Hemodynamic Effects of a Novel Myeloperoxidase Inhibitor With Exercise in Heart Failure With Preserved Ejection Fraction - A Randomized, Double-Blind, Placebo Controlled Proof of Principle Study
Phase: PHASE1
Conditions: Heart Failure
Sponsor: Mayo Clinic

Hemodynamic Effects of a 
Novel Myeloperoxidase 
Inhibitor with Exercise in Heart 
Failure with Preserved Ejection 
Fraction – A Randomized, 
Double-Blind, Placebo 
Controlled Proof of Principle 
Study 
 
NCT# 03611153 
 
January 11, 2022 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
Page 1 of 43 
Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
 
 
Hemodynamic Effects of a Novel Myeloperoxidase Inhibitor 
with Exercise in Heart Failure with Preserved Ejection 
Fraction – A Randomized, Double-Blind, Placebo Controlled 
Proof of Principle Study 
 
 
 
 
 
Regulatory Sponsor: 
Barry A. Borlaug, MD 
Mayo Clinic 
Department of Cardiovascular Medicine 
200 First Street SW 
Rochester, MN 55905 
 
Funding Sponsor: 
 
AstraZeneca 
Study Product: 
Oral myeloperoxidase (MPO) inhibitor (AZD4831) 
Protocol Number: (IRBe) 
17-002907 
IND Number: 
135800 
AB 
 
 
 
 
Initial version: 
09Jun2017 Version (1.0)  
Revised: 
03Aug2017 Version (2.0) 
Revised: 
20Sept2017 Version (3.0) 
Revised: 
29March2018 Version (4.0) 
Revised: 
15Jun2018 Version (5.0) 
Revised:        28 Jan 2019 Version (6.0) 
Revised: 
11Jun 2020 Version (7.0) 
Revised: 
24Dec2020 Version (8.0) 
Revised: 
16 Sept 2021 Version (9.0) 
Revised: 
11Jan2022 Version (10.0) 
(Add additional amendment dates as protocol is modified and update the version 
number.) 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
 
Table of Contents 
 
STUDY SUMMARY ...................................................................................................................................... 5 
INTRODUCTION ........................................................................................................................................... 6 
1.1 
BACKGROUND .................................................................................................................................... 6 
1.2 
INVESTIGATIONAL AGENT .................................................................................................................... 8 
1.3 
PRECLINICAL DATA ............................................................................................................................. 8 
1.4 
CLINICAL DATA TO DATE ..................................................................................................................... 8 
1.5 
DOSE RATIONALE ............................................................................................................................. 10 
1.6 
RISKS AND BENEFITS ........................................................................................................................ 11 
2 
STUDY OBJECTIVES ....................................................................................................................... 13 
3 
STUDY DESIGN ................................................................................................................................. 13 
3.1 
GENERAL DESCRIPTION .................................................................................................................... 13 
3.2 
PRIMARY STUDY ENDPOINTS ............................................................................................................ 16 
3.3 
SECONDARY STUDY ENDPOINTS ....................................................................................................... 16 
3.4 
PRIMARY SAFETY ENDPOINTS ........................................................................................................... 16 
3.5 
IDENTIFICATION OF SOURCE DATA ..................................................................................................... 17 
4 
SUBJECT SELECTION ENROLLMENT AND WITHDRAWAL ........................................................ 17 
4.1 
INCLUSION CRITERIA ......................................................................................................................... 17 
4.2 
EXCLUSION CRITERIA ....................................................................................................................... 17 
4.3 
SUBJECT RECRUITMENT, ENROLLMENT AND SCREENING .................................................................... 18 
4.4 
EARLY WITHDRAWAL OF SUBJECTS ................................................................................................... 18 
4.4.1 
When and How to Withdraw Subjects ................................................................................... 18 
4.4.2 
Data Collection and Follow-up for Withdrawn Subjects ........................................................ 18 
4.5 
PREGNANCY ..................................................................................................................................... 18 
4.5.1 
Maternal Exposure ................................................................................................................ 18 
4.6 
REPRODUCTIVE RESTRICTIONS ......................................................................................................... 19 
5 
STUDY DRUG .................................................................................................................................... 20 
5.1 
DESCRIPTION ................................................................................................................................... 20 
5.2 
TREATMENT REGIMEN ...................................................................................................................... 20 
5.3 
METHOD FOR ASSIGNING SUBJECTS TO TREATMENT GROUPS ............................................................ 20 
5.4 
PREPARATION AND ADMINISTRATION OF STUDY DRUG ....................................................................... 20 
5.5 
SUBJECT COMPLIANCE MONITORING ................................................................................................. 20 
5.6 
PRIOR AND CONCOMITANT THERAPY ................................................................................................. 21 
5.7 
PACKAGING ...................................................................................................................................... 21 
5.8 
MASKING/BLINDING OF STUDY .......................................................................................................... 21 
5.9 
RECEIVING, STORAGE, DISPENSING AND RETURN .............................................................................. 21 
5.9.1 
Receipt of Drug Supplies ....................................................................................................... 21 
5.9.2 
Storage .................................................................................................................................. 21 
5.9.3 
Dispensing of Study Drug ...................................................................................................... 21 
5.9.4 
Return or Destruction of Study Drug ..................................................................................... 22 
6 
STUDY PROCEDURES ..................................................................................................................... 22 
6.1 
VISIT 1 ............................................................................................................................................. 22 
6.2 
VISIT 2 ............................................................................................................................................. 23 
7 
STATISTICAL PLAN ......................................................................................................................... 26 
7.1 
SAMPLE SIZE DETERMINATION .......................................................................................................... 26 
7.2 
PRIMARY OUTCOME ANALYSIS .......................................................................................................... 26 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
8 
SAFETY AND ADVERSE EVENTS ................................................................................................... 26 
8.1 
DEFINITIONS..................................................................................................................................... 26 
8.2 
RECORDING OF ADVERSE EVENTS .................................................................................................... 28 
8.3 
REPORTING OF SERIOUS ADVERSE EVENTS AND UNANTICIPATED PROBLEMS ..................................... 29 
8.3.1 
Sponsor-Investigator reporting: notifying the Mayo IRB ....................................................... 29 
8.3.2 
Sponsor-Investigator reporting: Notifying the FDA................................................................ 30 
8.3.3 
Sponsor-Investigator reporting: Notifying AstraZeneca ........................................................ 30 
8.4 
UNMASKING/UNBLINDING PROCEDURES ............................................................................................ 30 
8.5 
STOPPING RULES ............................................................................................................................. 31 
8.6 
MEDICAL MONITORING ...................................................................................................................... 31 
9 
DATA HANDLING AND RECORD KEEPING ................................................................................... 31 
9.1 
CONFIDENTIALITY ............................................................................................................................. 31 
9.2 
SOURCE DOCUMENTS ...................................................................................................................... 32 
9.3 
CASE REPORT FORMS ...................................................................................................................... 32 
9.4 
RECORDS RETENTION ...................................................................................................................... 32 
10 
STUDY MONITORING, AUDITING, AND INSPECTING .................................................................. 33 
10.1 
STUDY MONITORING PLAN ............................................................................................................ 33 
10.2 
AUDITING AND INSPECTING ........................................................................................................... 33 
11 
ETHICAL CONSIDERATIONS .......................................................................................................... 33 
12 
STUDY FINANCES ............................................................................................................................ 34 
12.1 
FUNDING SOURCE ........................................................................................................................ 34 
12.2 
CONFLICT OF INTEREST ................................................................................................................ 34 
12.3 
SUBJECT STIPENDS OR PAYMENTS ............................................................................................... 34 
13 
PUBLICATION PLAN ........................................................................................................................ 35 
14 
REFERENCES ................................................................................................................................... 35 
15 
ATTACHMENTS ................................................................................................................................ 39 
 
Appendix I: Schedule of Protocol Assessment 
 
Appendix II: Comprehensive ECG Data at Baseline 
 
 
 
 
 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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CONFIDENTIAL 
List of Abbreviations 
 
A-V O2 diff 
Arteriovenous Oxygen content difference 
AE 
Adverse Event/Adverse Experience 
ANCOVA 
Analysis of Covariance 
AUC 
Area Under the Curve 
CFR 
Code of Federal Regulations 
cGMP 
cyclic Guanosine Monophosphate 
CRF 
Case Report Form 
DSMB 
Data and Safety Monitoring Board 
EMR 
Electronic medical record 
EndoPAT 
Endothelial function peripheral arterial tonometry 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
GMP 
Good Manufacturing Practice 
HF 
Heart Failure 
HFpEF 
Heart Failure with preserved Ejection Fraction 
HIPAA 
Health Insurance Portability and Accountability Act 
HOCl 
Hypochlorous Acid 
IB 
Investigator’s Brochure 
IMP 
Investigational Medicinal Product 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
MAD 
Multiple Ascending Dose 
MMP 
Matrix Metalloproteinases 
MPO 
Myeloperoxidase 
NFTF 
Non Face to Face 
NOAEL 
No-observed-adverse-effect-level 
NO 
Nitric Oxide 
O2 
Oxygen 
PCWP 
Pulmonary Capillary Wedge Pressure 
PHI 
Protected Health Information 
PI 
Principal Investigator 
PK 
Pharmacokinetic 
ROS 
Reactive Oxygen Species 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SAD 
Single Ascending Dose 
SOP 
Standard Operating Procedure 
TAPSE 
Tricuspid Annular Plane Systolic Excursion 
VO2 
Oxygen consumption 
Ve/VCO2 
Ventilatory efficiency 
 
 
 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
Study Summary 
Title 
Hemodynamic effects of a Novel Myeloperoxidase Inhibitor with 
Exercise in Heart Failure with Preserved Ejection Fraction – A 
Randomized, Double-Blind, Placebo Controlled Proof of Principle  
Study 
Protocol Number 
IRB #17-002907 
AZ Study Code D6580C00005 
Phase 
Proof of Principle 
Methodology 
This study will be a Proof of Principle, randomized, blinded, 
placebo-controlled, clinical trial with all subjects undergoing 
exercise 
catheterization 
and 
randomized 
to 
either 
oral 
myeloperoxidase (MPO) inhibitor or placebo. 
Overall Study 
Duration 
1 year 
Subject 
Participation 
Duration 
9-14 days 
Single or Multi-Site  Single site 
Objectives 
 To determine the effect of a single dose of an oral acute MPO 
inhibitor on resting and exercise hemodynamics in patients with 
heart failure with preserved ejection fraction (HFpEF)  
Number of 
Subjects 
36 
Diagnosis and 
Main Inclusion 
Criteria 
HFpEF defined by clinical symptoms of HF (dyspnea, fatigue), 
normal EF (≥50%), and catheterization documented elevated left 
ventricular filling pressures with exercise (≥25 mmHg).  
Study Product, 
Dose, Route, 
Regimen 
Oral administration of MPO inhibitor (AZD4831 tablets 5 mg and 
matching AZD4831 Placebo tablets) at a dose of 30 mg during 
catheterization 
Duration of 
Administration 
Single administration 
Reference therapy 
Placebo 
Statistical 
Methodology 
The principal comparisons will be exercise hemodynamic 
parameters at 20 Watt workload after study drug relative to 
corresponding values at 20 Watts workload prior to study drug.  
Primary hypotheses will be tested using analysis of covariance. 
 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
Introduction 
 
Heart failure (HF) with preserved ejection fraction (HFpEF) has become the most 
common cause of HF. Morbidity and mortality are high and there is no proven effective 
pharmacological treatment, making this a huge unmet public health need. Recently, 
abnormalities in Nitric Oxide (NO) signaling and Reactive Oxygen Species (ROS) 
generation have been implicated as a unifying mechanism by which aging and 
associated systemic comorbidities drive the myocardial and peripheral limitations that 
constrain exercise capacity in HFpEF. Myeloperoxidase (MPO) is a leukocyte enzyme 
that plays a key role in host defense but has also recently been implicated in the 
pathophysiology of incident cardiovascular diseases, including HF. MPO plays an 
important role in the regulation of vascular tone and arterial conduit function, since 
vascular MPO activity results in NO scavenging. Because loss of NO activity is heavily 
implicated in the pathogenesis of HFpEF, MPO inhibition may represent a novel 
therapeutic approach to target the NO imbalance and vascular dysfunction that exists in 
HFpEF.  
 
