Use of Entresto (Sacubitril/Valsartan) for the Treatment of Peripheral Arterial Disease
Phase: PHASE2
Conditions: Peripheral Arterial Disease
Sponsor: University of Minnesota

University of Minnesota 
Clinical Research Study Protocol 
 
 
Use of Entresto (sacubitril/valsartan) for the treatment of peripheral arterial disease 
 
Principal Investigator: 
Otto Sanchez, PhD 
University of Minnesota 
Assistant Professor Nephrology Epidemiology 
Division of Renal Disease and Hypertension 
Department of Internal Medicine 
 
Co-Investigators: 
Daniel Duprez, M.D., PhD 
University of Minnesota 
Department of Medicine 
Division of Cardiology 
 
Biostatistician: 
David Jacobs, PhD 
University of Minnesota 
School of Public Health 
Division of Epidemiology and Community Health 
Version Date:   June 21, 2017 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 2 of 32 
 
 
Table of Contents 
<Right-click below to update TOC after protocol text has been added> 
1 
Introduction ........................................................................................................................................................ 6 
1.1 
Background and Significance .....................................................................................................................6 
1.2 
Study Aims/Objectives ...............................................................................................................................8 
1.3 
Hypothesis ..................................................................................................................................................8 
2 
Study Design ...................................................................................................................................................... 9 
2.1 
Study Design ..............................................................................................................................................9 
3 
Outcome Measures ............................................................................................................................................. 9 
3.1 
Ankle brachial index (ABI), exercise testing, questionnaires, anthropometric measurements and blood 
pressure monitoring ................................................................................................................................................9 
3.2 
Laboratory Procedures ................................................................................................................................9 
3.3 
Diagnostic Procedure for PAD ...................................................................................................................9 
3.4 
Determination of maximal and pain free walking duration and the 6 minute walk test .............................9 
3.5 
Measurement of peak oxygen consumption and cardiac output ...............................................................10 
3.6 
Mitochondrial functional testing with magnetic resonance spectroscopy (MRS) ....................................10 
3.7 
Measurement of tissue perfusion and oxygenation using functional MRI (fMRI) ...........................11 
3.8 
fMRI data acquisition ...............................................................................................................................11 
4 
Subjects ............................................................................................................................................................ 13 
4.1 
Study Setting ............................................................................................................................................13 
4.2 
Eligibility Criteria .....................................................................................................................................13 
4.3 
Recruitment ..............................................................................................................................................14 
4.4 
Early Withdrawal of Subjects ...................................................................................................................14 
4.5 
Stopping Criteria ......................................................................................................................................14 
4.6 
Data Collection for Withdrawn Subjects ..................................................................................................14 
5 
Study Drug ....................................................................................................................................................... 14 
5.1 
Entresto (sacubitril/valsartan) ...................................................................................................................14 
5.2 
Dosage and Administration ......................................................................................................................15 
5.3 
Method for Assigning Subjects to Treatment Groups ..............................................................................15 
5.4 
Subject Compliance Monitoring ...............................................................................................................16 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 3 of 32 
 
5.5 
Blinding of Study Drug ............................................................................................................................16 
5.6 
Receiving, Storing, Dispensing and Return ..............................................................................................16 
6 
Study Procedures .............................................................................................................................................. 16 
6.1 
Participant Timeline/Procedures ...............................................................................................................16 
Screening and Baseline ........................................................................................................................................ 16 
6.2 
Treatment Period ......................................................................................................................................18 
6.3 
Phone Calls ...............................................................................................................................................18 
6.4 
End of Treatment ......................................................................................................................................18 
6.5 
Follow-up Visits .......................................................................................................................................18 
6.6 
Schedule of Events ...................................................................................................................................19 
7 
Data Handling and Record Keeping ................................................................................................................. 21 
7.1 
Confidentiality ..........................................................................................................................................21 
7.2 
Types of Data ...........................................................................................................................................21 
7.3 
Record Retention ......................................................................................................................................21 
7.4 
Source Documents ....................................................................................................................................21 
7.5 
Protocol Deviations ..................................................................................................................................21 
8 
Study Auditing, and Inspecting ........................................................................................................................ 22 
8.1 
Auditing and Inspecting ...........................................................................................................................22 
9 
Safety and Adverse Events ............................................................................................................................... 22 
9.1 
Adverse Event Definitions and Reporting Guidelines ..............................................................................22 
10 Data Analysis ................................................................................................................................................... 24 
10.1 Statistical Methods ...................................................................................................................................24 
10.2 Power analysis ..........................................................................................................................................25 
11 Clinical Management ....................................................................................................................................... 25 
12 Risks/Benefits ................................................................................................................................................... 25 
12.1 Potential Risks ..........................................................................................................................................25 
12.2 Methods to Minimize Risks ......................................................................................................................25 
12.3 Emergency Procedures .............................................................................................................................26 
12.4 Unblinding Procedures in Case of Emergency .........................................................................................26 
13 Ethical Considerations ...................................................................................................................................... 26 
13.1 Good Clinical Practice ..............................................................................................................................26 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 4 of 32 
 
13.2 Institutional Review Board (IRB) Review and Informed Consent ...........................................................26 
13.3 Confidentiality ..........................................................................................................................................26 
13.4 Declaration of Interests .............................................................................................................................27 
13.5 Publication Policy .....................................................................................................................................27 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 5 of 32 
 
Abbreviatio
ns 
 
AA 
Amino Acid 
AE 
Adverse Event 
ABI 
Ankle Brachial Index 
ABPM 
Ambulatory Blood Pressure Monitoring 
AHA 
American Heart Association 
ANP 
A-type Natriuretic Peptide 
ATP 
Adenosine Triphosphate 
BNP 
B-type Natriuretic Peptide 
CARDIA 
Coronary Artery Risk  Development in Young Adults 
cGMP 
Cyclic Guanosine Monophosphate 
CGM 
Continuous Glucose Monitoring 
CKD 
Chronic Kidney Disease 
CNP 
C-type Natriuretic Peptide 
CRF 
Case Report Forms 
CT 
Computed Tomography 
CTSI 
Clinical and Translational Science Institute 
DNP 
Dendroaspis Natriuretic Peptide 
DSMB 
Data Safety Monitoring Board 
ECG 
Electrocardiogram 
EF 
Effect Size 
fMRI 
Functional Magnetic Resonance Imaging 
GRE-EPI 
Gradient Recall Echo Planar [MRI] 
GXT 
Graded Exercise Test 
HIPAA 
Health Insurance Portability and Accountability Act 
HF 
Heart Failure 
IC 
Intermittent Claudication 
ICMJE 
International Committee Of Medical Journal Editors 
IDS 
Investigative Drug Pharmacy 
IRB 
Institutional Review Board 
MCRU 
Masonic Clinical Research Unit 
MEBRL 
Molecular Epidemiology and Biomarker Research Laboratory 
MG 
Medial Gastrocnemius 
MRI 
Magnetic Resonance Imaging 
MRS 
Magnetic Resonance Spectroscopy 
MWD 
Maximal Walking Distance 
NPs 
Natriuretic Peptides 
NPR 
Natriuretic Peptide Receptor 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 6 of 32 
 
NT-proBNP 
N-terminal pro B-type natriuretic peptide 
PAD 
Peripheral Artery Disease 
PAD-QOL 
Quality of Life 
PCr 
Phosphocreatine 
PFWD 
Pain Free Walking Distance 
PHI 
Protected Health Information 
PI 
Principal Investigator 
PKG 
Protein Kinase G 
Pro-BNP 
Prohormone Brain Natriuretic Peptide 
PTA 
Percutaneous Transluminal Angioplasty 
ROI 
Region of Interest 
SAE 
Serious Adverse Event 
SBP 
Systolic Blood Pressure 
SD 
Standard Deviation 
SF-36 
36-item Short Form 
SI 
[MR] Signal Intensities 
SNR 
Signal to Noise Ratio 
Sol 
Soleus 
SQ 
Subcutaneous 
TE 
Echo Time 
UPIRTSO 
Unanticipated Problems Involving Risk to Subjects or Others 
WIQ 
Walking Impairment Questionnaire 

 
1 
Introduction 
1.1 Background and Significance 
Peripheral arterial disease (PAD) affects about 12 million individuals in the United States 
1,2. The 
functional physical limitations observed in patients with PAD progressively deteriorate from a 
reduction in mobility to a complete loss of independence 
3. Unfortunately, the available therapeutic 
options are limited in success rate, adherence and long-term benefits 
4. Functional physical limitations 
observed in patients with PAD are associated with impaired mitochondrial and microvascular 
function 5-7. Because natriuretic peptides have been shown to improve oxygen consumption and blood 
flow in skeletal muscles 
8,9, we propose that increasing the levels of natriuretic peptide (NPs) using 
sacubitril/valsartan [LCZ696 (Entresto), Novartis Pharmaceuticals Corp.] initially designed to treat 
patients with heart failure 10, could be used as novel therapeutic option. These are the two most 
important biological mechanisms implicated in the functional limitations observed in PAD and could 
provide significant functional beneficial effects to this type of patient. 
 
The mitochondrial and renal effects of NP’s are exerted predominantly through activation of NP 
receptors A (NPR-A), while the endothelial, vascular and anti-fibrotic effects are related to activation 
of NPR-B 
11. Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) act through 
activation of NPR-A and C-type natriuretic peptide (CNP) activates NPR-C 12. Because sacubitril 
inhibits the enzyme that breakdown ANP, BNP as well as CNP, then the activity levels of all NPs 
should be increased. Therefore, the potential effects of sacubitril should include mitochondrial genesis, 
promotion of vascular growth, improvement of endothelial function and anti-fibrotic effects. 
 
1.1.1 
Peripheral Arterial Disease (PAD) pathophysiological mechanisms 
PAD is caused by a decrease in oxygen delivery and utilization that is unable to meet the metabolic 
demands of active skeletal muscles. Obstruction of large arteries by atherosclerotic plaques prevents 
adequate delivery of oxygen 
5 and the resulting dysfunctional microvasculature 
7,8 is unable to 
distribute blood to those active muscle fibers. Mitochondrial dysfunction, independent of oxygen 
delivery, cannot regenerate ATP and PCr to satisfy the metabolic demands of those active skeletal 
muscle fibers 
5. In addition, a decrease in arterial elasticity due to atherosclerosis also impairs blood 
flow in patients with PAD 
13.Therefore, treatment for patients with PAD should be designed to target 
the mechanisms that impair oxygen delivery, utilization and decrease arterial stiffness. 
 
1.1.2 
Treatment options for patients with PAD 
The low success rate of the drugs that are used in the treatment of PAD is probably related to the fact 
that none of them directly targets the mechanisms implicated in PAD 14. Surgical revascularization with 
percutaneous transluminal angioplasty (PTA) or with stents leads to significant improvements in blood 
flow, maximal walking distance (MWD) and pain free walking distance (PFWD) 15,16. However, 
despite the initial high success rate 15, restenosis occurs in 63% and 37% of cases within the first year 
following primarily successful PTA or stent, respectively 15. Another option, therapeutic angiogenesis, 
is still in the experimental phase and has not had the expected success in humans 17. Exercise 

rehabilitation programs are more successful than PTA in maintaining MWD and PFWD, as long as 
patients continue to exercise 18. Unfortunately, patients with pain at rest are unable to participate in 
exercise rehabilitation programs and 2 years patient are only of 36% and 68% in center and home 
based programs, respectively 19. 
 
