii)). Although we expect to be underpowered for 
incident mobility disability, we collect these data and compare estimates of event rates to those obtained in SA 
group LIFE participants that would have been eligible for ENRGISE (Aim a (ii)). While power is limited, we also 
examine whether there is sub-additivity and interaction of interventions in stratum 3, which supports the 
decision of whether to proceed with a factorial or parallel group design in Phase 3 (Aim a (ii)). . 
 
We integrate all available information to make a determination about which approaches to carry forward 
into the main study. Weighed in this decision is recruitment yield (is it practical to consider a full size trial?), 
safety and tolerability (do participants tolerate the interventions?), changes in IL-6 in the pilot studies, the 
relationship of change in IL-6 with change in walking speed, the conclusions from our data analysis of other 
studies (e.g. LIFE, Look AHEAD, Health ABC, CLIP), and a revised literature search. Finally, we combine all 
information to estimate the sample size and cost of the main trial (Aim a (ii)). For example, for a two-arm 
study, we would need 2,084 people to have 90% power to detect a 20% (HR=0.8) reduction in MMD assuming 
a two-sided test at the 5% level, a control group event rate of 18.3%/year, LTFU of 5%/year, 18 months of 
recruitment, and a total study length of 4 years. To detect a 25% effect (HR=0.75), we would need 1,296 
people. These calculations use the estimated MMD rate of 18.3%/year in the control group among people in 
LIFE-P whose baseline IL-6 was between 2.5 and 10 pg/ml. 
 
 
7. 
Possible Discomforts and Risks 
7.1. Confidentiality  
Lack of maintenance of confidentiality is a potential risk. The information below relates to all 
collaborating performance sites for the proposed study. Data are used only in aggregate and no identifying 
characteristics of individuals will be published or presented. Results of testing are sent to participant's private 
physicians if participants agree to this. Alert values for medically relevant procedures (e.g., blood pressure, 
pulse rate, MMSE, blood tests) are sent to participants and participants’ physicians, depending on the urgency 
of the values.  
Confidentiality of data is maintained by using research identification numbers that uniquely identify each 
individual. Safeguards are established to ensure the security and privacy of participants’ study records. 
Appropriate measures are taken to prevent unauthorized use of study information. The research ID number is 
used. The research records are kept in a locked room in the Field Center. The files matching participants' 
names and demographic information with research ID numbers are kept in a locked file. Only trained and 
certified study personnel has access to these files. After the study is completed, local data are stored with 
other completed research studies in a secured storage area.  
In compliance with the Health Insurance Portability and Accountability Act (HIPAA) and the Standards 
for Privacy of Individually Identifiable Health Information of the Department of Health and Human Services, we 
access personal health information and medical records only after receiving signed informed consent. 
  

 
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7.2. Biological samples repository 
We comply with the Office of Extramural Research requirements and guidelines related to the research use of 
stored biological specimens. The repository is subject to the oversight of the University of Vermont IRB which 
reviews and approves the protocol, training documents, and an informed consent document, according to the 
elements described above, for distribution to collector-investigators and their local IRBs.  
 
7.3. Safety, risks, risk protection and dose adjustment related to drug administration is discussed 
above in section 6. 
 
 
7.4. Safety measures during the assessments 
 
All study assessments are done by staff trained and certified in Good Clinical Practice, study 
procedures and assessments, and safety procedures. The study nurse/medical safety officer reviews all health 
assessments, vital signs, medical history, medication use, and blood tests. All assessors receive CPR training 
and training on management of acute events including syncope, chest pain, acute dyspnea and abnormal vital 
signs. All sites have on-call access to contact numbers for emergency services.  
 
Phlebotomy. The risks of drawing blood from a vein or finger stick include discomfort at the site of puncture; 
possible bruising and swelling around the puncture site; rarely an infection; and, uncommonly, faintness from the 
procedure. 
 
Blood pressure measurement. The risks of placing a blood pressure cuff on a participant’s arms are that it may 
cause pinching or slight bruising.  
 
400 m walk test. The 400 m walk test may be associated with the risk of falling or development of chest 
discomfort due to coronary ischemia or dyspnea due to heart failure or lung disease. Rarely, falling during the 
400 m walk test may result in a fracture. 
 
Research staff members that collect data are trained and certified in 400 m walk testing before they 
work with study participants. Study staff members are trained not to administer the 400 m walk test if they feel 
or the participant feels that testing is unsafe. Safety precautions are taken during the 400 m walk test by 
applying standardized stopping criteria. If the participant reports pain, tightness or pressure in the chest, 
significant shortness of breath, feeling faint, lightheaded or dizzy, or significant other medical problems the test 
is stopped. In addition, participants are asked whether they feel the test is safe. Those who state it may be 
unsafe are not allowed to complete the test. Staff members are trained to protect against falling and are trained 
in CPR. They are trained in activating the local emergency response system. In our experience conducting 
thousands of 400 m walk tests on individuals within various studies, the risk of falling is less than 1/500 and the 
risk of a fracture-associated fall is less than 1/5000. 
In the LIFE study we have performed approximately 10,000 400 m walk tests with no safety concerns raised by 
the DSMB.  
 
SPPB assessment. Similar to the 400 m walk test, completion of the SPPB may be associated with the risk of 
falling or development of chest discomfort due to coronary ischemia or dyspnea due to heart failure or lung 
disease. Rarely, falling during the SPPB test may result in a fracture. Research staff members who collect data 
are trained and certified in SPPB measurement.  
 