Accordingly, our guiding hypothesis is that an oral MPO inhibitor (AZD4831) will 
improve cardiac hemodynamics at rest and during exercise in relation to enhanced NO 
bioavailability. This Proof of Principle study will assess the effect of a single dose of an 
oral acute MPO inhibitor on resting and exercise hemodynamics in patients with HFpEF 
referred to the catheterization lab. In addition, we will evaluate NO bioavailability, cyclic 
Guanosine Monophosphate (cGMP) response and forearm flow mediated vasodilation 
as surrogate markers for improving endothelial function with MPO inhibition. 
 
This document is a protocol for a human research study. This study will be carried out in 
accordance with applicable United States government regulations and Mayo Clinic 
research policies and procedures.  
 
1.1 
Background 
 
Heart failure (HF) with preserved ejection fraction (HFpEF) is the most common 
cause of heart failure, with both aging and comorbidity burden being directly 
proportional to risk of incident HFpEF 1. Despite the high prevalence and socio-
economic burden, no pharmacological treatment options are currently available. 
This may be due to phenotypic heterogeneity and failure to target the final 
common pathway in this syndrome 2. Recently, abnormalities in Nitric Oxide 
(NO) signaling and Reactive Oxygen Species (ROS) generation have been 
implicated as a unifying mechanism by which aging and associated systemic 
comorbidities drive both the systemic inflammation and myocardial/peripheral 
limitations central to symptomatic HFpEF 3. Current investigative strategies 
targeting this NO pathway focus on exogenous replacement but do not address 
the underlying systemic inflammatory process driving the endothelial 
dysfunction and effective NO deficiency.  

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
 
MPO is a leukocyte enzyme involved in the generation of hypochlorous acid 
(HOCl) and ROS. While these actions are critical for host defense via beneficial 
bactericidal effects 4, recent evidence points to a deleterious role for excessive 
MPO activity in a number of cardiovascular diseases including HF 5, 
atherosclerosis 6, 7, 8, 9, 10, 11, 12 and endothelial dysfunction 13, 14, 15, 16. In 
normal physiology, MPO also plays a role in the regulation of vascular tone and 
arterial conduit function 15, 17 by oxidizing and thereby inactivating NO 4, 13, 18, 
19, 20, 21, and inhibiting endothelial NO synthase (Figure 1)22. Furthermore, MPO 
is also found in the subendothelial matrix and plays a role in tyrosine nitration 
with resultant adverse cardiac and tissue remodeling via modulation of matrix 
metalloproteinases (MMP) and other proteins 23, 24, 25, 26, 27, 28, 29. Accordingly, 
MPO inhibition may represent a novel therapeutic approach not only 
addressing the NO imbalance and vascular dysfunction that exists in HFpEF, 
but may also directly suppress the inflammatory ROS cascade driving cardiac 
and peripheral adverse remodeling and functional NO/cGMP deficiency in this 
syndrome. 
 
 
 
 
 
Figure 1. Scheme of the proposed mechanism of vascular distribution of MPO and MPO-
dependent catabolism of NO. 
 
In this study, we will evaluate whether an oral MPO inhibitor (AZD4831, 
currently under development by AstraZeneca) will exert beneficial effects on 
cardiac hemodynamics through improvement in NO bioavailability. This study 
will assess the effect of a single dose of an oral acute MPO inhibitor on resting 
and exercise hemodynamics in patients with HFpEF referred to the 
catheterization lab.  
 
 
 

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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
1.2 
Investigational Agent 
 
Code Name: 
AZD4831 
Molecular 
Formula: 
C15H15ClN4OS 
Molecular 
Weight: 
335 g/mol 
 
1.3 Preclinical Data 
 
AZD4831 is a potent mechanism-based MPO-inhibitor, acting on purified MPO 
in vitro with an IC50 of approximately 1 nmol/L. In human PMA-stimulated 
neutrophils in vitro, AZD4831 inhibits extracellular peroxidase activity with an 
IC50 of 12 nmol/L, whereas the intragranular peroxidase activity is inhibited with 
an IC50 of 2.7 μmol/L. In vitro, AZD4831 dose-dependently inhibits MPO-driven 
formation of urate and allantoin from xanthine, whereas it does not inhibit 
xanthine oxidase activity (at concentrations up to 30 μmol/L) or URAT-1 
mediated transport of urate (at concentrations up to 150 μmol/L). In vivo, 
AZD4831 dose-dependently reduces local peroxidase activity at doses down to 
0.033 mg/kg (yielding a plasma exposure of 1.7 nmol/L at 4h) and systemic 
MPO levels at 17 and 83 mg/kg in mouse peritonitis models. 
1.4 Clinical Data to Date 
 
MPO has long been regarded as a mediator of bactericidal activity through its 
generation of HOCl and ROS. However, with recent evidence linking MPO to 
incident cardiovascular disease and NO catabolism with vasoconstriction, there 
has been renewed interest in exploring its role in various disease states. 
HFpEF is an enormous public health problem with no current treatment options. 
According to current disease paradigms, a primary driver for this syndrome is 
increased oxidative stress, ROS generation, endothelial dysfunction and 
relative NO deficiency. Thus HFpEF represents a high yield target for MPO 
inhibition; to not only improve exercise capacity through NO augmentation, but 
also to directly suppress the oxidative stress and endothelial dysfunction that 
promotes progression of the disease.  
Our group at Mayo Clinic and others have demonstrated that endothelial 
function is impaired in HFpEF 30, 31 and is correlated with pulmonary vascular 
disease 32 as well as death and HF hospitalization 33. Thus a therapy such as 
MPO inhibition that improves endothelial function is likely to be beneficial in 
preventing adverse outcomes, in addition to improving aerobic capacity and 
symptoms. Further supporting this approach of NO augmentation, recent trials 
of nitrite supplementation in HFpEF have demonstrated a reduction in exercise 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
filling pressures, ventricular performance, pulmonary vasodilation, exercise 
capacity and cardiac output response 34, 35, 36, 37 further supporting an 
approach of MPO inhibition to augment NO bioavailability. 
There is preliminary evidence that MPO plays a role in vasoconstriction of 
muscular and conduit arteries 17 through a reduction in NO bioavailability. This 
appears to be mediated in part by vascular MPO scavenging of NO, since 
acute administration of heparin displaces subendothelial MPO and this results 
in enhanced NO activity 38. Thus the development of an inhibitor of MPO 
represents a logical next step in the assessment of vascular and hemodynamic 
changes in humans, and HFpEF represents a high yield target to demonstrate 
potentially beneficial effects from these changes.  
The presence of dynamic reserve limitations in HFpEF makes assessment of 
exercise hemodynamics a very sensitive and physiologically relevant way to 
evaluate pharmacologic effects. Our group has conducted and published 
numerous studies in HFpEF over the past 10 years using high fidelity 
micromanometer catheters. Specifically in the invasive hemodynamic 
laboratory, we have published numerous studies examining exercise 
hemodynamics and/or acute drug effects in patients with HFpEF 34, 35, 39. Most 
recently, a similar trial design to the one proposed here was employed for the 
evaluation of sodium nitrite therapy demonstrating significant hemodynamic 
benefits 34, 35.  
We have typically been able to enroll 2-3 patients per month over the past few 
years for these invasive hemodynamic exercise studies given the large volume 
of exercise right heart catheterizations performed at Mayo annually (>200 per 
year). The current proposal will enroll a much lower sample of 36 subjects over 
one year, further enhancing the likelihood of success.  
 
 
Safety 
 
AZD4831 has been administered to 59 healthy subjects in completed studies in 
single doses up to 405 mg, and in repeated doses up to 15 mg for up to 14 
days. There were no deaths or SAEs and all subjects except one completed the 
studies. The most common adverse events were headache and generalized 
maculopapular rash. Dosing in 45 mg cohort in the Multiple Ascending Dose 
(MAD) study was stopped because of self-reported throat tightness in one 
subject that was considered intolerable by the Principal Investigator (PI) and 
Safety Review Committee (SRC). This subject also had a more long-lasting 
rash of moderate intensity. Otherwise AZD4831 has been well tolerated in 
healthy subjects. No clinically meaningful differences for changes over time in 
clinical laboratory tests, vital signs or ECGs were observed between subjects 
who received AZD4831 and those who received placebo. 
 
 
 

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Principal Investigator: Barry Borlaug, MD 
 
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Since the SAD and MAD studies conducted in healthy volunteers noted above, 
a new Phase 2a study (NCT03756285) has been undertaken in patients with 
HFpEF, as are being enrolled in this study.  Thus far, 24 participants have been 
randomized to AZD4831 and 13 to matching placebo.  
 