1.1.3 
Natriuretic peptides: a potential treatment option 
The benefits in functional clinical parameters maximal walking duration (MWD) and pain free walking 
duration (PFWD) with exercise training are related to improvements in endothelial function, and 
increases in capillary and mitochondrial density
20. These changes improve oxygen delivery and 
utilization and clearance of metabolic waste products. A potential  treatment option that would target 
two of the most important pathophysiological mechanisms in PAD are NPs. ANP and BNP are 
synthesized in the atrium and ventricle of the heart in response to pressure and volume overload 
inducing natriuresis and vasodilation and regulating blood pressure 
21, CNP is synthesized by 
endothelial cells and renal epithelial cells 
22. NPs act by binding to particulate membrane bound 
guanylyl cyclase-coupled receptors called natriuretic peptide receptor A and B (NPR-A and B) 
11. Once 
activated, they act by increasing the intracellular messenger cyclic guanosine monophosphate (cGMP), 
which then acts on a cGMP-dependent protein kinase (PKG) mediating specific physiological 
functions 
22. ANP and BNP, but not CNP, bind specifically to NPR-A and only CNP binds to NPR-B 
22 
(Figure 1). ANP and BNP acting by activating NPR-A appears to be related to angiogenesis and 
mitochondrial genesis 11,22 and CNP acting through NPR-B is implicated in vasodilation, angiogenesis 
and anti-fibrotic activity 
10,11. The activation of NPR-A is most likely responsible for the increased 
perfusion of tissues, for increasing muscle oxygen consumption and improvements in insulin 
sensitivity observed in transgenic mice that overexpressed BNP or were infused with Carperitide, a 
homolog of ANP 
8,23. Activating NPR-B would induce vasodilation, increase angiogenesis and may 
decrease fibrosis in the peripheral vasculature, which would improve arterial elasticity. 
 
 
 
1.1.4 
Entresto (sacubitril/valsartan) biochemical characteristics and physiological properties 
Sacubitril is a molecule that inactivates neprilysin, a neutral endopeptidase responsible for degrading 
natriuretic peptides (Figure 1). The combination sacubitril/valsartan given to patients with heart failure 
raises serum and urinary levels of cGMP, a marker of NPs activity, by 35% and 25%, respectively 
24, 
due potentially to an increase in ANP, BNP and CNP (Figure 1). 
Sacubitril was proposed as a treatment option for patients with heart failure due to the diuretic and 
vasodilatory effects of natriuretic peptides. Sacubitril cannot be given alone as it raises the levels of 
angiotensin II and for this reason is combined with valsartan, an angiotensin II receptor blocker. This 
combination, sacubitril/valsartan, has been shown to decrease mortality for patients with heart failure 
25 and it is currently approved by the FDA and it is being used for that purpose 
26. In addition, a similar 
compound, Omapatrilat, which inhibits both neprilysin and angiotensin converting enzyme (ACE) has 
been shown to prevent endothelial cell permeability and atheroma formation in a rabbit model of 
atherosclerosis 
27. The effects of natriuretic peptides on microvascular and mitochondrial function 

have not been studied in human skeletal muscle. This study would provide us with the supporting 
evidence to determine if raising natriuretic peptides improves microvascular and mitochondrial 
function and if this is positively associated with exercise tolerance. Having a therapeutic option to 
improve microvascular and mitochondrial function would be clinically important because it could lead 
to effective therapeutic strategies for patients with peripheral arterial disease, diabetes and in elderly 
subjects who have limited exercise capacity due to low perfusion and oxygen utilization in skeletal 
muscles. 
 
1.1.5 
Clinical trial with carperitide, what do we still need to learn? 
The effects on pain free walking duration (PFWD) of 2 weeks of intravenous infusion of carperitide, a 
homolog of ANP, were tested in PAD patients with different degrees of severity 
8. That study showed a 
decrease in pain at rest, an increase in ankle/brachial blood pressure index and in PFWD with minimal 
side effects. However, the study only had 13 patients, PFWD was assessed in 4 patients and did not have 
a placebo group and the functional and structural changes in mitochondria and the microvasculature 
were not determined. Assessing those parameters would provide a sound biological basis for the effects 
of increasing the activity levels of NPs on the functional clinical limitations and for recommending 
Entresto for the treatment of PAD. 
 
1.1.6 
Significance 
Although there are an estimated 12 million individuals in the United States with PAD, this is an 
underestimation because about 50% of patients with PAD are undiagnosed 
2. Furthermore, patients 
with PAD have an 80% higher risk of disability than those without PAD 
28. Among patients with PAD, 
there is a higher risk of disability in those with poor functional performance at baseline 
29 and with 
lower ABI 
30. In addition, those with the greatest rate of decline in mobility had a 3 fold higher rate of 
disability when compared to those with the lowest rate of decline 
31 The inability to be mobile and 
participate in exercise rehabilitation programs leads to further deterioration culminating in limb 
amputation, loss of independence and greater risk of mortality 
31. Furthermore, total annual costs 
associated with vascular-related hospitalizations in patients with PAD were estimated at 21 billion in 
the year 2004 
32, this greatly increases when the cost for rehabilitation are included 
33. Entresto may be 
beneficial maintaining and improving mobility, which is a very important step in the prevention of 
disability, in lowering the risk of mortality and decreasing the costs associated with PAD. 
 
1.2 Study Aims/Objectives 
Primary Objective: 
1. To assess the effects of 12 weeks of Entresto on the functional clinical parameters maximal 
walking duration, pain free walking duration and 6 minute walk test of patients with PAD.  
 
Secondary Objective: 
1. To assess if changes in functional clinical parameters are related to improvements in 
microvascular and mitochondrial function and to assess the effects of increasing NP activity on 
arterial elasticity. 
2. To assess the time required for functional improvements (6 minute walk test) to return to baseline 

values following 12 weeks of Entresto. 
 
1.3 Hypothesis 
We hypothesize that the combined activation of both NPR- A and B will lead to enhanced vascular 
and mitochondrial function, improving oxygen delivery and utilization leading to substantial 
improvements in the functional clinical parameters of patients with PAD. We also hypothesize that 
the combined activation of NPR-A and B results in increased delivery, distribution, diffusion and 
utilization of oxygen by active skeletal muscle fibers, thus improving clinical functional limitations in 
patients with PAD. 
2 
Study Design 
2.1    Study Design 
This is a double blind, randomized, controlled clinical trial with a goal of enrolling 30 subjects. 
Individuals who meet the eligibility criteria will be admitted into the study and will be randomly 
assigned to 12 weeks of Entresto or the placebo group. A variable block randomization design will be 
used to endure a balance in sample size between the two groups 28. Each patient will be given a series 
of life style and health questionnaires, ankle brachial index will be measured and they will perform a 
graded exercise test to determine, pain free and maximal walking duration. In addition they will also 
perform the 6 minute walking test. They will also have a fMRI and MRS at pre and post 12 weeks of 
the clinical trial. 
 
Following the 12 weeks of the treatment period, patients will be evaluated at weeks 4 and 8 to assess if 
any changes occurred in PFWD MWD and the 6 minute walking test, life style and health 
questionnaires and the presence or absence of adverse events following withdrawal of Entresto or 
placebo. 
 
3 
Outcome Measures 
3.1 Ankle brachial index (ABI), exercise testing, questionnaires, anthropometric 
measurements and blood pressure monitoring 
Patients will have their ABI determined following standard procedures described by the American 
Heart Association 
34. If the patient’s ABI meets the inclusion criteria, then an exercise testing 
procedure to determine, PFWD and MWDt will be performed. In addition, on that same day the Mini 
Mental State Examination (MMSE), the Walking Impairment Questionnaire (WIQ),  the NIH toolbox 
assessment test for memory and cognitive function will perform, and Quality of Life Questionnaire 
(PAD-QOL) 
35 will be given to each participant. Body height and weight will be measured using 
standard anthropometric procedures 
36.. Ambulatory blood pressure monitors using the Omron 
BP742N (©Omron Healthcare Inc. Lake Forest, IL) will be provided and each participant will measure 
and record their own arm blood pressure three times a day for the duration of the study immediately 
after waking up, at noon before lunch and in the night before going to bed. 
 
3.2 Laboratory Procedures 
Participants will come following an 10 hour fast to provide a venous blood sample for measurements 

of plasma Na+, K+,  glucose, creatinine, NT-proBNP , BNP, serum insulin and  also urinary cGMP 
and creatinine. Urine pregnancy testing will be performed at each visit on women of childbearing 
potential. Before the blood draw, trained personnel will measure BP three times and the average of 
those measurements will be recorded. 
 
3.3 Diagnostic Procedure for PAD 
Diagnosis of PAD is described in detail in inclusion criteria. Patients will be asked for symptoms 
suggestive of intermittent claudication (IC) and will also have their brachial and ankle systolic blood 
pressure measured at both arms and legs. Patients will lie supine and will rest quietly for 5 minutes and 
systolic blood pressure (SBP) will be measured once in each arm following standard procedures 
37. 
Systolic blood pressure at the brachial artery and at the dorsalis pedis and posterior tibialis artery will 
be measured using an 8 MHz doppler vascular probe and the highest blood pressure at those sites will 
be recorded. A ratio of highest SBP measured at the ankle divided by the highest SBP measured at the 
brachial (ABI) < 0.90 will meet the diagnostic criteria for PAD. If there are symptoms of IC, but ABI is 
> 0.90 than patients will perform a walking test using the Gardner protocol and if highest systolic BP at 
the ankle decreases by > 25 mmHg that is also diagnostic of PAD. 
 
3.4 Determination of maximal and pain free walking duration and the 6 minute walk test 
Participant will undergo a baseline symptom-limited treadmill test as described below.  The treadmill 
test will be used to determine MWD and PFWD and will be repeated at follow-up. The graded exercise 
test (GXT) is initiated at 2.0 mph (3.2 km/h) at 0% grade, and every 2 minutes the grade is increased 
by 2% 
38. The time at which the participant first starts feeling claudication symptoms is defined as the 
pain free walking duration and the time at which participants has to stop walking is designated as the 
maximal walking duration. During the GXT, participants will rate their claudication pain severity 
every 30 seconds, using the following claudication pain scale: 1 = no pain, 2 = initial onset of mild 
claudication pain, 3 = moderate pain, 4 = submaximal and 5 = maximal claudication pain (point at 
which the participant will request to stop). Recent evidence suggest that the 6 minute walk test is an 
accurate predictor of the response to treatment, mortality rate and mobility loss in patients with PAD 
39. 
Therefore, we will also perform the 6 minute walk test because it offers the possibility of an 
inexpensive, accurate and valid tool to assess the effects of Entresto. The 6 minute walk test will be 
performed in a 20 meter corridor 
40. Patients will be asked to walk as quickly as they can along the 
corridor for 6 minutes to cover as much distance as possible. They will be allowed to stop if they have 
to, but will be encouraged to continue again as soon as they are able. During the time that the 
participant stops walking the clock will continue to run. At the end of test the distance covered is 
measured. 
 
. 
 
