Conditions: Hypertension
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 4
Healthy volunteers: no

Brief summary:
The purpose of this study is to test the hypothesis that combined angiotensin receptor blockade (ARB)/neprilysin (NEP) inhibition potentiates the effects of exogenous bradykinin, substance P, and brain natriuretic peptide (BNP) on forearm blood flow or endothelial tissue-type plasminogen activator (t-PA) release compared to ARB alone. A secondary goal is to determine if there is an interactive effect of ARB/NEP inhibition and dipeptidyl peptidase 4 (DPP4) inhibition on responses to these peptides.

Detailed description:
After informed consent is obtained, subjects will undergo a screening history and physical exam, and anti-hypertensive medications will be withdrawn. During this period, blood pressure (BP) will be measured every one to three days.

After subjects have been off anti-hypertensive medications for three weeks (four for spironolactone), they will be randomized to four-week treatment with valsartan 160 mg bid (80 mg bid for one week, then 160 mg bid) or LCZ696 200 bid (100 mg bid for one week, then 200 mg bid) in a double-blind fashion. On the morning of the 28th day of study drug, subjects will report to the Vanderbilt Clinical Research Center (CRC) after an overnight fast. Subjects will be studied in the supine position in a temperature-controlled room. They will be instrumented for intra-arterial infusions. Subjects will be given their last dose of study drug. One hour after drug administration, we will measure forearm blood flow (FBF) and give bradykinin, substance P, or BNP. Each peptide will be infused in three graded doses for five minutes. After administration of all three peptides, subjects will be allowed to rest for an hour. Then they will be given a single oral dose of sitagliptin 200 mg and be allowed to rest for 90 minutes. We will repeat baseline measurements and the peptide infusions with an intervening rest period. The four-week study treatment and protocol will be repeated after a three-week washout, until participants complete both arms.

Arms:
arm 1: After four-week treatment with valsartan, participants will receive intra-arterial infusions of bradykinin, substance P, and BNP in the presence and absence of sitagliptin.

Then, after three-week washout and four week therapy with LCZ696, participants will receive intra-arterial infusions of bradykinin, substance P, and BNP in the presence and absence of sitagliptin. arm 2: After four-week treatment with LCZ696, participants will receive intra-arterial infusions of bradykinin, substance P, and BNP in the presence and absence of sitagliptin.

Then, after three-week washout and four week therapy with valsartan, participants will receive intra-arterial infusions of bradykinin, substance P, and BNP in the presence and absence of sitagliptin.

Interventions: intervention 1: Valsartan intervention 2: LCZ696 intervention 3: Bradykinin intervention 4: Substance P intervention 5: BNP intervention 6: Sitagliptin
Intervention descriptions: intervention 1: oral valsartan intervention 2: oral LCZ696 intervention 3: Intra-arterial bradykinin at three graded doses intervention 4: Intra-arterial substance P at three graded doses intervention 5: Intra-arterial BNP at three graded doses intervention 6: oral sitagliptin

Protocol document:
Prot_SAP_000.pdf: 
                 1                                                                           2/11/19 
 
 
 
 
 
 
 
Mechanism(s) Underlying Cardiovascular Effects of ARB/NEP 
Inhibition – Aim 1 
 
 
Nancy J. Brown, M.D. 
D-3100 Medical Center North 
Vanderbilt University Medical Center 
Nashville, TN, 37232 
 
 
 
 
 
 
 
 
 
 
 
 


                 2                                                                           2/11/19 
 
 
 
Table of Contents: 
 
Study Schema 
1.0 
Background 
2.0 
Rationale and Specific Aims 
3.0 
Animal Studies and Previous Human Studies 
4.0 
Inclusion/Exclusion Criteria 
5.0 
Enrollment/Randomization 
6.0 
Study Procedures 
7.0 
Risks of Investigational Agents/Devices (side effects) 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to 
Participants or Others 
9.0 
Study Withdrawal/Discontinuation 
10.0 
Statistical Considerations 
11.0 
Privacy/Confidentiality Issues 
12.0 
Follow-up and Record Retention 
13.0 
Standard Techniques 
 
 
 