The safety results of this Phase 2a study in HFpEF are as follows:  
 
• 
AZD4831 was generally well tolerated 
• 
Of the 24 patients randomised to AZD4831, 23 completed 90 days 
treatment 
• 
There were no deaths. Two SAEs (8.3%) were reported in AZD4831 
group and one (7.7%) in the placebo group 
• 
One patient (4.2%) in AZD4831 group was discontinued from treatment 
due to generalized maculopapular rash (CTCAE grade 3) as 
prespecified in the protocol. No patients on placebo were discontinued 
from treatment. 
• 
AEs were balanced between groups and as expected for this patient 
population 
• 
Overall, 16 (66.7%) patients experienced at least one AE after receiving 
treatment AZD4831 and 7 [53.8%] patients after receiving placebo.  
• 
No clinically significant trends were observed in vital signs, clinical 
laboratory results, 12-lead ECGs 
 
 
1.5 Dose Rationale  
 
This is an oral single dose study in HFpEF patients and the first time AZD4831 
will be dosed in patients with heart failure. Oral administration is the intended 
route of administration for AZD4831 at an expected dose of 7 mg. 
In healthy subjects, when dosed from 5 to 405 mg AZD4831, a significant and 
dose dependent decrease in serum uric acid was observed at 135 mg and 405 
mg AZD4831, respectively.  
In addition, when dosed with 5 mg once daily for 10 days in healthy subjects, 
an approximate 30% reduction in MPO activity was observed at day 10 and 
steady state conditions.  
In the same study, the pharmacokinetic analysis showed an accumulation of 
approximately 3. In other words, plasma levels of AZD4831 were approximately 
3 fold higher at steady state with daily dosing as compared to single dose 
administration of AZD4831.  
Assuming similar PK in healthy subjects and HFpEF patients, a single dose of 
30mg AZD4831 is predicted to yield exposure seen at steady state following 
once daily administration of 10 mg AZD4831. Thus, a single dose of 30 mg 
provided in this acute, proof of concept trial, should replicate the degree of 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
MPO inhibition that will be seen with the predicted therapeutic dose of 7 mg 
AZD4831.  
The single dose of 30mg AZD4831 in this study is predicted to yield an AUC 
and Cmax of 4.7 µmol*h/L and 0.24 µmol/L, respectively.  These exposures are 
substantial lower than the AUC and Cmax achieved after a single dose of 405 
mg AZD4831 in healthy subjects and 40-60 fold lower than the AUC and Cmax 
exposures achieved at the NOAEL (20 mg/kg) in the one-month dog toxicity 
study. 
1.6 Risks and Benefits 
 
While we expect AZD4831 to improve hemodynamics, the short term nature of 
the study does not provide a meaningful opportunity for clinical benefit. 
 
A number of potential risks have been identified and are primarily based on 
preclinical safety studies with AZD4831 that include repeated dosing up to 1 
month in rats and dogs.  
 
The key adverse effects identified in the toxicology and safety pharmacology 
program for AZD4831 were:  
 
• BP reduction and reflex tachycardia because of α1A adrenoceptor block, 
which can be monitored by frequent BP, HR and ECG examination.  
 
• Inhibition of thyroid peroxidase: subjects with active thyroid disease will not 
be included in the study 
 
Theoretical risks 
The following risks are based on results from humans and animals with partial 
or total MPO deficiency. The relevance of the effects observed in genetic 
deficiency models as compared to administration of a MPO inhibitor is not 
known. 
 
Host-defense impairment - infections 
A theoretical risk is that treatment with an MPO inhibitor could impair host-
defense mechanisms. The MPO in neutrophils generate ROS to fight infecting 
microorganisms. MPO deficient mice show impaired fungicidal activity but no 
other special abnormalities compared to wild type mice. Humans with total or 
partial MPO deficiency generally do not have an increased susceptibility to 
infections but the incidence of Candida infections may be increased. In studies 
with the other MPO inhibitors AZD3241 and AZD5904, no increased incidence 
of infections, severe infections or increased inflammation has been observed 
although treatment duration and number of patients have been limited. 
 
Mitigation: Special considerations will be given to adverse events (AEs) related 
to frequency of infections, duration of infections, severity of infections and 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
inflammatory responses in clinical trials. The high-sensitivity C-reactive protein 
(hsCRP) will be measured as a general marker of inflammation prior to study 
drug and at the follow up visit. 
 
Agranulocytosis and allergic reactions 
Agranulocytosis is a serious side effect of drugs containing a thiourea motif that 
can be irreversible. Theoretical risks based on a thiourea motif of AZD4831 
molecule are agranulocytosis and allergic reactions, which are known liabilities 
for registered anti-thyroid drugs with similar thiourea motifs as AZD4831 
although the frequency is low (0.3 to 0.6%). 
 
There are no good preclinical models available to study agranulocytosis. There 
were no consistent findings on hematological or bone marrow parameters in the 
1 month studies in rats and dogs. In studies with the other MPO inhibitors 
AZD3241 and AZD5904 which also carry the thiourea motif, no cases of 
agranulocytosis were observed although treatment duration and number of 
patients were limited. 
 
Mitigation: Hematological parameters including white blood cell (WBC) count 
will be monitored as safety laboratory parameter prior to receiving study drug 
and again at the follow up visit. Special considerations will be given to the AEs 
of fever, sore throat and flu-like symptoms. Agranulocytosis by antithyroid drugs 
may be responsive to treatment with granulocyte colony-stimulating factor but 
data suggests that there is no benefit to this approach (Yang et al., Thyroid 
2016). 
 
Identified risks from the SAD and MAD studies Maculopapular rash was an 
identified risk in the SAD study for AZD4831. Noteworthy, subjects in both SAD 
and MAD (multiple ascending dose) studies with a previous MPOi in 
development by AstraZeneca AZD5904, also developed maculopapular rash at 
about the same frequency as AZD4831 that resolved during continued dosing. 
 
The maculopapular rash was self-limited in all cases and developed at 7-9 days 
post dose with AZD4831. The rash was generalized and classified as 
moderate. One rash occurred at the 45 mg, one at 135 mg and 2 at the 405 mg 
dose group. No rash was reported in the placebo, 5 mg or 15 mg dose groups. 
 
As described above, a generalized maculopapular rash was also noted in 1/24 
patients with HFpEF in the ongoing Phase 2a trial of AZD4831 
(NCT03756285). This occurred 5 days after increasing the dose of AZD4831 
from 2.5 to 5 mg daily, and resolved after discontinuation of study drug and 
symptomatic treatment using oral anti-histamines and corticosteroid. 
 
Mitigation: A lower dose of AZD4831 will be used that in early dosing studies has not 
been associated with rash.  
 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
Full skin examination will be performed at the follow up videoconference visit in all 
participants to evaluate for rash.  This will be performed remotely from the participants 
home, employing the clinical video platform (Zoom) that is currently used at the Mayo 
Clinic for non-face-to-face (NFTF) patient visits in our clinical practice. This system was 
developed to respond to travel restriction in the setting of the COVID19 pandemic and 
has proven very effective to manage symptoms and signs that are evident from video-
conference evaluation, as with a skin rash.  
 
If a concerning rash is identified, dermatology consultation will be obtained, with biopsy 
if deemed clinically appropriate by the consulting dermatologist.  Patients will be 
instructed to report back immediately if they develop a rash prior to the follow up visit. 
 
One subject self-reported throat tightness in the 45mg dose group which could not be 
verified by examination.  . 
 
2 Study Objectives 
Aim 1: Determine whether acute administration of oral MPO inhibitor 
(AZD4831) at a dose of 30 mg attenuates the pathologic elevation in exercise 
Pulmonary Capillary Wedge Pressure (PCWP) in HFpEF.  
Our primary hypothesis is that acute administration of an oral MPO inhibitor will 
augment NO signaling and thereby attenuate the pathologic elevation in exercise 
PCWP in HFpEF, compared to placebo. 
 
Aim 2: Determine whether treatment with an oral MPO inhibitor (AZD4831) at a 
dose of 30 mg improves other resting and exercise hemodynamics, peripheral 
oxygen extraction, and endothelial function in HFpEF.  
Our secondary hypotheses are that acute administration of an oral MPO inhibitor 
will improve exercise cardiac output reserve, reduce biventricular filling pressures at 
rest, enhance peripheral oxygen extraction, improve endothelial function, and lower 
pulmonary arterial pressure in HFpEF, compared to placebo. 
 
3 Study Design 
 
3.1 
General Description 
 
This is a Proof of Principle, randomized, double-blind, placebo-controlled trial 
testing whether an oral MPO inhibitor, as compared to placebo, can attenuate the 
pathologic elevations in PCWP during exercise in subjects with HFpEF.  
 
Brief Synopsis of Methods (Figure 2):  

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
1) Enroll subjects with normal EF referred to the cath lab for evaluation of dyspnea. 
Perform safety lab sampling (complete blood count, creatinine, blood urea 
nitrogen, sodium, potassium, TSH and T4, aspartate aminotransferase, alanine 
aminotransferase, total, direct and indirect bilirubin) hs c-RP, NTproBNP, 
echocardiography, arterial tonometry for pulse wave analysis, and assessment of 
endothelial function (EndoPAT) prior to administration of study drug. 
2) Assess baseline exercise capacity and hemodynamics at rest and during 
exercise using gold standard invasive techniques in patients with HFpEF. 
Venous, arterial and coronary sinus blood samples obtained at rest and during 
exercise. Focused echocardiography examination with measurement of LV and 
RV tissue Doppler echocardiography and cardiac volumes will be performed. 
3) Provide study drug (AZD4831 or matching placebo) orally following the invasive 
exercise test with the patient waiting for 30 minutes in the cath lab to facilitate 
therapeutic concentration. Vascular access will be maintained. 
4) Repeat resting and exercise invasive hemodynamics with blood sampling and 
echocardiography exactly as in (2). 
5) Measure biomarkers of oxidative stress, NO activity and PK before and after 
study drug (Soluble ST2, TnI, Gal-3, GDF15, NT-proCIII, C-telopeptide CI, 
Calprotectin, 
MMPx, 
hypoxanthine, 
xanthine, 
urate 
and 
allantoin, 
Arg/ADMA/SDMA). 
6) At the conclusion of the second exercise test, when all primary and secondary 
endpoints have been assessed, a single bolus dose of heparin (70 units/kg body 
weight) will be given to measure plasma MPO before and after. The change in 
MPO (amount and activity) with heparin will then be related to hemodynamic 
effects of AZD4831 observed in the earlier part of the trial. 
7) Subjects will be transferred to the catheterization lab recovery area, where 
sheaths will be removed and hemostasis verified according to our standard 
clinical practice. 
8) Repeat assessment of endothelial function will be performed in the recovery 
area, prior to dismissal. 
9) The primary analysis will compare resting and exercise hemodynamic response 
after AZD4831 or placebo relative to the initial assessment 2-way ANCOVA 
(using the baseline value as the covariate). 
10) A follow up, non-face-to-face videoconference visit will be scheduled between 
days 9-14 after dosing.  History and visual physical exam (including full skin 
exam) will be repeated.  Patients will be instructed to report back immediately if 
they develop a rash prior to the follow up visit. 
 

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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
 
Innovation:  
• The use of exercise hemodynamics to measure an intervention’s effect is highly 
novel and clinically meaningful because exercise is the time where symptoms 
develop and hemodynamics are most perturbed in response to heightened 
physiological demand.  Very few centers in the world could carry out this study. 
• The hypothesis that NO availability is limited in part by increased vascular MPO 
activity is novel and has not been tested in HFpEF. 
• No previous therapy in HFpEF has directly targeted the vascular processes that 
result in both decreased NO and increased ROS that are believed to be the 
underlying driver of global cardiac and peripheral reserve limitations in HFpEF.  
• Myocardial efficiency, assessed by myocardial O2 consumption (using coronary 
sinus sampling) in relation to myocardial performance, is a highly sensitive 
method of detecting drug effect on myocardial function. 
 