 

3.6 
Mitochondrial functional testing with 
magnetic resonance spectroscopy (MRS) 
The bioenergetics of the medial 
gastrocnemius (MG) and soleus (Sol) muscle 
(Figure 2) will be assessed using MRS before, 
during and following a 24 seconds isometric 
maximal voluntary contraction (MVC). MRS 
will be performed in a 7 Tesla/90 cm whole-
body actively shielded human scanner 
(Siemens AG, Erlangen, Germany). Optimal 
positioning of the muscle volume in the 
isocenter of the magnet will be determined 
using scout image scans with three orthogonal 
slices and multislice gradient echo images 
(matrix size = 108 × 128, 14 slices, 7 mm 
slice thickness, field of view = 13 × 16 × 20 
cm, echo time (TE) = 5 ms, TR = 0.4 s) 37. 
These scans will be used to identify the MG 
and the Sol muscle (Figure 2) and positioned near the center of the 31P coil for acquisitions of a time-
resolved 31P MR spectra. Acquisition of MRS will be performed using a pulse-acquire 31P MRS 
sequence with a small 31P surface coil (6 cm diameter) tuned to the resonance 
frequency of phosphorus at 7T (120.7 MHz). A nominal 90° square excitation pulse (pulse width  of 
300 ms) with TR of 1 s, an acquisition bandwidth of 5000 Hz and 2,048 data points will be used to 
acquire phosphorus data 42,43. MRS data will be quantified with jMRUI using the AMARES time 
domain fit routine 
44. The following parameters will be acquired by MRS: PCr content and pH at rest 
and at the end of a 16 s isometric contraction, the rate constant of PCr recovery (KPCr) and maximal 
rate of oxidative phosphorylation (Vmax). 
 
3.6.1 
Advantage of high field MRS 
Figure 3 shows the 31P spectra acquired using the 7T at the Center for Magnetic Resonance Research 
of the University of Minnesota by Dr. Wei Chen and his collaborators 
45. Collecting 31P-MRS data at 
7T offers more than twice as much signal to noise ratio (SNR) per unit time thus reducing 
measurement time compared to 3T magnet 
46. In addition, the increase in SNR allows the measurement 
of MRS parameters in muscles of interest and with high temporal resolution (1 s). This is important 
because the Sol, as a postural muscle, is predominantly a slow twitch muscle (slow fatigue, primary 
source of ATP is oxidative phosphorylation) and the gastrocnemius muscles, are more active during 
locomotion, is a 50% fast twitch muscle (fast fatigue and primary source of ATP is anaerobic 
glycolysis). The effects of PAD on each muscle may be different and the benefits from treatment will 
depend on the metabolic characteristics of each muscle. Therefore, measuring oxidative 
phosphorylation in each muscle separately will provide us with a more accurate assessment of the 
effects of Entresto on the bioenergetics of skeletal muscles in patients with PAD. 
 

3.6.2 
MRS Exercise protocol 
Individuals will perform a 24 s maximal isometric voluntary plantar flexion, while inside the magnet, 
to lower PCr resting levels by 50% 
47. MRS will be performed for 1-min before the contraction, during 
the 16 s isometric contraction and throughout a 3 min recovery period. Maximal voluntary contraction 
will be measured in each individual while in the magnet using an MRI compatible device that is 
composed of a foot holder attached to a force transducer 
48. 
 
3.6.3 
Rationale for measuring oxidative phosphorylation using MRS 
Phosphocreatine resynthesis rates have been used as a surrogate measure of mitochondrial function 
49 
and has been studied non-invasively using MRS 
50. MRS measured PCr resynthesis rate is independent 
of arterial occlusion 51 and has  been used to study the effects of exercise training and age on 
mitochondrial function 
47,52. Furthermore, the rate of PCr resynthesis in patients with PAD is 2/3 lower 
than age matched non-PAD individuals, partially explaining their limitations with mobility 
51,53,54. 
Therefore, measuring PCr resynthesis non-invasively is especially advantageous in patients with PAD 
in whom performing serial biopsies can cause wound healing complications. We expect that in patients 
receiving Entresto the rate of resynthesis of PCr will be increased and it will be associated with 
improvements in mobility. 
 
 
3.7 
Measurement of tissue perfusion and oxygenation using functional MRI (fMRI) 
Validations studies using near infrared spectroscopy showed that changes in tissue oxygenation and 
blood volume can be assessed using differences in the MR signal intensities acquired using a dual echo 
gradient recall echo-echo planar (GRE-EPI) MRI procedure 
55,56. This technique found differences in 
microvascular function between type 2 diabetic patients and healthy individuals 
48. 
 
3.8 fMRI data acquisition 

fMRI and MRS data will be acquired 
on the same magnet during a 
procedure that lasts about 90 
minutes. MR signal intensities (SI) 
will be acquired using a custom 
made 6 cm diameter surface 
proton/phosphorus coil. The same 
anatomical site used for the MRS 
data will be used to acquire the 
fMRI. Muscle functional MRI 
(fMRI) data will be acquired using a 
GRE, echo- planar imaging sequence 
for 30 s before, during a 3 min cuff-
occlusion, and for the following  210 
s.  . The same procedure will be 
performed again after a 5 minute rest 
period using a spin echo (SE) 
sequence with similar acquisition 
parameters except with a TE = 21 
ms. The functional images will be acquired using an acquired matrix = 128 × 128, slice thickness = 10 
mm, repetition time = 1000 ms (defining the time resolution of the measurements), and TE = 15 ms. In 
addition, MRS data will be acquired using a global signal using a free induction decay and adjusting the 
parameters to acquired preferentially from the MG muscle using a TR = 6 s Ernst angle = 77º and a a 
T1 of PCr in muscle for 7T = 4 s.  
A couple of blood pressure measurement will be taken following a 5 minute rest and before entering the 
MRI scanner. The highest blood pressure value will be used to determine the level of cuff inflation 
during the cuff occlusion procedure. The cuff will be inflated using a Hokanson rapid cuff inflation 
system, which inflates the cuff around the thigh in a couple of seconds. The cuff will be inflated 75 
mmHg above the systolic blood pressure or to a max of 275 mmHg. Before initiating the scanning 
session, blood pressure cuff will be inflated for about 10 seconds to the target pressure to show 
participants what they will feel and if they are ok with that level of pressure. Cuff pressure can be 
lowered by 10 mmHg for patient comfort, if necessary. Images will be acquired from the leg with the 
more severe claudication symptoms independent of the ABI values. If the patient has a stent or a bypass 
graft in one leg, images will be acquired from the contralateral leg. If there are stents or bypass graft in 
both legs then the patient may be excluded from the study. This will be discussed on an individual basis 
with the Co-PI Dr. Daniel Duprez and the study physician Jiwan Thapa. 
 
3.8.1 
Functional MRI data analysis 
Images will be analyzed using custom-written Matlab version 7.0.1 (The Mathworks, Natick, MA) 
routines. The functional images will be co-registered to the anatomical image using a rigid registration 
procedure 
57. Then the anatomical image will be used to identify the MG and Sol muscles and regions 
of interest (ROIs) will be specified separately around those two muscles. Resolved connective tissues 
and blood vessels will be excluded from the ROIs. The mean functional image SI for the ROI will be 

calculated as a function of time and divided by the corresponding mean pre contraction SI value. An 
average SI time course will be obtained by averaging the SI time course from two trials, as previously 
described 
56. 
The averaged SI time course will be fitted to a polynomial function, and inflections in the derivative of 
this polynomial will be used to identify the minimum and maximum post contraction SI values. Figure 
4 shows post contraction signal intensity indicating change in oxygenation plotted as a function of 
time. Data obtained at University of Minnesota CMRR 7T, unpublished observation. The change in SI 
(ΔSI = b - a) will be determined for the Sol and MG muscle reflecting the average post contraction 
changes in oxygenation at the muscles microvascular bed. 
 
3.8.2 
Rationale for functional MRI measurements 
A 12 week supervised exercise training program for patients with PAD showed that increases in peak 
VO2 was correlated with increases in capillary density and to a 70% improvement in peak leg blood 
flow measured using cuff occlusion plethysmography 
58. These results show that the improvements in 
functional clinical parameters are related to an enhanced tissue perfusion of the leg. The fMRI 
technique allows for the measurement of post contraction changes in tissue oxygenation with a high 
temporal and spatial resolution. The assessment of microvascular function in the MG and Sol muscle 
separately will allow us to determine if the effect of Entresto may be muscle fiber type dependent. 
Theoretical calculations indicate that at 7T, SE acquired images reflect tissue oxygenation surrounding 
small vessels, whereas images acquired using GRE are reflective of oxygenation status in tissues 
surrounding small and large vessels.  
4 
Subjects 
4.1 Study Setting 
This study will take place at the University of Minnesota. 
 
4.2 Eligibility Criteria 
Inclusion Criteria 
Subjects will be eligible to participate in the study if all of the following conditions exist: 
1. Age 18-80 years of age 
2. Intermittent claudication, such as exercise-induced pain, cramps, fatigue, or other 
equivalent discomfort, involving large muscle groups of the leg(s) (calf, thigh, 
buttocks), relieved by rest. 
3. Ankle-brachial index ≤ 0.90 acquired according to the American Heart Association 
guidelines. 
4. Highest ankle pressure reduced by at least 25 mm Hg after exercise compared to 
resting pressure (or loss of previously present Doppler signal for both the posterior 
tibial and anterior tibial arteries immediately after exercise if arteries were 
incompressible). 
5. Patients on medical treatment for PAD without significant improvement in 
intermittent claudication within the last 6 months. 
6. Absence of more than 60% renal artery stenosis within the last year. If a duplex 
ultrasound has not been perform within the last year to detect renal artery 

stenosis, it will be schedule during the screening visit. 
7. For women with reproductive potential, a willingness to use methods of contraception 
that will prevent the subject from becoming pregnant during the study. Adequate 
contraception methods include hormonal methods used for two or more menstrual 
cycles before Screening (eg, oral contraceptive pills, contraceptive patch, or 
contraceptive vaginal ring), barrier methods (eg, contraceptive sponge, diaphragm in 
conjunction with contraceptive foam or jelly, or condom in conjunction with 
contraceptive foam or jelly), intrauterine methods (IUD), sterilization (eg, tubal 
ligation or a monogamous relationship with a vasectomized partner) and abstinence. 
 