 
 
 
 
 
 
 
 
 
 


                 3                                                                           2/11/19 
1.0      Background 
 
The lifetime risk of developing heart failure (HF) in the United States exceeds 20%.1 Despite 
beneficial effects of angiotensin receptor blockers (ARB)s, angiotensin converting enzyme 
inhibitors (ACEi)s, beta blockers, and mineralocorticoid receptor (MR) antagonists on mortality, 
the five-year life expectancy of a patient with HF is 50%.1 The combined ARB/neutral 
endopeptidase (NEP)i LCZ696 was approved by the FDA after the Prospective Comparison of 
ARNi with ACEi to Determine Impact on Global Mortality and Morbidity in Heart Failure 
(PARADIGM-HF) showed a beneficial effect on mortality compared to enalapril in patients with 
HF and reduced ejection fraction (EF).2, 3 Nevertheless, many questions regarding the treatment of 
patients with an ARB/NEPi remain to be answered.4 
 
Beneficial effects of combined ARB/NEP inhibition have been attributed to potentiation of 
the vasodilatory, natriuretic, sympatholytic, antiproliferative, and antihypertrophic effects 
of natriuretic peptides (Figure 1).5 In HF, decreased sensitivity to the natriuretic peptides results 
from altered processing of proBNP to BNP, as well as altered clearance.6-8 LCZ696 decreases NT-
proBNP, but increases BNP,2, 9, 10 providing indirect evidence that BNP degradation is decreased; 
unfortunately commercially available assays cross-react with degradation fragments,11 leaving 
interpretation of these data open. 
 
 
 
Studies using exogenous natriuretic peptides in humans provide conflicting evidence 
regarding potentiation by NEP inhibition, however. For example, the NEPi thiorphan does not 
alter the vasodilator response to intra-arterial atrial natriuretic peptide (ANP) in HF patients taking 
an ACEi, but potentiates the response to bradykinin.12 NEP inhibition enhances the effects of 
intravenous BNP on plasma cGMP and blood pressure, but does not alter urinary cGMP or sodium 
excretion in patients with left ventricular impairment.13 In general, C-type natriuretic peptide 
(CNP) has minimal effect on vasodilation or natriuresis in humans.14-18 Dalzell reported no effect 
of thiorphan, captopril, or omapatrilat on a weak vasodilator response to CNP in small resistance 
arteries from patients with CAD;19 in one study, thiorphan potentiated the effect of intra-arterial 
CNP in healthy non-smokers.20 In this study, we will investigate the effects of NEP inhibition on 
the vasodilator response to intra-arterial BNP based on 1) our prior data showing a significant 
vasodilator response to BNP,21 2) indirect evidence for decreased degradation of BNP in HF 
patients taking LCZ696 mentioned earlier,9, 10 and 3) the potential for interaction between NEP 
and a dipeptidyl peptidase-4 (DPP4)i,22 used in the treatment of diabetes, on the degradation of 
BNP (Figure 2). 
 
Figure 1: LCZ696 inhibits neprilysin 
(NEP) and blocks the angiotensin type 1 
receptor 
(AT1R). 
Ang 
I 
indicates 
angiotensin I; Ang II, angiotensin II; 
ARB, angiotensin receptor blocker; ACE, 
angiotensin-converting enzyme; APP, 
aminopeptidase P; ANP, atrial natriuretic 
peptide; BNP, brain natriuretic peptide; 
CNP, C-type natriuretic peptide; DPP4, 
dipeptidyl peptidase 4 


                 4                                                                           2/11/19 
In addition to cleaving the natriuretic peptides, NEP cleaves peptides such as bradykinin, 
substance P, vasoactive intestinal peptide, glucagon, neurotensin, adrenomedullin, and 
amyloid β peptide.23 In fact, the Kcat/Km (a measure of catalytic efficiency) of NEP for 
bradykinin or substance P (69 and 158.7 min-1 µM-1, respectively)24 is significantly higher than 
those for ANP, BNP, and CNP (5.1, 0.53, and 7.58 min-1 µM-1, respectively).25 In small resistance 
arteries from patients with CAD and preserved EF, captopril, thiorphan, and omapatrilat all 
significantly potentiated the vasodilator response to bradykinin.19 The response to substance P was 
not potentiated by ACE, NEP, or combined ACE/NEP inhibition in this study, but NEP inhibition 
potentiates effects of substance P in human vascular smooth muscle cells in vitro.26 Patients given 
LCZ696 should not be taking an ACEi, but could be taking a DPP4i for the treatment of diabetes. 
As illustrated in Figure 2, there is potential for an interactive effect of NEP inhibition and DPP4 
inhibition on the degradation of substance P and/or BNP.  
 