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 Significance:   
• HFpEF afflicts millions worldwide, and with the aging population the prevalence 
is growing by 1% per year.  This makes identification of effective treatments a 
major unmet public health need with the potential to change clinical practice. 
• MPO inhibition has the potential to uniquely target the underlying hyperactive 
oxidative stress and inflammation that is believed to represent a common 
pathway driving the phenotypically diverse HFpEF syndrome.  
• In addition, augmentation of NO bioavailability by decreasing its oxidation 
through MPO inhibition has the potential to improve exercise capacity. This is a 
clinically relevant end point in this population defined by exercise intolerance. 
• Identification of a beneficial effect of MPO inhibition on hemodynamics would 
provide critical preliminary data to support a larger scale clinical trial in HFpEF 
focused on surrogate markers of HF severity, exercise tolerance or cardiac 
remodeling. 
3.2 
Primary Study Endpoints 
 
The primary endpoint will be the PCWP at 20 Watts exercise after study drug 
relative to the PCWP at 20 Watts exercise in the initial assessment prior to study 
drug. 
3.3 
Secondary Study Endpoints 
 
Secondary exploratory endpoints will be changes in resting PCWP after study 
drug as well as rest and exercise changes in right atrial pressure, pulmonary 
artery pressure, pulmonary vascular resistance and compliance, coronary sinus 
saturation, systemic BP, heart rate, cardiac output, A- V O2 content difference 
during exercise, VO2, Ve/VCO2 slope and Borg effort/dyspnea scores. Increase 
in cGMP in response to MPO inhibition along with changes in other biomarkers 
and MPO described above will also be assessed. 
3.4 
Primary Safety Endpoints 
 
Safety and tolerability will be measured by the incidence of SAEs, AEs and 
clinical changes in ECG, vital signs and clinical laboratory evaluations. 
 
A maculopapular skin rash can occur which will be monitored. The characteristics 
of the rash will be documented. Photos including a body overview photo plus a 
more detailed photo of a representative affected skin area will be taken, if rash 
occurs. Dermatologic evaluation with biopsy if deemed necessary will be 
obtained for significant rashes.   
 

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Principal Investigator: Barry Borlaug, MD 
 
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3.5 
Identification of Source Data 
 
The following source data will be directly recorded on the Case Report Form 
(CRF):   
• Echo findings at baseline, during cath  
• EndoPAT 
• History and Physical Exam 
• Hemodynamic data from all study phases 
• Clinical data and Demographics 
 
The following source data will not be directly collected in the Case Report Form 
(CRF), but will be captured in supportive documentation (study source 
documents, EMR): 
• Laboratory results and clinical interpretation of the values. 
• ECG records and clinical significance of observations (when applicable) 
 
4 Subject Selection Enrollment and Withdrawal 
4.1 
Inclusion Criteria 
 
1. 
Males and females of non-childbearing potential confirmed at screening by 
fulfilling one of the following criteria: 
• Postmenopausal defined as amenorrhoea for at least 12 months or 
more following cessation of all exogenous hormonal treatments and 
follicle-stimulating hormone (FSH) levels in the postmenopausal range 
• Documentation of irreversible surgical sterilisation by hysterectomy, 
bilateral oophorectomy or bilateral salpingectomy but not tubal ligation 
2. 
Age ≥ 30 years 
3. 
Symptoms of dyspnea (II-IV) at the time of screening 
4. 
EF ≥ 50% as determined on imaging study within 12 months of enrollment  
5. 
Catheterization documented elevated filling pressures at rest (PCWP ≥15) 
or with exercise (PCWP ≥25)  
4.2 
Exclusion Criteria 
 
1. 
Use of nitrates, phosphodiesterase 5 inhibitors or other NO-providing 
therapy in the past 24 hours of screening 
2. 
Significant valvular disease (>moderate left-sided regurgitation, >mild 
stenosis) 
3. 
Requirement of intravenous heparin at the start of case 
4. 
Severe pulmonary parenchymal disease 
5. 
Acute coronary syndrome or coronary disease requiring revascularization 
in the judgement of investigators 
6. 
Resting systolic blood pressure < 100 mmHg  
7. 
Constrictive pericarditis 

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Principal Investigator: Barry Borlaug, MD 
 
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8. 
Infiltrative, restrictive, or hypertrophic obstructive cardiomyopathies 
9. 
Previous anaphylaxis to any drug 
10. Pregnancy or breastfeeding mothers 
11. High Output heart failure 
12. Active thyroid disease 
13. Treatment with a new chemical entity (defined as a compound which has 
not been approved for marketing) within the preceding 3 months 
14. Patients with any prior allergy to propylthiouracil  
4.3 
Subject Recruitment, Enrollment and Screening 
 
Eligible patients will be identified from screening of the patients listed for 
exercise right heart catheterization as well as patients with known HFpEF at the 
Mayo Clinic in Rochester, Minnesota.    
4.4 
Early Withdrawal of Subjects 
4.4.1 When and How to Withdraw Subjects 
 
Development of any condition that requires study withdrawal 
related to safety, disease progression, subject decision or failure to 
adhere to protocol requirements could qualify as reason to 
withdraw from the study. 
4.4.2 Data Collection and Follow-up for Withdrawn Subjects 
 
Subjects withdrawing from the study will be contacted 2 weeks 
following study drug administration to ensure that there have been 
no adverse events.  Participants that withdraw may be replaced to 
allow for achievement of target enrollment. 
 
4.5 
Pregnancy 
 
All pregnancies and outcomes of pregnancy should be reported to the sponsor 
and AstraZeneca. 
 
4.5.1 Maternal Exposure 
 
Pregnancy itself is not regarded as an AE unless there is a suspicion that 
the IP under study may have interfered with the effectiveness of a 
contraceptive medication.  Congenital abnormalities/birth defects and 
spontaneous miscarriages should be reported and handled as SAEs.  
Elective abortions without complications should not be handled as AEs.  

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Principal Investigator: Barry Borlaug, MD 
 
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The outcome of all pregnancies (including spontaneous miscarriage, 
elective termination, and ectopic pregnancy) will be documented.  
 
If any pregnancy occurs in the course of the study, then the Investigator or 
other site personnel informs the sponsor’s representatives within 1 day of 
when he/she became aware of it. 
 
4.6 
Reproductive Restrictions 
 
It is important that women of childbearing potential, who are the partners 
of male subjects, do not become pregnant during the study and for a total 
period of 3 months after subjects have taken the last dose of IMP.  
 
Non-sterilized males who are sexually active with a female partner of 
childbearing potential must use condom and spermicide from Day 1 
through and for a total period of 3 months after subjects have taken the 
last dose of IMP. Because male condom and spermicide is not a highly 
effective contraception method it is strongly recommended that female 
partners of male study subjects also use a highly effective method of 
contraception throughout this period.  
 
Highly effective contraception is defined as having a low failure rate, i.e., 
less than 1% per year when used consistently and correctly. Highly 
effective contraception forms of birth control include: 
 
Tubal occlusion 
  
• Copper T intrauterine device  
• Levonorgestrel-releasing intrauterine system (e.g., Mirena®)  
• Medroxyprogesterone injections (e.g., Depo-Provera®)  
• Etonogestrel implants (e.g., Implanon®, Norplan®)  
• Combined pills  
• Norelgestromin/ethinyl estradiol transdermal system (e.g., Ortho Evra®)  
• Intravaginal device (e.g., NuvaRing®)  
• Cerazette® pill  
 
Male subjects who have been sterilized are required to use 1 barrier 
method of contraception (condom).  
 
Sperm Donation  
Male subjects should not donate sperm for the duration of the study and 
for at least 3 months after the last day of IMP administration. 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
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Pregnancy  
Subjects will be instructed that if their partner becomes pregnant during 
the study this should be reported to the Investigator. The Investigator 
should also be notified of pregnancy occurring during the study but 
confirmed after completion of the study. In the event that a subject’s 
partner is subsequently found to be pregnant after the subject is included 
in the study, then consent will be sought from the partner and if granted 
any pregnancy will be followed and the status of mother and/or child will 
be reported to the sponsor after delivery.  
 
A pregnancy notification form and follow-up will be completed. 
 
5 Study Drug  
5.1 
Description 
 
The IMP (Investigational Medicinal Product) is a white film-coated tablet 
containing 5 mg of AZD4831. A matched placebo (PTM AZD4831 film-coated tablet 
5 mg) is also presented. The IMP will be manufactured by AstraZeneca in 
accordance with Good Manufacturing Practice (GMP). 
5.2 
Treatment Regimen 
 
A single administration of MPO inhibitor at a dose of 30 mg or matched placebo, 
will be given orally following baseline exercise right heart catheterization, if 
patients meet the above eligibility criteria. 
5.3 
Method for Assigning Subjects to Treatment Groups 
 
Patients will be randomized 1:1 in a blinded fashion to AZD4831 or placebo with 
blinding and randomization performed by the Mayo Clinic Research Pharmacy. 
5.4 
Preparation and Administration of Study Drug 
 
The IMP will be provided in bulk from AstraZeneca, to be packed into study 
specific containers with study specific labels by the pharmacy, according to the 
bottling instruction provided by AstraZeneca. 
 
The AZD4831 film-coated tablet 5 mg and PTM AZD4831 film-coated tablet 5 mg  will be 
supplied in induction-sealed high-density polyethylene bottles with silica desiccant. 
5.5 
Subject Compliance Monitoring 
 

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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
This will be a single dose, directly observed dosing study so formal checks for 
long term compliance are not necessary 
5.6 
Prior and Concomitant Therapy 
 
Participants’ chronic medications will not be changed for the purposes of this 
study. They will continue taking their home medications as prescribed up until 
the morning of the procedure. Data for baseline medication usage will be 
obtained. 
5.7 
Packaging 
AZD4831 film-coated tablet 5 mg and PTM AZD4831 film-coated tablet 5 
mg will be delivered in bulk from AstraZeneca. Empty bottles, caps and 
desiccant will be delivered separately. Each bottle should be filled with 35 
tablets, with 2 desiccant unit added, and be capped and labelled appropriately, 
including information of expiry date in accordance with bulk information. 
5.8 
Masking/Blinding of Study  
 
Only research pharmacy staff will be aware of randomization scheme and all 
study personnel and subjects will remain blinded to the identity of study drug.   
5.9 
Receiving, Storage, Dispensing and Return 
5.9.1 Receipt of Drug Supplies 
 
Upon receipt of the study treatment supplies, an inventory must be 
performed and a drug receipt log filled out by the person accepting the 
shipment.  It is important that the designated study staff counts and 
verifies that the shipment contains all the items noted in the shipping 
invoice. Any discrepancies, damaged or unusable study drug in a given 
shipment (active drug or comparator) will be documented in the study files. 
The 
sponsor-investigator 
must 
be 
notified 
immediately 
of 
any 
discrepancies, damaged or unusable products that are received. 
5.9.2 Storage 
 
The IMP will be stored in a secure facility under appropriate storage 
conditions. The IMP should be stored in room temperature (<30°C) 
protected from light prior to use. Details of storage conditions will be 
provided on the label of the IMP. 
 