Exclusion Criteria 
Subjects will be excluded from participation in the study if any of the following conditions exist: 
1. 
Patients with physician diagnosed heart (HF) stage > II, liver failure or cancer other than 
skin cancer. 
2. 
Estimated glomerular filtration rate (eGFR) < 30 mL/min/1.73 m2. 
3. 
Severe limitations in mobility due to osteomuscular disorders. 
4. 
Dementia or other mental disorders that prevent patients from following a research 
protocol. 
a. Subjects that score ≤ 23/30 in the Mini Mental State Examination. 
5. 
Patients engaged in an exercise rehabilitation program. 
6. 
Patients schedule to undergo a revascularization procedure during the study. 
7. 
History of angioedema related to use of angiotensin convertase enzyme inhibitor 
(ACEinh) or angiotensin receptor blocker (ARB). 
8. 
Current use of ACEInh or ARB 
9. 
Concomitant use with aliskiren (renin inhibitors) in patients with diabetes. 
10. 
Renal artery stenosis of more than 60% occlusion in any of the renal arteries by duplex 
ultrasound. 
11. 
Patients on therapy with lithium compounds. 
12. 
Women who are pregnant or nursing. Women who are not postmenopausal (≥ 12 
months of non-therapy- induced amenorrhea) or surgically sterile are defined as being 
of child-bearing potential, must have a negative result from a urine pregnancy test at 
Visits 1 and 2. Women with the potential to become pregnant who are not willing to 
use an acceptable form of birth control, defined as abstinence or use of an intrauterine 
device (IUD), oral contraceptive, barrier and spermicide, or hormonal implant 
throughout the study period. 
13. 
Patients with implantable cardiac defibrillators 
14. 
Patients with hyperkalemia (K+ > 5.2 mEq/L) and patients with conditions that are the 
most frequent cause of hyperkalemia, such as CKD and ESRD 
15. 
Patients on potassium sparing diuretics 
 
 
4.3 Recruitment 
Recruitment will be performed with the help of the Clinical and Translational Science Institute (CTSI) 

of the University of Minnesota recruiting services and by advertisement in clinics and other 
institutions with a predominantly elderly population. Patients with PAD who have completed a 
cardiac rehab programs will receive previously IRB approved letters inviting them to participate in 
our study. In addition, from the Institute of Health and Informatics, a list of patients with PAD, 
containing information regarding the date of their upcoming appointment, will be made available to 
the PI at the secure data shelter. The PI will confirm if the patient meets the inclusion and none of the 
exclusion criteria on Epic and will send an email to the provider through Epic, requesting that the 
patient be informed about this clinical trial.  If the patient is interested, they will contact the PI to 
request more information. Our research group has ample experience in recruiting patients with PAD, 
in maintaining high adherence rate and successfully completing clinical trials 
38,59. Although PAD is 
more frequent in African Americans males, we will make an effort to recruit across all racial and 
ethnic groups and from both genders. 
 
4.4 Early Withdrawal of Subjects 
Any subject may discontinue participation in the study for any reason at any time. Subjects who elect to 
withdraw may resume other treatments as determined appropriate by their physician. Reasons and 
circumstances surrounding withdrawal will be documented in the study records. 
 
When a pregnancy has been confirmed in a subject during maternal exposure to treatment, the study 
drug will be discontinued immediately and the subject will be referred to her primary physician. In the 
event that any subject discontinues study drug and, subsequently, withdraws from the study before 
completion, regardless of reason, reasonable efforts will be made to have the subject return for an early 
termination visit and have the end of treatment procedures completed. If a subject is unable to return to 
the clinic for an early termination visit, attempts will be made to contact the subject by phone to assess 
AEs. Subjects will be asked to return to the clinic for further assessments if the PI deems it appropriate. 
 
4.5 Stopping Criteria 
Reasons for terminating the study may include but are not necessarily limited to the following: 
• 
The incidence or severity of AEs indicates a potential health hazard to subjects. 
• 
Advice of DSMB 
• 
Subject enrollment is unsatisfactory. 
If the study is discontinued, all assessments listed in the end of treatment visit should be completed. 
 
4.6 Data Collection for Withdrawn Subjects 
Data of withdrawn subjects will be collected up to the point of withdrawal. This data will still be 
included in the pooled data of the study for further analysis. For subjects who withdraw from further 
data collection, there will not be replacement with more subjects. When study drug is discontinued 
(regardless of the reason), the investigator will encourage that all of the evaluations required at the End 
of Treatment Visit be performed and that any additional evaluations be completed that may be 
necessary to ensure that the patient is free of untoward effects. The patient will be encouraged to seek 
appropriate follow-up for any continuing health problems. 
 
5 
Study Drug 

5.1 Entresto (sacubitril/valsartan) 
Sacubitril is a molecule that inactivates neprilysin, a neutral endopeptidase responsible for degrading 
natriuretic peptides. Sacubitril cannot be given alone as it raises the levels of angiotensin II 62-64 and 
for this reason is combined with valsartan, an angiotensin II receptor blocker 65. This combination, 
sacubitril/valsartan [LCZ696 (Entresto), Novartis Pharmaceuticals Corp.], has been given to patients 
with heart failure raising serum and urinary levels of cGMP, a marker of NPs activity, by 35% and 
25%, respectively 24 and decreases mortality in this type of patients 25. Entresto is currently approved 
by the FDA for the treatment of patients with heart failure NYHA Class II-IV 26. 
 
5.1.1 
Description 
ENTRESTO contains a complex comprised of anionic forms of sacubitril and valsartan, sodium 
cations, and water molecules in the molar ratio of 1:1:3:2.5, respectively. Following oral 
administration, the complex dissociates into sacubitril (which is further metabolized to LBQ657) and 
valsartan. The complex is chemically described as Octadecasodiumhexakis(4-{[(1S,3R)- 1-([1,1´-
biphenyl]-4-ylmethyl)-4-ethoxy-3-methyl-4-oxobutyl]amino}-4-oxobutanoate)hexakis(N-pentanoyl-
N-{[2´-(1H- tetrazol-1-id-5-yl)[1,1´-biphenyl]-4-yl]methyl}-L-valinate)—water (1/15). Its empirical 
formula (hemipentahydrate) is C48H55N6O8Na3 2.5 H2O. Its molecular mass is 957.99 M. 
 
5.2    Dosage and Administration 
Entresto will be given orally at the recommended starting dose of 49/51 mg twice-daily. The dose 
of Entresto should be doubled after 2 to 4 weeks to the target maintenance dose of 97/103 mg twice 
daily, as tolerated by the patient 
25,60. 
 
12 Weeks of Treatment 
We will follow the recommended dosages as reported in the Entresto prescribing information sheet 
distributed by: Novartis Pharmaceuticals Corporation East Hanover, New Jersey 07936. Subjects will 
initiate with the recommended dose of 49/51 mg (sacubitril/valsartan) twice daily for two weeks. For 
patients not currently taking an angiotensin-
converting enzyme inhibitor (ACEi) or an 
angiotensin II receptor blocker (ARB) or 
previously taking a low dose of these agents 
we will initiate treatment with the lowest 
dose available (24/26) (sacubitril/valsartan) 
twice daily. After two weeks of treatment 
the study physician will progressively 
increase to the target maintenance dose of 
97/103 mg (sacubitril/valsartan). Each 
participant will receive a package with 
enough pills containing Entresto/placebo 
that will last until the following schedule 
visit. To assure adherence to the treatment, 
patients will return all the packages with the empty vials at each study visit. Participating patients will 
be encouraged to follow their standard health care treatment and lifestyle recommendations prescribed 
Table 1. Criteria to lower dosage or discontinuation of 
Entresto 
1. Hypotension (symptomatic hypotension 
or orthostatic hypotension, or hypotension 
that requires treatment) 
2. Renal failure. Increase in creatinine > 50% of    
baseline value. 
3. Hyperkalemia. K+ > 5.2 mEq/L 
4. Falls (related to hypotensive episodes) 
5. Angioedema 
Hypotension = a decrease from baseline in sitting 
systolic blood pressure (SBP) of ≥ 20 mm Hg and a 
decrease in sitting SBP to < 90 mm Hg or symptoms.  

by their attending physician. 
5.2.1 
Dose Adjustment 
Dose of Entresto is started at the middle level of 49/51 mg twice-daily and if well tolerated by the 
patient then dosage is increased to the target maintenance dose described above. If manifestations of side 
effects of Entresto appear (Table 1), dose can be lowered to the lowest dose of 24/26 mg twice- daily. If 
at the lowest dose level adverse events continue then patient will be withdrawn from the study. Dose of 
Entresto will be administered following the recommendation as written in the Entresto prescribing 
information sheet from Novartis Pharmaceuticals Corporation East Hanover, New Jersey 07936 as 
revised by the FDA on August, 2015 (Patient information sheet attached). The study physician Dr. Jiwan 
Thapa will be responsible for all dose changes in subjects participating in this study. Specific guidelines 
on dosage change are detailed below. 
 
Dosage modifications guidelines:  
 
Any patient with a serum potassium > 5.2 mEq/L after randomization requires regular, repeated checks of 
potassium concentration (beyond that prescribed in the protocol) until it is clear that the potassium 
concentration is stable and not rising into the range of concern (≥ 5.5 and < 6.0 mEq/L) or potential 
danger (≥ 6.0 mEq/L).  
 
Corrective action for management of hyperkalemia.  
 
Serum potassium > 5.2 and less than or equal to 5.5 mEq/L 
 
Confirm potassium concentration in a non-hemolyzed sample. 
 
Reinforce low potassium diet and restriction of food/drinks with high potassium content (e.g. 
orange juice, melon, bananas, low-salt substitutes etc.). 
 
Review medical regimen (including dietary supplements and over-the-counter medications) for 
agents known to cause hyperkalemia. Consider reduction in dose or discontinuation of these 
agents: 
• 
Potassium supplements, e.g., potassium chloride. 
• 
Salt substitutes. 
• 
Non-steroidal anti-inflammatory drugs (NSAIDs) 
• 
Herbal Supplements: 
o For example, Noni juice, alfalfa (Medicago sativa), dandelion (Taraxacum 
officinale), horsetail (Equisetum arvense), nettle (Urtica dioica), milkweed, lily 
of the valley, Siberian ginseng, hawthorn berries. 
 
Repeat serum potassium measurement within 3 to 5 days. 
 
If serum potassium remains > 5.2 and ≤ 5.5 mEq/L, regularly monitor serum potassium levels to 
ensure stability (suggested once monthly). 
 
Consider down-titration of study medication, according to study physician medical judgment. 
 
Serum potassium > 5.5 and < 6.0 mEq/L 
 
Confirm potassium concentration in a non-hemolyzed sample. 
 
Consider down-titration or temporarily discontinue study drug according to study physician 
medical judgment. 

 
Apply all measures outlined for serum potassium > 5.2 and ≤ 5.5 mEq/L. 
 
Repeat serum potassium measurement after 2-3 days. 
 
If serum potassium < 5.5 mEq/L, consider resumption of study drug at lower dose with repeat 
potassium within 5 days. 
 
Serum potassium greater than or equal to 6.0 mEq/L 
 
Immediately discontinue study drug. 
 
Confirm potassium concentration in a non-hemolyzed sample. 
 
Urgently evaluate patient and treat hyperkalemia as clinically indicated. 
 
Apply all measures outlined for serum potassium > 5.2 and < 6.0 mEq/L. 
 
No resumption of study drug without individualized case discussion with and permission from the 
DSMB. 
 
Guidelines for the management of renal dysfunction 
 
If, at any time after randomization, eGFR decreases by ≥ 25% from baseline (or if serum creatinine 
concentration increase to 2.5 mg/dL or by 50% of baseline value, the investigator will check for 
potentially reversible cases of renal dysfunction such as: 
 
Non-steroidal anti-inflammatory drug intake, antibiotics, or other treatments known to affect 
creatininemia 
 
Volume decrease 
 
Urinary infection 
 
Urinary tract obstruction 
 
Study medication 
 
If a patient eGFR decreases by ≥ 40% from baseline (or if serum creatinine concentration rises above 3 
mg/dL, the investigator will check for potentially reversible causes of renal dysfunction (see above). If the 
study physician judges that the study medications needs to be stopped, the principal investigator needs to 
be informed and the DSMB contacted.  
 
Guidelines for the management of blood pressure 
If symptomatic hypotension occurs: 
 
Correct any treatable cause, e.g. hypovolemia. 
 