Strength measures, may rarely cause pain or muscle sprain. In our experience conducting thousands of 
measures, the testing has demonstrated to be safe.  
 
Questionnaire administration.  
Participation includes a risk of loss of confidentiality of personal health information. A number of methods are 
employed to maintain confidentiality of participants. First, questionnaire data are collected in secure spaces 
where the interview cannot be overheard. Secondly, only study investigators and key research staff (i.e. study 

 
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programmers and biostatisticians) have access to the study database. Third, participants are assigned a 
unique study identifier. Individual names are removed from the study database and only the unique study 
identifier is used to distinguish participants in the database. Fourth, collected data are maintained in locked 
computer files and file cabinets to which only study investigators have access. Collected data are used only for 
research purposes. Published data will not contain any individual identifiers.  
 
Participant burden and fatigue 
  
The duration of clinic visits for screening, baseline, and follow-up range between 15 min to 3 hours. If 
a participant becomes too fatigued we make arrangements for a second visit or to complete the testing in the 
participant’s home. Based on our experience with LIFE-Pilot, LIFE and other RCTs in the elderly (SHEP),194 
with observational studies in healthy older persons (CHS195 and Health ABC196), and with disabled elderly 
women (WHAS),197 visits lasting between 2 and 4 hours did not negatively affect annual follow-up retention, 
which was >90%. We closely monitor the potential impact of visit burden on retention. This information will be 
used to promptly implement strategies to promote retention and adherence.  
 
  
 
7.5. Data Safety and Monitoring Plan 
Monitoring adverse events 
 
Safety of the study participants is always our major concern. The Intervention and Safety Committee 
provides early monitoring of adverse events and ensures standardization of clinical practice and safety issues 
across all sites.  
An adverse event is any untoward medical occurrence associated with the use of a drug in humans, 
whether or not considered drug related. 
 The event is serious if it results in death, is life-threatening, requires inpatient hospitalization or 
prolongs an existing hospitalization, results in a persistent or significant disability/incapacity, or congenital 
anomaly/birth defect.  
Safety related events are reported in a timely fashion, as required by the NIH, FDA, DSMB and the 
IRBs that are responsible for study oversight.  
The classification of potential relationship to the intervention is as follows. 
Definite   
Temporal pattern + Known or expected AE response pattern + Confirmed by 
stopping the intervention + Reappearance of AE on re-challenge 
 
Possible   
Temporal pattern + Known or expected AE response pattern + Could have been 
produced by a number of other factors 
 
Not related  AE for which sufficient information exists to indicate that the cause is unrelated to 
the study intervention 
ENRGISE study participants are comprised of a frail, elderly population expected to be at high risk for 
acute and chronic comorbid health events.  
A Data and Safety Monitoring Plan (DSMP) is implemented to ensure the safety of all participants 
involved in the study and to ensure the validity and integrity of the data. The Principal Investigators with the 
advice and assistance of the Intervention and Safety Committee described below and the Steering committee 
monitor all aspects of safety. The Intervention and Safety Committee reviews all Serious, Unexpected, and On-
site adverse events and makes recommendations to the Steering Committee for any changes in reporting, 
consent or study activities. 
A Data Safety Monitoring Board (DSMB) is established, with responsibility to monitor all aspects of the 
study, including those that require access to any masked data. The DSMB and its chair are named and 
approved by the NIA. It is planned that the DSMB meets by conference call as determined by the DSMB and 
the NIA. The DSMB has access to all study data, documents and progress. The Intervention and Safety 
Committee, comprised of safety personnel from each site, the Chair, and a representative of the 
DMAQC reports to the DSMB for issues related to participant safety.  
The DSMB has the following charges: 

 
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• 
Review the study protocol. 
• 
Review data (including masked data) over the course of the trial. 
• 
Identify problems relating to safety over the course of the study.  
• 
Identify needs for additional data relevant to safety issues and request these data from the study 
investigators. 
• 
Propose appropriate analyses and periodically review developing data on safety and endpoints. 
• 
Make recommendations regarding recruitment, treatment effects, retention, compliance, safety issues 
and continuation of the study. 
• 
At any time, the DSMB may recommend discontinuation of any component/treatment group of the study 
 
Finally, the NIA makes the final decision on whether or not to accept the DSMB’s recommendation about 
discontinuation of any component of the study. Any serious adverse event that might be due to the study 
intervention are reported to the DSMB, the IRBs, and to the NIA Project Office. The exact timeline for reporting 
serious adverse events is determined by the DSMB, IRBs, and NIA.  
 
8. Potential benefits of the proposed research to subjects and to others  
There is direct benefit to the study participants, including study blood tests and a comprehensive assessment 
of physical function. All study participants are encouraged to communicate the results from the study to their 
primary care providers.  
 
ENRGISE will provide critical information regarding the efficacy of the study interventions in averting low-grade 
chronic inflammation and mobility impairments in vulnerable older adults. The results of ENRGISE will have 
relevant clinical and public health implications, and will fill an important gap in knowledge for practicing 
evidence-based geriatric medicine.  
 
We believe that the aforementioned risks of this study are minimal and reasonable, when compared to 1) the 
scientific knowledge to be gained by performing these studies as well as 2) the potential benefits to study 
subjects. 
 
 
 
9. 
Conflict of Interest 
None 
 
 
 
 
 

 
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