5.9.3 Dispensing of Study Drug 
 

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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
Regular study drug reconciliation will be performed to document drug 
assigned, drug dispensed, drug returns, and drug remaining.  This 
reconciliation will be logged on the drug reconciliation form, and signed 
and dated by the study team. 
5.9.4 Return or Destruction of Study Drug 
 
At the completion of the study, there will be a final reconciliation of drug 
shipped, drug dispensed, drug returns, and drug remaining.  This 
reconciliation will be logged on the drug reconciliation form, signed and 
dated.  Any discrepancies noted will be documented and investigated, 
prior to return or destruction of unused study drug.  Drug destroyed on site 
will be documented in the study files. 
 
6 
Study Procedures 
6.1 Visit 1 
 
At Visit 1, subjects will provide written informed consent.  Medications will be 
documented.  Safety blood tests (complete blood count, creatinine, blood urea 
nitrogen, sodium, potassium, TSH and T4, aspartate aminotransferase, alanine 
aminotransferase, total, direct and indirect bilirubin) hs c-RP, NTproBNP, 
assessment of endothelial function, radial tonometry for pulse wave analysis, 
and comprehensive echocardiography will be performed before catheterization.   
 
Invasive right heart catheterization with simultaneous limited echocardiography 
and expired gas analysis will then be performed at rest and during 20 watts of 
ergometer exercise as per standard practice for the evaluation of unexplained 
dyspnea (5 minute exercise duration). Subjects will be given study drug 
(AZD4831 or matching placebo) orally followed by 30 minutes waiting period in 
the cath lab to facilitate therapeutic concentration.  
 
Patients will then repeat resting and exercise invasive catheterization at 20 W 
in exactly the same fashion as prior to study drug.  Blood samples will be 
collected before and after study drug to measure biomarkers of oxidative 
stress, NO activity and PK.   
 
Following completion of both rest-exercise runs, participants will receive a bolus 
of intravenous heparin (70 U/kg) to displace tissue MPO. By comparing MPO 
concentrations and activity prior to study drug, following study drug and then 
following heparin, we will be able to relate MPO activity to the 
pharmacodynamic effects of study drug on central hemodynamics.  
 
Subjects will be transferred to the catheterization lab recovery area as per 
standard clinical practice, where intravascular sheaths will be removed and 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
hemostasis obtained.  Participants will be monitored according to standard 
practice guidelines following exercise right heart catheterization procedures and 
discharged on the same day.  A repeat EndoPAT test will be performed while 
the patients are in the recovery area. 
 
6.2 Visit 2 
 
Visit 2 will be scheduled on day 9-14 after dosing and will be a virtual, NFTF 
(non-face to face) visit, performed through use of Zoom, similar to other NFTF 
visits performed clinically at Mayo Clinic. The focus at the virtual NFTF visit will 
be to assess for any symptoms or adverse events and to perform a full skin 
examination by video, given the potential for maculopapular rash. If a clinically 
significant rash is identified dermatologic consultation will be obtained with 
biopsy if deemed appropriate by the consulting dermatologist. Patients will be 
instructed to report back immediately if they develop a rash prior to the follow 
up visit. 
   
 
Study Assessments (Figure 3): 
 
Right 
heart 
catheterization 
will 
be 
performed 
using 
high 
fidelity 
micromanometers at rest and during supine exercise with simultaneous expired 
gas analysis (MedGraphics, St. Paul, MN, USA).  A 9 Fr Internal jugular 
catheter for continuous right atrial pressure and to allow introduction of a 
balloon tipped fluid filled 
catheter will be placed in 
the right internal jugular 
vein. A high Fidelity 
micromanometer 
wire 
will be placed in the 
balloon wedge catheter 
to 
enable 
accurate 
pressure recording with 
exercise. A 4 Fr radial 
arterial cannula will also 
be placed for continuous 
arterial 
pressure 
measurement 
and 
arterial saturation with 
exercise (all standard 
care). The coronary sinus is cannulated at select times to measure myocardial 
biomarkers and indicators of metabolism.  Figure 3 shows the assessments 
performed in the protocol. The protocol is rest—passive leg elevation—20 
Watts exercise for 5 minutes. Hemodynamics, venous, arterial and coronary 
sinus blood samples, arterial and mixed venous blood gas and expired gas 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
data are acquired at rest, and during exercise. Detailed list of assessments and 
timing is found in Appendix I.  
Exercise is not performed to exhaustion, but we have previously shown that 80-
90% of the increases in cardiac filling pressures that will occur with exercise 
occur at 20W workload in HFpEF, and the requirement to perform 2 studies in 
the same day dictates that this lower workload is used uniformly in both tests. 
Of note, this workload is close to maximal in most patients with HFpEF, and 
most relevant to the workload of activities of daily living with affected patients. 
Perceived symptoms of dyspnea and fatigue will be quantified using the Borg 
dyspnea and effort scores during exercise.  
After the initial exercise study, the oral study drug (30 mg AZD4831 or placebo) 
will be provided. After a 30 minute waiting period for absorption and therapeutic 
concentration, subjects will repeat resting and exercise hemodynamics with 
expired gas data acquired exactly as in the initial run including venous, arterial 
and coronary sinus sampling at rest and exercise.  
Heparin will not be used during the studies due to its known effect on liberating 
vascular MPO and increasing NO bioavailability.38 At the conclusion of the 
study, a single bolus of heparin will be administered (70 units/kg) to evaluate 
vascular MPO levels and activity present in subjects. This will provide a more 
sensitive measure of tissue MPO activity after drug inhibition to correlate with 
hemodynamics. Both MPO level and activity will be measured, to allow 
quantification of specific activity. The blood sample collection for MPO 
assessment will be done at rest and following exercise, both before and after 
study drug. In addition following the exercise portion of the study it will be 
measured before and after heparin administration. Protamine will be given 
following final laboratory assessments prior to sheath removal to minimize risk 
of bleeding. 
Comprehensive echocardiography will be performed before catheterization for 
descriptive purposes (appendix II). 
Aortic pulse wave analysis will be determined from invasive radial artery 
pressure waveforms obtained during the cardiac catheterization procedure.  
Focused echocardiography will be done simultaneously with cardiac 
catheterization focusing on cardiac volumes and measures of biventricular 
systolic and diastolic function using strain and Tissue Doppler (appendix III).  
Measurements will be repeated at rest and during exercise before and after the 
study drug. 
Blood samples will be collected during catheterization. Up to 150 cc of blood 
will be taken to measure arterial and venous blood gases, saturations, and 
cyclic guanosine monophosphate (cGMP) levels at rest and exercise. Baseline 
blood samples will be obtained for MPO activity (MPO concentration and 
activity, Arg/ADMA/SDMA) level and biomarkers of inflammation/oxidative 
stress (cRP, troponin I, GDF-15, NO metabolites), fibrosis/remodeling (C-

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
telopeptide CI, sST2, MMP, galactin 3, Pro-collagen III N-terminal peptide) and 
neurohormonal activation (NT proBNP).  
Purine metabolites (hypoxanthine, xanthine, urate and allantoin) will also be 
quantified at these time points as this may provide important information 
regarding the mechanisms behind MPO-driven urate reduction. High sensitivity 
troponin will be assessed as an indicator of myocardial stress. The metabolites 
and troponin levels will be quantified in samples drawn from the radial artery, 
superior vena cava, the coronary sinus and from the main pulmonary artery to 
determine impact of AZD4831 on the different vascular beds. All metabolites 
should increase in the circulation at exercise. If MPO is a “vasomotor controller, 
as is believed, then pretreatment with AZD4831 should decrease the 
concentration of all metabolites. If MPO inhibition only affects the oxidation of 
hypoxanthine and xanthine, the concentration of these metabolites should be 
unaffected/increased and urate and allantoin decreased. If AZD4831 unloads 
the heart, this may reduce troponin levels in the circulation, particularly in the 
coronary sinus samples. A detailed list of blood sample testing is included 
below. 
 
 
Endothelial function testing will be assessed by pulse wave analysis using the 
Endo PAT (Peripheral Arterial Tone) 40 system beforestudy drug. Indirect 
serological surrogates of endothelial function including plasma cGMP and NO 
metabolites will also be assessed before and after study drug.  The Endo PAT 
will be performed for baseline values before catheterization in preparation room 
and for follow-up values in the recovery area following the procedure, but prior to 
discharge.    

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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7 Statistical Plan 
7.1 Sample Size Determination 
 
No data is available on the effect of an oral MPO inhibitor on exercise PCWP in 
humans. Based on our previously published data on IV sodium nitrite, the 
decrease in exercise PCWP was 2±5 with placebo and 11±5 with sodium nitrite 
35. To detect a decrease in PCWP magnitude of this magnitude or greater 
compared to placebo with 80% power at an α=0.05, only 6 subjects would be 
required in each group.  
However, since we expect the magnitude of hemodynamic effect of an oral 
MPO inhibitor to be at least 25% less than that of IV sodium nitrite (which would 
still be a meaningful decrease in PCWP); we will enroll 18 patients in each arm 
for a total of 36 patients. This will allow for 80% power to detect a reduction in 
exercise PCWP of 5.3 mmHg or greater, which would be considered to be 
clinically significant. 
7.2 Primary Outcome Analysis 
 
Primary Statistical Hypotheses: 
 
Ha: Exercise PCWP values at 20 Watt workload after study drug relative to 
corresponding values at 20 Watts workload prior to study drug will be lower 
after study drug administration compared to placebo 
 
Analysis Summary: 
The effect of AZD4831 on the primary endpoint of exercise PCWP will be 
assessed by analysis of covariance, using the initial exercise PCWP measured 
before study drug infusion and exercise PCWP following study drug as the 
covariates. Statistical testing for the primary analysis will be two-sided at the 
alpha=0.05 level of significance.  Other details of the statistical approach will be 
included in the Statistical Analysis Plan (SAP).   
 
8 
Safety and Adverse Events 
8.1 Definitions 
Unanticipated Problems Involving Risk to Subjects or Others (UPIRTSO) 
Any unanticipated problem or adverse event that meets the following three 
criteria:  
• 
Serious: Serious problems or events that result in significant harm, (which 
may be physical, psychological, financial, social, economic, or legal) or 
increased risk for the subject or others (including individuals who are not 
research subjects). These include: (1) death; (2) life threatening adverse 
experience; 
(3) 
hospitalization 
- 
inpatient, 
new, 
or 
prolonged; 
(4) 

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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
disability/incapacity - persistent or significant; (5) birth defect/anomaly; (6) 
breach of confidentiality and (7) other problems, events, or new information (i.e. 
publications, DSMB reports, interim findings, product labeling change) that in 
the opinion of the local investigator may adversely affect the rights, safety, or 
welfare of the subjects or others, or substantially compromise the research 
data, AND 
• 
Unanticipated: (i.e. unexpected) problems or events are those that are not 
already described as potential risks in the protocol, consent document, not 
listed in the Investigator’s Brochure, or not part of an underlying disease. A 
problem or event is "unanticipated" when it was unforeseeable at the time of its 
occurrence. A problem or event is "unanticipated" when it occurs at an 
increased frequency or at an increased severity than expected, AND 
• 
Related: A problem or event is "related" if it is possibly related to the 
research procedures. 
 