If hypotension persists, the study drug should be down-titrated or withdrawn at the judgment of 
the study physician.  
5.3 Method for Assigning Subjects to Treatment Groups 
A variable block randomization design will be used to endure a balance in sample size between the two 
groups 
5.4 Subject Compliance Monitoring 
Compliance to drug administration will be documented at all study visits. Subjects will be asked to 
bring in their medication at each study visit for monitoring purposes. 
5.5 Blinding of Study Drug 
Blinding of the drug will be done by the investigational Drug Services (IDS) at the University of 
Minnesota. Once a subject is eligible to participate in the study, the PI will inform IDS and they will 

be in responsible for randomizing the subject into one of the two groups and preparing the first two 
weeks of treatment. Capsules containing pills with either placebo or Entresto will be placed in a 
container identified with a code, which will be kept by IDS pharmacy unless it is requested as part of 
an emergency response or study termination. Each individual pill will be packaged in identical 
capsules by IDS pharmacy staff. Entresto comes in pills of three dosages: 24/26 mg, 49/51mg and 
97/103 mg. Capsules for each of the three dosages (placebo or Entresto) will be prepared by IDS and 
the study physician (blinded) will provide the capsules following the dosing procedures as described 
in 5.2.1. The subject will only be unblinded if there is a medical situation where it is necessary to 
know if the subject is on drug or placebo. 
 
5.6 Receiving, Storing, Dispensing and Return 
The Investigational Drug Services (IDS) pharmacy staff will in charge of preparing the pills and 
the placebo capsules and dispensing the drug for this study. 
 
6 
Study Procedures 
6.1 Participant Timeline/Procedures 
 
Screening and Baseline 
Pre-Screening Phone 
Interview 
An initial phone 
interview will be used to 
pre-screen subjects for 
potential eligibility. Subjects 
that meet all inclusion 
criteria and none of the 
exclusion criteria as 
described in 4.2, will be 
invited to participate in the 
study. If potential 
participants are interested, 
they will be asked to come in to discuss the study further and if they wish to participate they will be 
asked to sign the consent form. 
Visit 1 (Screening/Baseline 1) 
After consent has been obtained, the following tests and procedures will be done to determine 
whether the potential subject is eligible to participate. 
• 
Patients will be asked a series of questions about symptoms suggestive of intermittent 
claudication (IC) 
• 
Brachial and ankle systolic blood pressure will be measured with both arms and legs. 
Patients will lie supine and will rest quietly for 5 minutes and systolic blood pressure 
(SBP) will be measured once in each arm following standard procedures. Patients will 
have their ankle brachial index (ABI) determined following standard procedures 
described by the American Heart Association 
34. Systolic blood pressure at the ankles will 
Weeks 
0 
2 
4 
6 
12 
16 
20 
Days  
1 and 
2. 
4 hours 
Medication 
adjustments and 
monitoring 
visits.  
Days  
3 and 4. 
4 hours 
each 
Baseline 
visits 
Post 
treatmen
t visits 
Post treatment 
followup visits.  
90 min each 
Timeline and duration of activities 

be measured at the dorsalis pedis and posterior tibialis artery using an 8 MHz doppler 
vascular probe and the highest blood pressure at those sites will be recorded. A ratio of 
highest SBP measured at the ankle divided by the SBP measured at the brachial (ABI) < 
0.90 will meet the diagnostic criteria for PAD. If there are symptoms of IC, but ABI is > 
0.90 than patients will perform a walking test using the Gardner protocol and if highest 
systolic BP at the ankle decreases by > 25 mmHg that is also diagnostic of PAD. 
• 
10 hour fasting venous blood sample for measurements of plasma Na+, K+,  
glucose, creatinine, serum insulin and urinary cGMP and creatinine. Measure BP 
three times and the average of those measurements will be recorded. 
• 
will be collected for measurement of plasma potassium and creatinine/eGFR 
• 
Women of child bearing potential will have a urine pregnancy test to verify they are not 
pregnant. 
• 
Patients will perform the mini mental state evaluation to test. Patients must have a score of 
≥23/30 to be included in the study. 
 
Individuals evaluated by the study physician or the main PI that meet all the inclusion and none of the 
exclusion criteria will continue with the following tests and procedures. 
 
• 
Arterial elasticity testing using applanation tonometry in the radial artery. Patients in the 
supine position will have electrodes placed in their chest to record an EKG, will have a 
blood pressure taken and arterial pulse wave will be recorded for 30 seconds in the radial 
artery. 
• 
A graded exercise testing procedure to determine pain free and maximal walking duration 
will be performed. Participant’s blood pressure and EKG will be measured before, during 
and for 6 minutes following the exercise test. Contraindications outlined by the American 
College of Sports Medicine to perform and to stop an exercise test will be followed. 
• 
The 6 minute walk test will be performed in a 20 meter corridor. Patients will be asked to 
walk as quickly as they can along the corridor for 6 minutes to cover as much distance as 
possible. They will be allowed to stop if they have to, but will be encouraged to continue 
again as soon as they are able. During the time that the participant stops walking the clock 
will continue to run. At the end of test the distance covered is measured. 
• 
A Walking Impairment Questionnaire (WIQ), a 36-item short-form health survey (SF-
36), and Quality of Life Questionnaire (PAD-QOL) will be given to each participant. 
• 
Body height and weight will be measured using standard anthropometric procedures. 
• 
Total body lean and fat mass will be determined using bioelectrical impedance from 
Valhalla Scientific body comp scale®. 
 
Visit 2 (Baseline 2/Randomization) 
The second screening visit will take place within 48 hours of Visit 1. The following tests and procedures 
will be performed: 
• 
Women of child bearing potential will have a urine pregnancy test to verify they are not 
pregnant. 
• 
MRS and fMRI will be performed in a 7 Tesla/90 cm whole-body actively shielded human 

scanner. 
• 
IDS will randomize and dispense the treatment drug or placebo. 
• 
The subject will be given instructions on how to take the drug and will be provided a diary 
to keep track of any adverse events they are experiencing. 
• 
Ambulatory blood pressure monitors using the Omron BP742N (©Omron Healthcare Inc. 
Lake Forest, IL) will be provided and each participant will be asked to measure and 
record their own arm blood pressure three times a day for the duration of the treatment 
period. Subjects will be asked to measure their blood pressure immediately after waking 
up, at noon before lunch and in the night before going to bed. Participants will be 
instructed on the use of the ambulatory blood pressure monitoring (ABPM) and will have 
a practice session. 
6.2 Treatment Period 
Visits 3, 4 and 5 (Treatment visits at Weeks 2, 4 and 6) 
Subjects will be required to return to the clinic at weeks 2, 4 and 6 of treatment to perform the 
following procedures and tests: 
• 
Dose modification to target dose at week 2 and lowering of dose per direction of physician at 
weeks 4 and 6. 
• 
Blood glucose, creatinine and potassium levels. 
• 
Women of child bearing potential will have a urine pregnancy test to verify they are not 
pregnant. 
• 
Blood pressure measurement and reading of recording on the blood pressure monitor. 
• 
Review of daily diaries for any side effects. 
• 
Collect and assess study medication; dispense study medication. 
 
6.3 Phone Calls 
The PI or Study Coordinator will call subjects every two weeks during the treatment period in order 
to assess AEs. The phone calls will occur at Week 1, 3, 5 and 7. 
 
6.4 End of Treatment 
Visit 6 (End of treatment visit 1) 
At the end of the 12 week treatment period, subjects will be asked to return for testing. Subjects will 
be asked to return within 24 hours of taking their last study pill. The following tests and procedures 
will be performed: 
• 
Ankle Brachial Index (ABI) 
• 
GXT will be done to determine pain free and maximal walking distance 
• 
A Walking Impairment Questionnaire (WIQ), a 36-item short-form health survey (SF-
36), and Quality of Life Questionnaire (PAD-QOL) will be given 
• 
Body height and weight will be measured using standard anthropometric procedures 
• 
Total body lean and fat mass will be determined 
• 
Blood pressure measurement and reading of recording on the blood pressure monitor. 
• 
Review of daily diaries for any side effects. 

• 
Collect and assess study medication.  
• 
Women of child bearing potential will have a urine pregnancy test to verify they are not 
pregnant. 
 
Visit 7 (End of treatment visit 2) 
Visit 7 will occur within 48 hours of visit 6. At that visit the following tests and procedures will be done: 
• 
10 hour fasting venous blood sample for measurements of plasma Na+, K+,  glucose, 
creatinine, NT-proBNP, BNP, serum insulin and  also urinary cGMP and creatinine.   
• 
Women of child bearing potential will have a urine pregnancy test to verify they are 
not pregnant. The 6 minute walk test will be performed in a 20 meter corridor. 
• 
Measure BP three times and the average of those measurements will be recorded. 
• 
fMRI and MRS will be performed in a 7 Tesla/90 cm whole-body actively shielded human 
scanner. 
6.5 Follow-up Visits 
Visits 8 (Follow-up, Week 6 after End of Treatment Visit) 
Subjects will return  6 weeks after their treatment has ended to assess if any changes have occurred in pain 
free walking duration, maximal walking duration during the 6 minute walking test. Subjects will have a 
Walking Impairment Questionnaire (WIQ) and Quality of Life Questionnaire (PAD-QOL) administered. 
The presence or absence of adverse events following withdrawal of study drug will be assessed.  

Schedule of Events 
 
Schedule 
of Events 
Visit 1 
Screening 
Visit 2 
Baseline 
Study Visits 3-7 
Follow-up Visit 8 
Time Point 
Day 0 
Week 2 
Week 4 
Week 8 
Week 12 
Week 12 
Week 18 
Time estimates 
4 hours 
4 hours 
1 hour 
1 hour 
1 hour 
4 hours 
4 hours 
45 min  
Obtain Consent 
x 
Ankle brachial Index 
x 
x 
Duplex ultrasound1 
x 
 
 
 
Arterial elasticity 
X 
X 
 
 
Questionnaires 
x 
x 
x 
Memory test 
X 
 
 
 
 
Height, weight, lean 
and fat mass 
X 
 
X 
 
 
Treadmill test 
x 
 
x 
 
x 
6 minute walk 
x 
x 
x 
x 
MRI 
x 
x 
Blood draw for labs 
X 
x 
x 
x 
x 
x 
x 
Urine test 
x 
x 
Blood pressure2 
x 
x 
x 
x 
x 
x 
x 
x 
Pregnancy test3 
x 
x 
X 
X 
X 
X 
X 
X 
Receive 
drug/placebo 
x 
x 
x 
x 
Review blood 
pressure records and 
diary 
x 
x 
x 
x 
 
 
1. Screening visit could be 5 hours long if duplex ultrasound is required, if not it will last 4 hours. 
2. Visit should occur within 24 hours of last dose of drug.  
3. Blood draw at screening and end of treatment visit 1are following 10 hour fasting to assess serum: creatinine and K+, sodium, glucose, and 
serum insulin. Study visits at weeks 2, 4 and 6 are also fasting 10 hours prior to blood draw to assess: serum glucose, creatinine and 
potassium. 
4. Duplex ultrasound will be perform only on those patients without a duplex ultrasound for the diagnosis of renal artery stenosis within the last 
year. 
5. NIH toolbox for cognitive function. 

6. The treadmill GXT test will be used to determine pain free walking distance, maximal walking distance.  
7. Subjects will be instructed to measure and record their blood pressure at home, 3 times daily during the treatment period. 
8. For women of childbearing potential.

7. Data Handling and Record Keeping 
 
6.6 Confidentiality 
Information about study subjects will be kept confidential and managed according to the requirements of the 
Health Insurance Portability and Accountability Act of 1996 (HIPAA). Those regulations require a signed 
subject authorization informing the subject of the following: 
• 
What protected health information (PHI) will be collected from subjects in this study 
• 
Who will have access to that information and why 
• 
Who will use or disclose that information 
• 
The rights of a research subject to revoke their authorization for use of their PHI. 
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by regulation, retains the 
ability to use all information collected prior to the revocation of subject authorization. For subjects that have 
revoked authorization to collect or use PHI, attempts should be made to obtain permission to collect at least vital 
status (i.e. that the subject is alive) at the end of their scheduled study period. 
 
6.7 Types of Data 
Data for this study will include safety, laboratory and outcome measures. 
 
6.8 Record Retention 
Per University of Minnesota policy all documents concerning the use of human subjects in research will be 
maintained for 3 years from completion of IRB-related work and 6 years for HIPAA. No record will be destroyed 
without the written consent of the sponsor-investigator. 
 
The sponsor-investigator will permit authorized representatives of the University of Minnesota and regulatory 
agencies to examine (and when required by applicable law, copy) clinical records for the purposes of clinical site 
monitoring, quality assurance reviews, audits, and evaluation of the study safety and progress. 
 
6.9 Source Documents 
A source document is defined as the location where study-related data are initially recorded. Source documents 
for this study will include hard copy paper and/or electronic forms, laboratory printouts, and medical records 
onto or into which data will first be recorded. 
 
6.10 Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol. The noncompliance may be either on 
the part of the subject, the investigator, or the study site staff. As a result of deviations, corrective actions are to 
be developed by the site and implemented promptly. 
 
When a deviation from the protocol is necessary for an individual subject, the Investigator must complete a 
description of the deviation from the protocol and justification on the Protocol Deviation Form. 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 28 of 32 
 
7 
Study Auditing, and Inspecting 
7.1 Auditing and Inspecting 
This study will be monitored according to FDA/GCP guidelines. The investigator will allocate adequate time for 
such monitoring activities. The Investigator will also ensure that the monitor or other compliance or 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 monitoring visit. 
 
8 
Safety and Adverse Events 
8.1 Adverse Event Definitions and Reporting Guidelines 
 
9.1.1 
Adverse Event 
An adverse event (AE) is any symptom, sign, illness or experience that develops or worsens in severity during 
the course of the study. 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 
 
9.1.1.1 
Serious Adverse Events (SAE) 
 
A serious adverse event (SAE) is any adverse event that is: 
• 
Fatal 
• 
Life-threatening 
• 
Requires or prolongs a hospital stay 
• 
Results in persistent or significant disability or incapacity 
• 
A congenital anomaly or birth defect 
 
Important medical events are events that may not be immediately life-threatening, but are clearly of major clinical 
significance and may be SAEs. They may jeopardize the subject, and may require intervention to prevent one or 
the other serious outcomes noted above. 
 
Hospitalization 
Hospitalization shall include any initial admission (even if less than 24 hours) to a healthcare facility as a result 
of a precipitating clinical adverse event; to include transfer within the hospital to an intensive care unit. 
Hospitalization or prolongation of hospitalization in the absence of a precipitating, clinical adverse event (e.g., 
for a preexisting condition not associated with a new adverse event or with a worsening of the preexisting 
condition; admission for a protocol- specified procedure) is not, in itself, a serious adverse effect. 
 
9.1.1.2 
Expected Adverse Events (AE) 
Expected adverse events are those that are known to be associated with or have the potential to arise as a 
consequence of participation in the study. 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 29 of 32 
 
 
9.1.2 
Unexpected Adverse Event 
An adverse event or suspected adverse reaction is considered unexpected if it is not listed in the Protocol at the 
specificity or severity that has been observed. 
Unanticipated Problems Involving Risk to Subjects or Others (UPIRTSO) 
An adverse event that in the opinion of the Principal Investigator is unexpected and related to the investigational 
agent. 
Assessment of Severity 
Categories 
Definition 
Definitely related 
This relationship suggests that a definite 
causal relationship exists between the 
administration of the investigational agent 
and the AE, and other conditions 
(concurrent illness, progression/expression 
of disease state, or concurrent medication 
reaction) do not appear 
to explain the event. 
Probably related 
This relationship suggests that a reasonable 
temporal sequence of the event with 
investigational agent administration exists 
and, based upon the known or previously 
reported adverse reactions, or judgment 
based on the investigator’s clinical 
experience, the association of the event with 
the investigational agent 
seems likely. 
Possibly related 
This relationship suggests that treatment with 
the investigational agent may have caused or 
contributed to the AE (i.e., the event follows a 
reasonable temporal sequence from the time 
of investigational agent administration and/or 
follows a known response pattern to the 
investigational agent but could also have been 
produced by other factors.) 
Not related 
This relationship suggests that there is no 
association between the investigational agent 
and the reported event. 
 
The sponsor-investigator will make an assessment of severity for each AE and SAE reported during 
the study. The assessment will be based on the sponsor-investigator’s clinical judgment. 
 
Assessment of Causality 
The sponsor-investigator will estimate the relationship between the investigational agent and the occurrence of 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 30 of 32 
 
each AE or SAE by using his best clinical judgment. Other elements, such as the history of the underlying 
disease, concomitant therapy, other risk factors, and the temporal relationship of the event to administration of 
the investigational agent, will be considered and investigated. 
 
An SAE may be recorded when the sponsor-investigator has minimal information to include in the initial report. 
The sponsor-investigator may change his opinion of the causality in light of follow-up information, with 
subsequent amendment of the SAE report. 
 
Recording of Adverse Events 
At each contact with the subject, the investigator must seek information on adverse events by specific 
questioning and, as appropriate, by examination. Information on all adverse events should be recorded 
immediately in the source document, and also in the appropriate adverse event module of the case report form 
(CRF). All clearly related signs, symptoms, and abnormal diagnostic procedures results should be recorded in 
the source document, though should be grouped under one diagnosis. A adverse event monitoring form will be 
use to interview patients during the biweekly phone call to assess presence or absence of adverse events.   
 
All adverse events occurring during the study period must be recorded.  The clinical course of each event should 
be followed until resolution, stabilization, or until it has been determined that the study treatment or 
participation is not the cause. Serious adverse events will be collected for the six month period following study 
product administration. Serious adverse events that are still ongoing at the end of the study period must be 
followed up to determine the final outcome. Any serious adverse event that occurs after the study period and is 
considered to be possibly related to the study treatment or study participation should be recorded and reported 
immediately. 
 
 
9.1.2.1 
Data and Safety Monitoring Board (DSMB) 
Before recruitment of participants, an independent DSMB will be formed in accord with guidelines published by 
the U.S. Department of Human Health Services 
61 and the National Heart Lung and Blood Institute 
62. This 
board will be composed by a physician expert in the management of patients with PAD, a biostatistician and at 
least two investigators with expertise in current clinical trials conduct and methodology. Board members will be 
independent from the study and at least one will not be affiliated with the University of Minnesota. None of 
them will have any financial, proprietary, professional, or other interests that may affect impartial, independent 
decision-making that constitute a potential conflict of interest. This board will be in charge of 1) periodically 
reviewing and evaluate the accumulated study data for participant safety, study conduct and progress, and, when 
appropriate, efficacy, and 2) make recommendations to the principal investigators (PIs) and the Internal Review 
Board concerning the continuation, modification, or termination of the trial. A DSMB will be selected and meet 
before the start of recruitment and will design a plan of action, frequency of meetings and elaborate detail safety 
guidelines for this study. 
 
9 
Data Analysis 
9.1 Statistical Methods 
General baseline characteristics will be described using mean and proportions. Smirnov-Kolmogorov tests will 
be used to test normality of distribution of variables. For variables that do not follow the normal distribution, log 
transformation will be performed. Linear simple and multiple regressions will be performed to assess 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 31 of 32 
 
association between the effects of Entresto on metabolic and physiological changes with clinical functional 
variables. Differences between the placebo and the 
Entresto group will be assessed using 
independent sample t-test and Chi-square 
for quantitative and categorical variables, 
respectively. Change following the 12 
week clinical trial in metabolic and 
physiological parameters will be 
assessed using paired sample t-test. 
Repeated measures analysis of 
covariance will also be used to evaluate 
differences between groups as a function 
of time. A p value < 0.05 will be used to 
determine statistical significance. Effect 
size (EF) calculated as EF = (mean 
Entresto group – mean placebo 
group)/SD (control group) will be used to assess the clinical significance of the effects of Entresto 
63. The PI 
will be in charge of performing all statistical calculations using SAS v 9.3 by SAS Institute Inc., Cary, NC. Dr. 
David R. Jacobs Jr. Professor at the School of Public Health a statistician with more the 800 publications will 
be a consultant in statistical analysis of the data. 
 
9.2 Power analysis 
Sample size for a double blind randomized clinical trial was determined using SAS version 9.3 (SAS Institute 
Inc., Cary, NC, USA). Sample size is based on the mean (SD) values obtain in a clinical trial that measured the 
effects of carperitide on pain free walking distance in patients with PAD 5. The mean (SD) pre post change in 
pain free walking distance was of 278 (240) m. [We have estimated that for a power 0.85 we will need 15 
subjects per group to reject the null hypothesis with Type I error probability of 0.05] (Figure 6). 
 
10 Clinical Management 
Dr. Jiwan Thapa  Assistant Professor in the Department of Medicine Division of Kidney Diseases and 
Hypertension will be the study physician for clinical trial. This includes adjusting the dose of Entresto (based 
on information obtain from the Entresto information sheet, Novartis), development of safety procedures and 
treatment of medical complications. The main PI (Otto A. Sánchez MD PhD) will be the person directly in 
charge of admittance of patients into the study, performing and supervising the diagnostic tests and the 
metabolic, physiological and clinical functional testing. 
 
11 Risks/Benefits 
11.1 Potential Risks 
As part of the PARADIGM-HF clinical trial adverse reactions occurring at an incidence of ≥ 5% in patients 
who were treated with ENTRESTO in the double-blind period are shown in Table 2. 
 
 
 
 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 32 of 32 
 
Table 2. Adverse effects and percentage of cases in the PARADIGM-HF. 
Adverse effects 
Entresto 
Placebo + Enalapril 
Preventive or corrective action 
Angioedema, % 
0.5 
0.2 
Discontinue 
Hypotension, % 
 
18 
 
12 
Dose adjustment of blood 
pressure lowering medications 
Serious 
hypotension, % 
 
1.5 
 
1.5 
Dose adjustment of blood 
pressure lowering medications 
Renal failure, % 
5 
5 
Monitor creat and blood urea 
Hyperkalemia 
12 
14 
Monitor K+ 
Cough, % 
9 
13 
Modify dose 
Dizziness, % 
6 
9 
Prevent hypotension 
Falls, % 
1.90 
1.30 
Prevent hypotension 
 
 
11.2 Methods to Minimize Risks 
Patients will return to the clinic on weeks 2, 4 and 6 of the treatment period to assess for known adverse events 
and dosage adjustment if necessary. In addition, the clinical coordinator or the principal investigator will contact 
the subject by phone every other week to assess presence of adverse events and determine if an additional visit 
to the clinic is required. Patients will maintain a diary provided by the principal investigator to record blood 
pressure values and the date and time of the occurrence of known or unexpected adverse reactions. Subjects will 
be verbally warned and given a handout with a list of medications which are contraindicated or potentially cause 
an adverse interaction with Entresto. The medications on the list are on the Novartis information sheet on 
Entresto. The study physician and the PI will highlight these medications and the potential adverse events that 
can occur. 
 