 
Adverse Event 
An untoward or undesirable experience associated with the use of a medical 
product (i.e. drug, device, biologic) in a patient or research subject. 
Serious Adverse Event 
Adverse events are classified as serious or non-serious.  Serious 
problems/events can be well defined and include; 
• 
death 
• 
life threatening adverse experience 
• 
hospitalization 
• 
inpatient, new, or prolonged; disability/incapacity 
• 
persistent or significant disability or incapacity 
• 
birth defect/anomaly 
 
and/or per protocol may be problems/events that in the opinion of the sponsor-
investigator may have adversely affected the rights, safety, or welfare of the 
subjects or others, or substantially compromised the research data. 
 
All adverse events that do not meet any of the criteria for serious, should be 
regarded as non-serious adverse events.  
 
Adverse Event Reporting Period 
For this study, the study treatment follow-up period is defined as the period 
between Visit 1 and Visit 2. Visit 2 should occur 9 to 14 days after Visit 1. The 
study period during which adverse events must be reported is normally defined 
as the period from the initiation of any study procedures to the end of the study 
treatment follow-up.  
 
Preexisting Condition 

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A preexisting condition is one that is present at the start of the study.  A 
preexisting condition should be recorded as an adverse event if the frequency, 
intensity, or the character of the condition worsens during the study period. 
 
General Physical Examination Findings 
At screening, any clinically significant abnormality should be recorded as a 
preexisting condition.  At the end of the study, any new clinically significant 
findings/abnormalities that meet the definition of an adverse event must also be 
recorded and documented as an adverse event.  
 
Post-study Adverse Event 
All unresolved adverse events should be followed by the sponsor-investigator 
until the events are resolved, the subject is lost to follow-up, or the adverse 
event is otherwise explained.  At the last scheduled visit, the sponsor-
investigator should instruct each subject to report, to the sponsor-investigator, 
any subsequent event(s) that the subject, or the subject’s personal physician, 
believes might reasonably be related to participation in this study.  
 
Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization 
should be documented and reported as a serious adverse event unless 
specifically instructed otherwise in this protocol.  Any condition responsible for 
surgery should be documented as an adverse event if the condition meets the 
criteria for an adverse event.  
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are 
reported as an adverse event in the following circumstances:  
• 
Hospitalization or prolonged hospitalization for diagnostic or elective 
surgical procedures for a preexisting condition.  Surgery should not be reported 
as an outcome of an adverse event if the purpose of the surgery was elective or 
diagnostic and the outcome was uneventful. 
• 
Hospitalization or prolonged hospitalization for therapy of the target 
disease of the study, unless it is a worsening or increase in frequency of 
hospital admissions as judged by the clinical investigator. 
 
8.2 Recording of Adverse Events 
At each contact with the subject, the study team 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 section of the case report 
form (CRF).  All clearly related signs, symptoms, and abnormal diagnostic, 
laboratory or procedure results should recorded in the source document. 
 
All adverse events occurring during the study period must be recorded.  The 
clinical course of each event should be followed until resolution, stabilization, or 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
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until it has been ultimately determined that the study treatment or participation 
is not the probable cause.  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 at least possibly related to the study treatment or study participation 
should be recorded and reported immediately. 
 
8.3 Reporting of Serious Adverse Events and Unanticipated Problems 
 
When an adverse event has been identified, the study team will take 
appropriated action necessary to protect the study participant and then 
complete the Study Adverse Event Worksheet and log. In addition to this, all 
SAEs are to be submitted to the AstraZeneca Product Safety mailbox: 
AEMailboxClinicalTrialTCS@astrazeneca.com The investigator ensures that all 
the necessary information is provided to the AstraZeneca Product Safety 
mailbox within 1 calendar day of initial receipt for fatal and life threatening 
events and within 5 calendar days of initial receipt for all other SAEs. The 
sponsor-investigator will evaluate the event and determine the necessary 
follow-up and reporting required. 
 
8.3.1 Sponsor-Investigator reporting: notifying the Mayo IRB 
 
The investigator will report to the Mayo IRB any UPIRTSOs and Non-
UPIRTSOs according to the Mayo IRB Policy and Procedures. 
 
Information collected on the adverse event worksheet  
• Subject’s name:  
• Medical record number:  
• Disease (if applicable):  
• The date the adverse event occurred:  
• Description of the adverse event:  
• Relationship of the adverse event to the research (drug, procedure, or 
intervention):  
• If the adverse event was expected:  
• The severity of the adverse event:  
• If any intervention was necessary:  
• Resolution: (was the incident resolved spontaneously, or after 
discontinuing treatment) 
• Date of Resolution:  
 
The investigator will review all adverse event reports to determine if 
specific reports need to be made to the IRB and FDA.  The investigator 
will sign and date the adverse event report when it is reviewed.  For this 
protocol, only directly related SAEs will be reported to the IRB. 

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Principal Investigator: Barry Borlaug, MD 
 
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8.3.2 Sponsor-Investigator reporting: Notifying the FDA 
 
The sponsor-investigator will report to the FDA all unexpected, serious 
suspected adverse reactions according to the required IND Safety 
Reporting timelines, formats and requirements. 
 
Unexpected fatal or life threatening suspected adverse reactions where 
there is evidence to suggest a causal relationship between the study 
drug/placebo and the adverse event, will be reported as a serious 
suspected adverse reaction.  This will be reported to the FDA on FDA 
Form 3500A, no later than 7 calendar days after the sponsor-investigator’s 
initial receipt of the information about the event. 
 
Other unexpected serious suspected adverse reactions where there is 
evidence to suggest a causal relationship between the study drug/placebo 
and the adverse event, will be reported as a serious suspected adverse 
reaction.  This will be reported to the FDA on FDA Form 3500A, no later 
than 15 calendar days after the sponsor-investigator’s initial receipt of the 
information about the event. 
 
Any clinically important increase in the rate of serious suspected adverse 
reactions over those listed in the protocol or product insert will be reported 
as a serious suspected adverse reaction.  This will be reported to the FDA 
on FDA Form 3500A no later than 15 calendar days after the sponsor-
investigator’s initial receipt of the information about the event. 
 
Findings from other studies in human or animals that suggest a significant 
risk in humans exposed to the drug will be reported.  This will be reported 
to the FDA on FDA Form 3500A, no later than 15 calendar days after the 
sponsor-investigators initial receipt of the information about the event. 
 
8.3.3 Sponsor-Investigator reporting: Notifying AstraZeneca 
 
AstraZeneca will be notified of any maculopapular rash on an ongoing 
basis, within 7 days of each occurrence is known. 
 
8.4 
Unmasking/Unblinding Procedures 
 
The investigators will be given access to the treatment code for their 
Participants for emergency un-blinding.  Any suspected study drug-related 
events should be treated as though the Participant received active 
therapy.   

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
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Randomization data are kept strictly confidential, accessible only to 
authorized persons, until the time of un-blinding.  
8.5 
Stopping Rules  
 
The study may be terminated prematurely if:  
 
• The PI (sponsor) and AstraZeneca assess that the number and/or 
severity of AEs justify discontinuation of the study. For instance if there 
were a case of fatal SAE or 2 cases of other SAEs that were 
considered related by the Investigator and AstraZeneca.  
• AstraZeneca considers the applied doses of the study drug to be no 
longer relevant.  
• The PI (sponsor) decides to discontinue the study.  
• Data not known before become available and raise concern about the 
safety of study drug so that continuation would pose potential risks to 
the subjects.  
 
Premature termination of the study must be mutually agreed upon by the 
PI (sponsor) and AstraZeneca and must be documented. However, study 
results will be reported according to the requirements outlined in this 
clinical study protocol as far as applicable. 
 
8.6 
Medical Monitoring 
It is the responsibility of the Principal Investigator to oversee the safety of 
the study at his/her site.  This safety monitoring will include careful 
assessment and appropriate reporting of adverse events as noted above, 
as well as the construction and implementation of a site data and safety-
monitoring plan (see section 10  “Study Monitoring, Auditing, and 
Inspecting”).  Medical monitoring will include a regular assessment of the 
number and type of serious adverse events. 
 
9 
Data Handling and Record Keeping 
9.1 
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 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
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• 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 (long term survival status that the subject is alive) at 
the end of their scheduled study period. 
9.2 
Source Documents 
Source data is all information, original records of clinical findings, observations, 
or other activities in a clinical trial necessary for the reconstruction and evaluation 
of the trial.  Source data are contained in source documents.  Examples of these 
original documents, and data records include: hospital records, clinical and office 
charts, laboratory notes, memoranda, subjects’ diaries or evaluation checklists, 
pharmacy dispensing records, recorded data from automated instruments, copies 
or transcriptions certified after verification as being accurate and complete, 
microfiches, photographic negatives, microfilm or magnetic media, x-rays, 
subject files, and records kept at the pharmacy, at the laboratories, and at 
medico-technical departments involved in the clinical trial. 
9.3 
Case Report Forms 
 
The study case report form (CRF) is the primary data collection instrument for the 
study.  All data requested on the CRF will be recorded.  All missing data will be 
explained.   
 
9.4 
Records Retention 
 
The sponsor-investigator will maintain records and essential documents related 
to the conduct of the study.  These will include subject case histories and 
regulatory documents. 
 
The sponsor-investigator will retain the specified records and reports for; 
1. 
Up to 2 years after the marketing application is approved for the drug; or, if 
a marketing application is not submitted or approved for the drug, until 2 years 
after shipment and delivery of the drug for investigational use is discontinued and 
the FDA has been so notified. OR 
2. 
As outlined in the Mayo Clinic Research Policy Manual –“Retention of and 
Access to Research Data Policy” http://mayocontent.mayo.edu/research-
policy/MSS_669717 , whichever is longer. 
 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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10 Study Monitoring, Auditing, and Inspecting 
10.1 
Study Monitoring Plan 
 
This study will be monitored on a routine basis during the conduct of the trial.  
The Mayo Clinic Office of Research Regulatory Support will provide periodic 
study monitoring as a service for the sponsor-investigator, to verify the validity 
and integrity of the data and protection of human research subjects.  Written 
monitoring reports will be provided to the Sponsor-Investigator and should be 
provided to the IRB at the time of continuing review.   This will assist with 
compliance with Food and Drug Administration regulations and GCP 
Guidelines. 
 