11.3 Emergency Procedures 
Patients will be given detailed instructions of how to recognize any of the known serious or non-serious adverse 
events that may occur while receiving Entresto and also to report on previously unreported adverse events. If a 
non-life-threatening serious adverse events occurs, patients are advised to contact their primary care physician and 
then to contact the principal investigator.  If there is a life threatening situation, subjects are advised to call 911 and 
then to contact the principal investigator. Patients will be given an emergency identification card with the contact 
information of the principal investigator and the name of the drug, to help inform the health care provider that this 
patient is in a clinical trial and may have been randomized to either Entresto or a placebo. In case of a serious 
adverse event, Entresto will be discontinued or dosage may be adjusted depending on the judgement of the study 
physician. All adverse events will be reported to the DSMB and their recommendations will be followed.  
 
11.4 Unblinding Procedures in Case of Emergency 
The Investigational Drug Services (IDS) pharmacy will generate the randomization schedule and will keep 
the clinical trial code stored in a secure location. The inpatient IDS pharmacy will hold the code, which can 
be access 24 hours, 7 days a week if necessary. The study physician and the main PI can have access to the 
code in case of a clinical emergency. The code will be revealed on request by the data safety monitoring 
board (DSMB) and at the completion of the clinical trial. 
 
12 Ethical Considerations 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 33 of 32 
 
12.1 Good Clinical Practice 
The study will be conducted in accordance with the appropriate regulatory requirement(s). Essential clinical 
documents will be maintained to demonstrate the validity of the study and the integrity of the data collected. 
Master files should be established at the beginning of the study, maintained for the duration of the study and 
retained according to the appropriate regulations. 
 
12.2 Institutional Review Board (IRB) Review and Informed Consent 
The study will be conducted in accordance with ethical principles founded in the Declaration of Helsinki. The 
IRB will review all appropriate study documentation in order to safeguard the rights, safety and well-being of 
the patients. The study will only be conducted once IRB approval has been obtained. The protocol, informed 
consent, written information given to the patients, safety updates, annual progress reports, and any revisions to 
these documents will be provided to the IRB by the investigator. 
 
A signed consent form will be obtained from the subject. The consent form will describe the purpose of the 
study, the procedures to be followed, and the risks and benefits of participation. A copy of the consent form will 
be given to the subject, and this fact will be documented in the subject’s record. Research staff will ensure that 
candidates understand all elements of the consent form by addressing questions posed during the consent 
procedure and by asking for verbal confirmation that the candidate has no additional questions and verbally 
understands the purpose of the study, study intervention, basic procedures, risks, and that participation is 
voluntary. Subjects will be given as much time as they need to read and understand the consent form. 
 
12.3 Confidentiality 
All biologic specimens and clinical data obtained from this study will be linked to a study specific code and not 
to personal identifying information (e.g., name, social security number, medical record number). A key which 
will link the four-digit study code to the personal information will be maintained in a secure pass-word 
protected file maintained by the site study investigators. 
 
All sites are committed to ensuring that appropriate measures are taken to protect the privacy and 
confidentiality of all Protected Health Information (PHI) for which it is responsible. This includes compliance 
with all regulations set forth by the relevant jurisdictions. In the US, this includes the HIPAA Privacy Rule, as 
well as with existing US State and Federal laws pertaining to PHI. All study volunteers in US will be given a 
copy of the Notice of Privacy Practices. Good faith efforts will be made to obtain each individual’s written 
acknowledgement of receipt of the Notice(s). The rights mandated by the HIPAA Privacy Rule concerning an 
individual’s ability to access, amend, and disclose certain sections of his or her own PHI will be fully respected, 
and every effort will be made to assist patients who choose to exercise these rights. 
 
12.4 Declaration of Interests 
Novartis is not funding this study and will play no role in the collection, analysis and interpretation of the 
data or the conduct of the study. None of the researchers have a conflict of interest to disclose regarding 
Entresto. 
 
12.5 Publication Policy 
The International Committee of Medical Journal Editors (ICMJE) member journals have adopted a trials-
registration policy as a condition for publication. This policy requires that all clinical trials be registered in a 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 34 of 32 
 
public trials registry such as ClinicalTrials.gov, which is sponsored by the National Library of Medicine. This 
protocol will be registered on ClinicalTrials.gov. 
 
 
References 
 
1. 
Fowkes FG, Housley E, Cawood EH, Macintyre CC, Ruckley CV, Prescott RJ. Edinburgh Artery Study: 
prevalence of asymptomatic and symptomatic peripheral arterial disease in the general population. Int J Epidemiol 
1991;20:384-92. 
2. 
Hirsch AT, Criqui MH, Treat-Jacobson D, et al. Peripheral arterial disease detection, awareness, and 
treatment in primary care. JAMA 2001;286:1317-24. 
3. 
Widener JM. Peripheral arterial disease and disability from NHANES 2001-2004 data. J Vasc Nurs 
2011;29:104-12. 
4. 
Stewart KJ, Hiatt WR, Regensteiner JG, Hirsch AT. Exercise training for claudication. New England 
Journal of Medicine 2002;347:1941-51. 
5. 
Anderson JD, Epstein FH, Meyer CH, et al. Multifactorial determinants of functional capacity in 
peripheral arterial disease: uncoupling of calf muscle perfusion and metabolism. J Am Coll Cardiol 2009;54:628-
35. 
6. 
Englund EK, Langham MC, Li C, et al. Combined measurement of perfusion, venous oxygen saturation, 
and skeletal muscle T2* during reactive hyperemia in the leg. J Cardiovasc Magn Reson 2013;15:70. 
7. 
Wu WC, Mohler E, 3rd, Ratcliffe SJ, Wehrli FW, Detre JA, Floyd TF. Skeletal muscle microvascular flow 
in progressive peripheral artery disease: assessment with continuous arterial spin-labeling perfusion magnetic 
resonance imaging. Journal of the American College of Cardiology 2009;53:2372-7. 
8. 
Park K, Itoh H, Yamahara K, et al. Therapeutic potential of atrial natriuretic peptide administration on 
peripheral arterial diseases. Endocrinology 2008;149:483-91. 
9. 
Yamahara K, Itoh H, Chun TH, et al. Significance and therapeutic potential of the natriuretic 
peptides/cGMP/cGMP-dependent protein kinase pathway in vascular regeneration. Proc Natl Acad Sci U S A 
2003;100:3404-9. 
10. 
von Lueder TG, Sangaralingham SJ, Wang BH, et al. Renin-angiotensin blockade combined with 
natriuretic peptide system augmentation: novel therapeutic concepts to combat heart failure. Circ Heart Fail 
2013;6:594-605. 
11. 
Mangiafico S, Costello-Boerrigter LC, Andersen IA, Cataliotti A, Burnett JC, Jr. Neutral endopeptidase 
inhibition and the natriuretic peptide system: an evolving strategy in cardiovascular therapeutics. Eur Heart J 
2013;34:886-93c. 
12. 
Ichiki T, Burnett JC, Jr. Atrial Natriuretic Peptide- Old But New Therapeutic in Cardiovascular Diseases. 
Circ J 2017;81:913-9. 
13. 
Husmann M, Jacomella V, Thalhammer C, Amann-Vesti BR. Markers of arterial stiffness in peripheral 
arterial disease. Vasa 2015;44:341-8. 
14. 
Roset PN. Systematic review of the efficacy of cilostazol, naftidrofuryl oxalate and pentoxifylline for the 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 35 of 32 
 
treatment of intermittent claudication (Br J Surg 2012; 99: 1630-1638). Br J Surg 2013;100:1838. 
15. 
Kudagi VS, White CJ. Endovascular stents: a review of their use in peripheral arterial disease. Am J 
Cardiovasc Drugs 2013;13:199-212. 
16. 
Schillinger M, Sabeti S, Loewe C, et al. Balloon angioplasty versus implantation of nitinol stents in the 
superficial femoral artery. New England Journal of Medicine 2006;354:1879-88. 
17. 
Grochot-Przeczek A, Dulak J, Jozkowicz A. Therapeutic angiogenesis for revascularization in peripheral 
artery disease. Gene 2013;525:220-8. 
18. 
Perkins JM, Collin J, Creasy TS, Fletcher EW, Morris PJ. Exercise training versus angioplasty for stable 
claudication. Long and medium term results of a prospective, randomised trial. Eur J Vasc Endovasc Surg 
1996;11:409-13. 
19. 
Ashworth NL, Chad KE, Harrison EL, Reeder BA, Marshall SC. Home versus center based physical 
activity programs in older adults. Cochrane Database Syst Rev 2005:CD004017. 
20. 
Haas TL, Lloyd PG, Yang HT, Terjung RL. Exercise training and peripheral arterial disease. Compr 
Physiol 2012;2:2933-3017. 
21. 
Houben AJHM, van der Zander K, de Leeuw PW. Vascular and renal actions of brain natriuretic peptide in 
man: physiology and pharmacology. Fund Clin Pharmacol 2005;19:411-9. 
22. 
Woodard GE, Rosado JA. Natriuretic peptides in vascular physiology and pathology. Int Rev Cell Mol 
Biol 2008;268:59-93. 
23. 
Yamahara K, Itoh H, Chun TH, et al. Significance and therapeutic potential of the natriuretic 
peptides/cGMP/cGMP-dependent protein kinase pathway in vascular regeneration. Proceedings of the National 
Academy of Sciences of the United States of America 2003;100:3404-9. 
24. 
Kobalava Z, Kotovskaya Y, Averkov O, et al. Pharmacodynamic and Pharmacokinetic Profiles of 
Sacubitril/Valsartan (LCZ696) in Patients with Heart Failure and Reduced Ejection Fraction. Cardiovascular 
therapeutics 2016;34:191-8. 
25. 
McMurray JJ, Packer M, Desai AS, et al. Angiotensin-neprilysin inhibition versus enalapril in heart 
failure. N Engl J Med 2014;371:993-1004. 
26. 
FDA approves new drug to treat heart failure. U.S. Department of Health and Human Services, 2015. at 
http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm453845.htm.) 
27. 
Ichiki T, Izumi R, Cataliotti A, Larsen AM, Sandberg SM, Burnett JC, Jr. Endothelial permeability in vitro 
and in vivo: protective actions of ANP and omapatrilat in experimental atherosclerosis. Peptides 2013;48:21-6. 
28. 
Nitta A, Hozawa A, Kuriyama S, et al. Relationship between peripheral arterial disease and incident 
disability among elderly Japanese: the Tsurugaya project. J Atheroscler Thromb 2010;17:1290-6. 
29. 
McDermott MM, Guralnik JM, Tian L, et al. Baseline functional performance predicts the rate of mobility 
loss in persons with peripheral arterial disease. Journal of the American College of Cardiology 2007;50:974-82. 
30. 
Peacock JM, Keo HH, Duval S, et al. The incidence and health economic burden of ischemic amputation 
in Minnesota, 2005-2008. Prev Chronic Dis 2011;8:A141. 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 36 of 32 
 
31. 
McDermott MM, Liu K, Ferrucci L, et al. Decline in functional performance predicts later increased 
mobility loss and mortality in peripheral arterial disease. Journal of the American College of Cardiology 
2011;57:962-70. 
32. 
Mahoney EM, Wang K, Cohen DJ, et al. One-year costs in patients with a history of or at risk for 
atherothrombosis in the United States. Circ Cardiovasc Qual Outcomes 2008;1:38-45. 
33. 
Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes FG. Inter-Society Consensus for the 
Management of Peripheral Arterial Disease (TASC II). J Vasc Surg 2007;45 Suppl S:S5-67. 
34. 
Rooke TW, Hirsch AT, Misra S, et al. Management of patients with peripheral artery disease (compilation 
of 2005 and 2011 ACCF/AHA Guideline Recommendations): a report of the American College of Cardiology 
Foundation/American Heart Association Task Force on Practice Guidelines. Journal of the American College of 
Cardiology 2013;61:1555-70. 
35. 
Treat-Jacobson D, Lindquist RA, Witt DR, et al. The PADQOL: development and validation of a PAD-
specific quality of life questionnaire. Vasc Med 2012;17:405-15. 
36. 
Turkbey EB, McClelland RL, Kronmal RA, et al. The impact of obesity on the left ventricle: the Multi-
Ethnic Study of Atherosclerosis (MESA). JACC Cardiovasc Imaging 2010;3:266-74. 
37. 
Criqui MH, Denenberg JO, Bird CE, Fronek A, Klauber MR, Langer RD. The correlation between 
symptoms and non-invasive test results in patients referred for peripheral arterial disease testing. Vasc Med 
1996;1:65-71. 
38. 
Bronas UG, Hirsch AT, Murphy T, et al. Design of the multicenter standardized supervised exercise 
training intervention for the claudication: exercise vs endoluminal revascularization (CLEVER) study. Vasc Med 
2009;14:313-21. 
39. 
McDermott MM, Guralnik JM, Criqui MH, Liu K, Kibbe MR, Ferrucci L. Six-minute walk is a better 
outcome measure than treadmill walking tests in therapeutic trials of patients with peripheral artery disease. 
Circulation 2014;130:61-8. 
40. 
Gibbons WJ, Fruchter N, Sloan S, Levy RD. Reference values for a multiple repetition 6-minute walk test 
in healthy adults older than 20 years. J Cardiopulm Rehabil 2001;21:87-93. 
41. 
Warburton DE, Haykowsky MJ, Quinney HA, Humen DP, Teo KK. Reliability and validity of measures 
of cardiac output during incremental to maximal aerobic exercise. Part I: Conventional techniques. Sports 
Medicine 1999;27:23-41. 
42. 
Meyerspeer M, Robinson S, Nabuurs CI, et al. Comparing localized and nonlocalized dynamic 31P 
magnetic resonance spectroscopy in exercising muscle at 7 T. Magn Reson Med 2012;68:1713-23. 
43. 
Meyerspeer M, Scheenen T, Schmid AI, Mandl T, Unger E, Moser E. Semi-LASER localized dynamic 
31P magnetic resonance spectroscopy in exercising muscle at ultra-high magnetic field. Magn Reson Med 
2011;65:1207-15. 
44. 
Vanhamme L, van den Boogaart A, Van Huffel S. Improved method for accurate and efficient 
quantification of MRS data with use of prior knowledge. J Magn Reson 1997;129:35-43. 
45. 
Du F, Zhu XH, Qiao H, Zhang X, Chen W. Efficient in vivo 31P magnetization transfer approach for 
noninvasively determining multiple kinetic parameters and metabolic fluxes of ATP metabolism in the human 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 37 of 32 
 
brain. Magn Reson Med 2007;57:103-14. 
46. 
Bogner W, Chmelik M, Schmid AI, Moser E, Trattnig S, Gruber S. Assessment of (31)P relaxation times 
in the human calf muscle: a comparison between 3 T and 7 T in vivo. Magn Reson Med 2009;62:574-82. 
47. 
Larsen RG, Callahan DM, Foulis SA, Kent-Braun JA. In vivo oxidative capacity varies with muscle and 
training status in young adults. J Appl Physiol (1985) 2009;107:873-9. 
48. 
Sanchez OA, Copenhaver EA, Chance MA, et al. Postmaximal contraction blood volume responses are 
blunted in obese and type 2 diabetic subjects in a muscle-specific manner. Am J Physiol Heart Circ Physiol 
2011;301:H418-27. 
49. 
Walter G, Vandenborne K, McCully KK, Leigh JS. Noninvasive measurement of phosphocreatine 
recovery kinetics in single human muscles. American Journal of Physiology 1997;272:C525-34. 
50. 
Smith SA, Montain SJ, Zientara GP, Fielding RA. Use of phosphocreatine kinetics to determine the 
influence of creatine on muscle mitochondrial respiration: an in vivo 31P-MRS study of oral creatine ingestion. J 
Appl Physiol (1985) 2004;96:2288-92. 
51. 
Taylor DJ, Amato A, Hands LJ, et al. Changes in energy metabolism of calf muscle in patients with 
intermittent claudication assessed by 31P magnetic resonance spectroscopy: a phase II open study. Vasc Med 
1996;1:241-5. 
52. 
Lanza IR, Befroy DE, Kent-Braun JA. Age-related changes in ATP-producing pathways in human skeletal 
muscle in vivo. J Appl Physiol (1985) 2005;99:1736-44. 
53. 
Di Marzo L, Miccheli A, Sapienza P, et al. 31Phosphorus magnetic resonance spectroscopy to evaluate 
medical therapy efficacy in peripheral arterial disease. A pilot study. Panminerva Med 1999;41:283-90. 
54. 
Isbell DC, Berr SS, Toledano AY, et al. Delayed calf muscle phosphocreatine recovery after exercise 
identifies peripheral arterial disease. Journal of the American College of Cardiology 2006;47:2289-95. 
55. 
Damon BM. Repeatability of a dual gradient-recalled echo MRI method for monitoring post-isometric 
contraction blood volume and oxygenation changes. Magn Reson Med 2009;22:753-61. 
56. 
Damon BM, Hornberger JL, Wadington MC, Lansdown DA, Kent-Braun JA. Dual gradient-echo MRI of 
post-contraction changes in skeletal muscle blood volume and oxygenation. Magn Reson Med 2007;57:670-9. 
57. 
Wells WM, 3rd, Viola P, Atsumi H, Nakajima S, Kikinis R. Multi-modal volume registration by 
maximization of mutual information. Med Image Anal 1996;1:35-51. 
58. 
Duscha BD, Robbins JL, Jones WS, et al. Angiogenesis in skeletal muscle precede improvements in peak 
oxygen uptake in peripheral artery disease patients. Arterioscler Thromb Vasc Biol 2011;31:2742-8. 
59. 
Bronas UG, Treat-Jacobson D, Leon AS. Comparison of the effect of upper body-ergometry aerobic 
training vs treadmill training on central cardiorespiratory improvement and walking distance in patients with 
claudication. J Vasc Surg 2011;53:1557-64. 
60. 
Murphy TP, Cutlip DE, Regensteiner JG, et al. Supervised exercise, stent revascularization, or medical 
therapy for claudication due to aortoiliac peripheral artery disease: the CLEVER study. J Am Coll Cardiol 
2015;65:999-1009. 
61. 
Guidance for clinical trial sponsors: Establishment and operation of clinical trial data monitoring 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 38 of 32 
 
committees. In: Services USDoHaH, ed.2006. 
62. 
NHLBI Policy for Data and Safety Monitoring of Extramural Clinical Studies. National Heart, Lung, and 
Blood Institute, 2012. at http://www.nhlbi.nih.gov/research/funding/human-subjects/data-safety-monitoring-
policy.) 
63. 
Cohen J. A power primer. Psychol Bull 1992;112:155-9. 
 
 
 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 39 of 32 
 
Appendix A: Contraindications from the American College of Sports Medicine 
 
As most potential participants in this study will be a high-risk population for cardiovascular events, every 
exercise test is monitor using continuous EKG, blood pressure monitoring at rest, at each stage and for 6 minutes 
following the exercise test. The School of Nursing Exercise Laboratory is equipped with an automated 
defibrillator and has a standard procedure in place to respond to adverse events that may occur during the 
performance of an exercise test.  Staff and the main PI will rehearse the emergency procedures twice a year and 
will have up to date CPR certifications. The main PI will supervised all exercise tests and will be responsible for 
following the guidelines outline below. 
 
The following are absolute and relative contraindications to initiate an 
exercise test Absolute 
1. A recent significant change in the resting EKG suggesting significant ischemia, recent myocardial 
infarction (within two days), or other acute cardiac event. 
2. Unstable angina. 
3. Uncontrolled cardiac dysrhythmias causing symptoms or hemodynamic compromise. 
4. Symptoms of severe aortic stenosis. 
5. Uncontrolled symptomatic heart failure. 
6. Acute pulmonary embolus or pericarditis. 
7. Suspected or known dissecting aneurism. 
8. Acute systemic infection, accompanied by fever, body aches, or swollen lymph nodes. 
Relative 
1. Left main coronary stenosis 
2. Moderate stenotic valvular heart disease 
3. Electrolyte abnormalities 
4. Severe arterial hypertension (e.g., systolic blood pressure > 200 mmHg and/or diastolic blood 
pressure > 110 mmHg) at rest. 
5. Tachydysrhythmia or bradydysrthymia. 
6. Hypertrophic cardiomyopathy or any other form of outflow tract obstruction. 
7. Neuromuscular, musculoskeletal, or rheumatoid disorders that are exacerbated by exercise. 
8. High degree atrioventricular block. 
9. Ventricular aneurism. 
10. Uncontrolled metabolic disorders. 
11. Chronic infectious disease. 
12. Mental of physical impairment leading to inability to exercise adequately. 
 
 
 
 
 
 
 

Version 2.0 03/24/2017 
Entresto for PAD 
Page 40 of 32 
 
The following are absolute and relative indications to terminate an 
exercise test Absolute Indications 
 
1. Suspicion of a myocardial infarction or acute myocardial infarction (heart attack) 
2. Onset of moderate-to-severe angina (chest pain) 
3. Drop in systolic blood pressure (SBP) below standing resting pressure or drop in SBP with 
increasing workload accompanied by signs or symptoms 
4. Signs of poor perfusion (circulation or blood flow), including pallor (pale appearance to the skin), 
cyanosis (bluish discoloration), or cold and clammy skin 
5. Severe or unusual shortness of breath 
6. CNS (central nervous system) symptoms 
1. e.g., ataxia (failure of muscular coordination), vertigo (An illusion of dizzying movement), 
visual or gait (pattern of walking or running) problems, confusion 
7. Serious arrhythmias (abnormal heart rhythms) 
1. e.g.: second / third degree AV block, atrial fibrillation with fast ventricular response, increasing 
premature ventricular contractions or sustained ventricular tachycardia) 
8. Technical inability to monitor the ECG 
9. Patient's request (to stop) 
 
Relative Indications 
 
1. Any chest pain that is increasing 
2. Physical or verbal manifestations of shortness of breath or severe fatigue 
3. Wheezing 
4. Leg cramps or intermittent claudication (grade 3 on a 4-point scale) 
5. Hypertensive response (SBP >260 mm Hg; DBP>115 mm Hg) 
6. Pronounced ECG changes from baseline 
1. >2 mm of horizontal or down sloping ST- segment depression, or >2 mm of ST-segment 
elevation (except in aVR) 
7. Exercise-induced bundle branch block that cannot be distinguished from ventricular tachycardia 
8. Less serious arrhythmias (abnormal heart rhythms) such as supraventricular tachycardia