10.2 
Auditing and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by 
the IRB, the sponsor, and government regulatory agencies, of all study related 
documents (e.g. source documents, regulatory documents, data collection 
instruments, study data etc.).  The investigator will ensure the capability for 
inspections of applicable study-related facilities (e.g. pharmacy, diagnostic 
laboratory, etc.). 
 
Participation as an investigator in this study implies acceptance of potential 
inspection by government regulatory authorities and applicable compliance 
offices. 
11 Ethical Considerations 
This study is to be conducted according to United States government regulations and 
Institutional research policies and procedures. 
 
This protocol and any amendments will be submitted to a properly constituted local 
Institutional Review Board (IRB), in agreement with local legal prescriptions, for formal 
approval of the study.  The decision of the IRB concerning the conduct of the study will 
be made in writing to the sponsor-investigator before commencement of this study. 
 
All subjects for this study will be provided a consent form describing this study and 
providing sufficient information for subjects to make an informed decision about their 
participation in this study.  This consent form will be submitted with the protocol for 
review and approval by the IRB for the study.  The formal consent of a subject, using 
the Approved IRB consent form, must be obtained before that subject undergoes any 
study procedure.  The consent form must be signed by the subject or the subject’s 
legally authorized representative, and the individual obtaining the informed consent. 
 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
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12 Study Finances 
12.1 
Funding Source 
 
This study is financed through AstraZeneca.  Study drug including MPO inhibitor 
and placebo will be provided by AstraZeneca. 
12.2 
Conflict of Interest 
 
Any study team member who has a conflict of interest with this study (patent 
ownership, royalties, or financial gain greater than the minimum allowable by 
their institution, etc.) must have the conflict reviewed by a properly constituted 
Conflict of Interest Committee with a Committee-sanctioned conflict management 
plan that has been reviewed and approved by the study sponsor-investigator 
prior to participation in this study. 
12.3 
Subject Stipends or Payments 
 
$300 per subject and up to $200 for subject’s family/caregiver that may need to 
stay longer in town for hotel and meal expenses for the extra overnight stay 
requirement. 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
 
13 Publication Plan 
 
The primary responsibility for publication of the results lies with the principal 
investigator.  The trial will be registered on ClinicalTrials.gov. 
 
14 References 
 
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2. 
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WJ. Phenotype-specific treatment of heart failure with preserved ejection fraction: 
A multiorgan roadmap. Circulation. 2016;134:73-90 
3. 
Paulus WJ, Tschope C. A novel paradigm for heart failure with preserved 
ejection fraction: Comorbidities drive myocardial dysfunction and remodeling 
through coronary microvascular endothelial inflammation. J Am Coll Cardiol. 
2013;62:263-271 
4. 
Nicholls SJ, Hazen SL. Myeloperoxidase and cardiovascular disease. 
Arterioscler Thromb Vasc Biol. 2005;25:1102-1111 
5. 
Tang WH, Tong W, Troughton RW, Martin MG, Shrestha K, Borowski A, Jasper 
S, Hazen SL, Klein AL. Prognostic value and echocardiographic determinants of 
plasma myeloperoxidase levels in chronic heart failure. J Am Coll Cardiol. 
2007;49:2364-2370 
6. 
Brennan ML, Penn MS, Van Lente F, Nambi V, Shishehbor MH, Aviles RJ, 
Goormastic M, Pepoy ML, McErlean ES, Topol EJ, Nissen SE, Hazen SL. 
Prognostic value of myeloperoxidase in patients with chest pain. N Engl J Med. 
2003;349:1595-1604 
7. 
Zhang R, Brennan ML, Fu X, Aviles RJ, Pearce GL, Penn MS, Topol EJ, 
Sprecher DL, Hazen SL. Association between myeloperoxidase levels and risk of 
coronary artery disease. Jama. 2001;286:2136-2142 
8. 
Wong ND, Gransar H, Narula J, Shaw L, Moon JH, Miranda-Peats R, Rozanski 
A, Hayes SW, Thomson LE, Friedman JD, Berman DS. Myeloperoxidase, 
subclinical atherosclerosis, and cardiovascular disease events. JACC Cardiovasc 
Imaging. 2009;2:1093-1099 
9. 
Khan SQ, Kelly D, Quinn P, Davies JE, Ng LL. Myeloperoxidase aids 
prognostication together with n-terminal pro-b-type natriuretic peptide in high-risk 
patients with acute st elevation myocardial infarction. Heart. 2007;93:826-831 
10. 
Kataoka Y, Shao M, Wolski K, Uno K, Puri R, Murat Tuzcu E, Hazen SL, Nissen 
SE, Nicholls SJ. Myeloperoxidase levels predict accelerated progression of 
coronary atherosclerosis in diabetic patients: Insights from intravascular 
ultrasound. Atherosclerosis. 2014;232:377-383 
11. 
Baldus S, Heeschen C, Meinertz T, Zeiher AM, Eiserich JP, Munzel T, Simoons 
ML, Hamm CW. Myeloperoxidase serum levels predict risk in patients with acute 
coronary syndromes. Circulation. 2003;108:1440-1445 

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CONFIDENTIAL 
12. 
Meuwese MC, Stroes ES, Hazen SL, van Miert JN, Kuivenhoven JA, Schaub 
RG, Wareham NJ, Luben R, Kastelein JJ, Khaw KT, Boekholdt SM. Serum 
myeloperoxidase levels are associated with the future risk of coronary artery 
disease in apparently healthy individuals: The epic-norfolk prospective population 
study. J Am Coll Cardiol. 2007;50:159-165 
13. 
Baldus S, Heitzer T, Eiserich JP, Lau D, Mollnau H, Ortak M, Petri S, Goldmann 
B, Duchstein HJ, Berger J, Helmchen U, Freeman BA, Meinertz T, Munzel T. 
Myeloperoxidase enhances nitric oxide catabolism during myocardial ischemia 
and reperfusion. Free Radic Biol Med. 2004;37:902-911 
14. 
Vita JA, Brennan ML, Gokce N, Mann SA, Goormastic M, Shishehbor MH, Penn 
MS, Keaney JF, Jr., Hazen SL. Serum myeloperoxidase levels independently 
predict endothelial dysfunction in humans. Circulation. 2004;110:1134-1139 
15. 
Zhang H, Xu H, Weihrauch D, Jones DW, Jing X, Shi Y, Gourlay D, Oldham KT, 
Hillery CA, Pritchard KA, Jr. Inhibition of myeloperoxidase decreases vascular 
oxidative stress and increases vasodilatation in sickle cell disease mice. J Lipid 
Res. 2013;54:3009-3015 
16. 
Zhang C, Patel R, Eiserich JP, Zhou F, Kelpke S, Ma W, Parks DA, Darley-
Usmar V, White CR. Endothelial dysfunction is induced by proinflammatory 
oxidant hypochlorous acid. Am J Physiol Heart Circ Physiol. 2001;281:H1469-
1475 
17. 
Rudolph TK, Wipper S, Reiter B, Rudolph V, Coym A, Detter C, Lau D, Klinke A, 
Friedrichs K, Rau T, Pekarova M, Russ D, Knoll K, Kolk M, Schroeder B, 
Wegscheider K, Andresen H, Schwedhelm E, Boeger R, Ehmke H, Baldus S. 
Myeloperoxidase deficiency preserves vasomotor function in humans. Eur Heart 
J. 2012;33:1625-1634 
18. 
Abu-Soud HM, Hazen SL. Nitric oxide is a physiological substrate for mammalian 
peroxidases. J Biol Chem. 2000;275:37524-37532 
19. 
Abu-Soud HM, Hazen SL. Nitric oxide modulates the catalytic activity of 
myeloperoxidase. J Biol Chem. 2000;275:5425-5430 
20. 
Eiserich JP, Hristova M, Cross CE, Jones AD, Freeman BA, Halliwell B, van der 
Vliet 
A. 
Formation 
of 
nitric 
oxide-derived 
inflammatory 
oxidants 
by 
myeloperoxidase in neutrophils. Nature. 1998;391:393-397 
21. 
Eiserich JP, Baldus S, Brennan ML, Ma W, Zhang C, Tousson A, Castro L, Lusis 
AJ, Nauseef WM, White CR, Freeman BA. Myeloperoxidase, a leukocyte-derived 
vascular no oxidase. Science. 2002;296:2391-2394 
22. 
Zhang C, Reiter C, Eiserich JP, Boersma B, Parks DA, Beckman JS, Barnes S, 
Kirk M, Baldus S, Darley-Usmar VM, White CR. L-arginine chlorination products 
inhibit endothelial nitric oxide production. J Biol Chem. 2001;276:27159-27165 
23. 
Askari AT, Brennan ML, Zhou X, Drinko J, Morehead A, Thomas JD, Topol EJ, 
Hazen SL, Penn MS. Myeloperoxidase and plasminogen activator inhibitor 1 play 
a central role in ventricular remodeling after myocardial infarction. J Exp Med. 
2003;197:615-624 
24. 
Rudolph V, Andrie RP, Rudolph TK, Friedrichs K, Klinke A, Hirsch-Hoffmann B, 
Schwoerer AP, Lau D, Fu X, Klingel K, Sydow K, Didie M, Seniuk A, von Leitner 
EC, Szoecs K, Schrickel JW, Treede H, Wenzel U, Lewalter T, Nickenig G, 
Zimmermann WH, Meinertz T, Boger RH, Reichenspurner H, Freeman BA, 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
Eschenhagen T, Ehmke H, Hazen SL, Willems S, Baldus S. Myeloperoxidase 
acts as a profibrotic mediator of atrial fibrillation. Nat Med. 2010;16:470-474 
25. 
Vasilyev N, Williams T, Brennan ML, Unzek S, Zhou X, Heinecke JW, Spitz DR, 
Topol EJ, Hazen SL, Penn MS. Myeloperoxidase-generated oxidants modulate 
left ventricular remodeling but not infarct size after myocardial infarction. 
Circulation. 2005;112:2812-2820 
26. 
Sato E, Koyama S, Camhi SL, Nelson DK, Robbins RA. Reactive oxygen and 
nitrogen metabolites modulate fibronectin-induced fibroblast migration in vitro. 
Free Radic Biol Med. 2001;30:22-29 
27. 
Baldus S, Eiserich JP, Mani A, Castro L, Figueroa M, Chumley P, Ma W, 
Tousson A, White CR, Bullard DC, Brennan ML, Lusis AJ, Moore KP, Freeman 
BA. Endothelial transcytosis of myeloperoxidase confers specificity to vascular 
ecm proteins as targets of tyrosine nitration. J Clin Invest. 2001;108:1759-1770 
28. 
Fu X, Kassim SY, Parks WC, Heinecke JW. Hypochlorous acid oxygenates the 
cysteine switch domain of pro-matrilysin (mmp-7). A mechanism for matrix 
metalloproteinase 
activation 
and 
atherosclerotic 
plaque 
rupture 
by 
myeloperoxidase. J Biol Chem. 2001;276:41279-41287 
29. 
Fu X, Kassim SY, Parks WC, Heinecke JW. Hypochlorous acid generated by 
myeloperoxidase modifies adjacent tryptophan and glycine residues in the 
catalytic domain of matrix metalloproteinase-7 (matrilysin): An oxidative 
mechanism for restraining proteolytic activity during inflammation. J Biol Chem. 
2003;278:28403-28409 
30. 
Borlaug BA, Olson TP, Lam CS, Flood KS, Lerman A, Johnson BD, Redfield MM. 
Global cardiovascular reserve dysfunction in heart failure with preserved ejection 
fraction. J Am Coll Cardiol. 2010;56:845-854 
31. 
Marechaux S, Samson R, van Belle E, Breyne J, de Monte J, Dedrie C, Chebai 
N, Menet A, Banfi C, Bouabdallaoui N, Le Jemtel TH, Ennezat PV. Vascular and 
microvascular endothelial function in heart failure with preserved ejection 
fraction. J Card Fail. 2016;22:3-11 
32. 
Farrero M, Blanco I, Batlle M, Santiago E, Cardona M, Vidal B, Castel MA, Sitges 
M, Barbera JA, Perez-Villa F. Pulmonary hypertension is related to peripheral 
endothelial dysfunction in heart failure with preserved ejection fraction. Circ Heart 
Fail. 2014;7:791-798 
33. 
Matsue Y, Suzuki M, Nagahori W, Ohno M, Matsumura A, Hashimoto Y, Yoshida 
K, Yoshida M. Endothelial dysfunction measured by peripheral arterial tonometry 
predicts prognosis in patients with heart failure with preserved ejection fraction. 
Int J Cardiol. 2013;168:36-40 
34. 
Borlaug BA, Melenovsky V, Koepp KE. Inhaled sodium nitrite improves rest and 
exercise hemodynamics in heart failure with preserved ejection fraction. Circ 
Res. 2016;119:880-886 
35. 
Borlaug BA, Koepp KE, Melenovsky V. Sodium nitrite improves exercise 
hemodynamics and ventricular performance in heart failure with preserved 
ejection fraction. J Am Coll Cardiol. 2015;66:1672-1682 
36. 
Simon MA, Vanderpool RR, Nouraie M, Bachman TN, White PM, Sugahara M, 
Gorcsan J, 3rd, Parsley EL, Gladwin MT. Acute hemodynamic effects of inhaled 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
sodium nitrite in pulmonary hypertension associated with heart failure with 
preserved ejection fraction. JCI Insight. 2016;1:e89620 
37. 
Eggebeen J, Kim-Shapiro DB, Haykowsky M, Morgan TM, Basu S, Brubaker P, 
Rejeski J, Kitzman DW. One week of daily dosing with beetroot juice improves 
submaximal endurance and blood pressure in older patients with heart failure 
and preserved ejection fraction. JACC Heart Fail. 2016;4:428-437 
38. 
Baldus S, Rudolph V, Roiss M, Ito WD, Rudolph TK, Eiserich JP, Sydow K, Lau 
D, Szocs K, Klinke A, Kubala L, Berglund L, Schrepfer S, Deuse T, Haddad M, 
Risius T, Klemm H, Reichenspurner HC, Meinertz T, Heitzer T. Heparins 
increase endothelial nitric oxide bioavailability by liberating vessel-immobilized 
myeloperoxidase. Circulation. 2006;113:1871-1878 
39. 
Schwartzenberg S, Redfield MM, From AM, Sorajja P, Nishimura RA, Borlaug 
BA. Effects of vasodilation in heart failure with preserved or reduced ejection 
fraction implications of distinct pathophysiologies on response to therapy. J Am 
Coll Cardiol. 2012;59:442-451 
40. 
Axtell AL, Gomari FA, Cooke JP. Assessing endothelial vasodilator function with 
the endo-pat 2000. J Vis Exp. 2010 
 

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15 Attachments 
 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
Appendix I: Schedule of Assessments 
 
Visit 1 
(Day 1) 
NFTF 
Visit 2 
(Day 
9-
14) 
 
Baseline 
(Before 
Cath) 
Baseline Cath 
Study drug Cath 
Heparin phase Cath 
 
 
 
Rest 
 
20 
Watts 
 
Rest 
 
20 
Watts 
 
Recovery 
After Heparin 
 
Time relative to study 
drug  
-20 min 
-10 
min 
-5 min 
30 min 
35 min 
60 min  
 
Eligibility Review 
and Consent 
X 
 
 
 
 
 
 
 
 
 
History and physical 
exam 
X 
 
 
 
 
 
 
 
 
X 
Venous Blood 
samples 
 
 
 
 
 
 
 
 
 
 
Safety Labs# 
X* 
 
 
 
 
 
 
 
 
 
High sensitive CRP 
X* 
 
 
 
 
 
 
 
 
 
NT proBNP 
X* 
 
 
 
 
 
 
 
 
 
FSH for female, if not 
previously done 
X 
 
 
 
 
 
 
 
 
 
ProSeek panel† 
 
X** 
 
 
 
 
 
 
 
 
AZD4831 exposure 
 
X** 
 
X** 
X** 
X** 
 
MPO biomarkers ‡ 
 
X** 
X** 
X** 
X** 
X** 
 
cGMP and NO 
metabolites and hs 
troponin levels 
 
X** 
X** 
X** 
X** 
X** 
 
Pulmonary artery 
sample 
 
 
 
 
 
 
 
 
 
 
Pulmonary artery 
saturation 
 
X 
X 
X 
X 
 
 
 
 
 
MPO biomarkers ‡ 
 
X** 
X** 
X** 
X** 
 
 
 
 
 
cGMP and NO 
metabolites and hs 
troponin levels 
 
X** 
X** 
X** 
X** 
 
 
 
 
 
Pulmonary capillary 
sample 
 
 
 
 
 
 
 
 
 
 
Pulmonary capillary 
saturation 
 
X 
X 
X 
X 
 
 
 
 
 
MPO biomarkers ‡  
 
X** 
X** 
X** 
X** 
 
 
 
 
 
cGMP and NO 
metabolites and hs 
troponin levels 
 
X** 
X** 
X** 
X** 
 
 
 
 
 
Coronary sinus 
samples 
 
 
 
 
 
 
 
 
 
 

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Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
 
Visit 1 
(Day 1) 
NFTF 
Visit 2 
(Day 
9-
14) 
 
Baseline 
(Before 
Cath) 
Baseline Cath 
Study drug Cath 
Heparin phase Cath 
 
 
 
Rest 
 
20 
Watts 
 
Rest 
 
20 
Watts 
 
Recovery 
After Heparin 
 
Coronary sinus blood 
gas 
 
X 
X 
X 
X 
 
 
 
 
 
MPO biomarkers ‡ 
 
X** 
X** 
X** 
X** 
 
 
 
 
 
cGMP and NO 
metabolites and hs 
troponin levels 
 
X** 
X** 
X** 
X** 
 
 
 
 
 
Arterial blood 
samples 
 
 
 
 
 
 
 
 
 
 
Arterial blood gas 
 
X 
X 
X 
X 
 
 
 
 
 
MPO biomarkers ‡ 
 
X** 
X** 
X** 
X** 
 
 
 
 
 
cGMP and NO 
metabolites and hs 
troponin levels 
 
X** 
X** 
X** 
X** 
 
 
 
 
 
Endopat 
X 
 
 
 
 
 
 
 
 
 
Comprehensive 
echocardiogram 
X 
 
 
 
 
 
 
 
 
 
Limited 
echocardiography* 
 
X 
X 
X 
X 
 
 
 
 
 
Arterial Pulse Wave 
Analysis 
X 
 
 
 
 
 
 
 
 
 
PA pressure 
 
X 
X 
X 
X 
 
 
 
 
 
PCWP 
 
X 
X 
X 
X 
 
 
 
 
 
RA pressure 
 
X 
X 
X 
X 
 
 
 
 
 
A-V O2 content 
difference 
 
X 
X 
X 
X 
 
 
 
 
 
O2 consumption 
 
X 
X 
X 
X 
 
 
 
 
 
VE/VCO2, RER 
 
X 
X 
X 
X 
 
 
 
 
 
Systemic BP 
 
X 
X 
X 
X 
 
 
 
 
 
Heart rate/ 
ECG/telemetry 
 
X 
X 
X 
X 
X 
 
 
*  Measured at Mayo Clinic 
**  Measured by/at AstraZeneca 
***. 
†  Soluble ST2, TnI, Gal-3, GDF15, NT-proCIII, C-telopeptide CI, Elastase, Calprotectin, MMPx 
‡  Purine metabolites (hypoxanthine, xanthine, urate and allantoin), MPO concentration and activity, Arg/ADMA/SDMA 
#   Safety labs include: complete blood count, creatinine, blood urea nitrogen, sodium, potassium, TSH and T4, aspartate 
aminotransferase, alanine aminotransferase, total, direct and indirect bilirubin.  TSH and T4 will only be assessed at 
baseline. 

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CONFIDENTIAL 
 
Appendix II: Comprehensive Echocardiogram Data at Baseline 
 
 
Value 
LV Septal wall thickness  
 
LV posterior wall thickness  
 
LV End Diastolic Dimension  
 
LV End Systolic Dimension  
 
LVEDV  
 
LVESV  
 
LVEF  
 
LA Volume  
 
RV dimension base  
 
RV dimension mid  
 
Mitral E (m/sec) 
 
Mitral A (m/sec) 
 
Early diastolic mitral annular velocity (medial) (medial e’) 
 
Systolic mitral annular velocity (medial) (medial s’) 
 
Early diastolic mitral annular velocity (lateral) (lateral e’) 
 
Systolic mitral annular velocity (lateral) (lateral s’) 
 
RV early diastolic lateral annular velocity  (RV e’) 
 
RV systolic lateral annular velocity  (RV e’) 
 
TAPSE (mm) 
 
TR velocity (m/sec) 
 
RA Pressure estimate (mmHg) 
 
4 chamber, 2 chamber and 3 chamber view of LV for retrospective 
LV strain calculations 
 
4 chamber, 2 chamber and 3 chamber view of LA for retrospective 
atrial strain calculations 
 
 
 
 
 
 
 
 
 

Hemodynamic Effects of MPO inhibition on exercise in HFpEF 
 
Page 43 of 43 
Principal Investigator: Barry Borlaug, MD 
 
CONFIDENTIAL 
 
Appendix III: Echocardiography Data with Exercise 
 
 
Value 
LVEDV  
 
LVESV  
 
LVEF  
 
Mitral E (m/sec) 
 
Mitral A (m/sec) 
 
Early diastolic mitral annular velocity (medial) (medial e’) 
 
Systolic mitral annular velocity (medial) (medial s’) 
 
Early diastolic mitral annular velocity (lateral) (lateral e’) 
 
Systolic mitral annular velocity (lateral) (lateral s’) 
 
RV early diastolic lateral annular velocity  (RV e’) 
 
RV systolic lateral annular velocity  (RV e’) 
 
TAPSE (mm) 
 
RV Fractional area change