 
Decreased degradation of bradykinin and substance P by 
NEP could lead to beneficial effects on vascular relaxation 
and fibrinolysis. Studies from our group and others using the pharmacological bradykinin B2 
receptor blocker icatibant have shown that endogenous bradykinin contributes to the beneficial 
effects of ACEis on blood pressure (BP) and on endothelial fibrinolysis.27-29 Substance P also 
causes vasodilation and tissue-plasminogen activator (t-PA) release from the vascular 
endothelium, and administration of a substance P (NK1) receptor antagonist blocks these effects 
in humans.30, 31 Substance P also increases sympathetic activation,30 an effect that may be 
detrimental in HF patients.32  
 
ARB/NEPi use may be associated with angioedema.  
Treatment with the combined ACE/NEPi omapatrilat results in a three-fold increase in 
angioedema compared to ACE inhibition.33 The increased rate of angioedema has been attributed 
to simultaneous inhibition of two pathways involved in the degradation of bradykinin and 
substance P, major effectors of angioedema.34 Bradykinin is inactivated primarily by ACE, but 
also by NEP and aminopeptidase P (APP), while substance P is inactivated by ACE, NEP, and 
DPP4 (Figure 2).24, 35, 36 Consistent with a role for NEP in the pathogenesis of angioedema, a 
polymorphism in the gene encoding for NEP is associated with ACEi-associated angioedema.37 
Because LCZ696 inhibits NEP without inhibiting ACE, it has been suggested that the incidence 
of angioedema will be lower than that observed with omapatrilat.   
 
In PARADIGM-HF, there were 19 confirmed cases of angioedema in patients treated with 
LCZ696 compared to ten cases in patients treated enalapril (an incidence of 0.45% versus 0.23%, 
p=0.13).2 The PARADIGM-HF trial likely underestimates the risk of angioedema in patients 
taking LCZ696 in the real world setting. Seventy-seven percent of patients enrolled in the trial 
were previously treated with an ACEi, and all were exposed to enalapril and LCZ696 during a 
single-blind, run-in period. Including a high proportion of patients who tolerated exposure to an 
S P K MV Q G S G
C
F
G
R
K
M
D R I S S S
S
G
L
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C
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DPP4
NEP
IDE
BNP
P
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K
Q Q F F G L M
R
NEP
ACE
DPP4
Substance P
R P P G F S P F R
NEP
ACE
APPDPP4
Bradykinin
Figure 2: Cleavage of bradykinin, substance P, and brain natriuretic 
peptide (BNP) by neprilysin (NEP), angiotensin-converting enzyme (ACE), 
aminopeptidase P (APP), dipeptidyl peptidase-4 (DPP4), and insulin-
degrading enzyme (IDE). The dotted arrow indicates that bradykinin must 
be cleaved by (and is inactivated by) APP before it is a DPP4 substrate 


                 5                                                                           2/11/19 
ACEi may have led to a decreased incidence of angioedema. Moreover, just 5% of patients in 
PARADIGM were African American, a group at increased risk of ACEi- and vasopeptidase 
inhibitor-associated angioedema.2, 33, 38, 39 
 
Summary  
The prevalence of HF is increasing with the aging of the population, and mortality remains 
unacceptably high. A newly approved ARB/NEPi reduced mortality in a single trial, but its 
mechanism of action has been inadequately studied. NEP cleaves many vasoactive peptides in 
addition to the natriuretic peptides, including bradykinin and substance P, two peptides that 
contribute to beneficial and adverse effects of ACEis.  Understanding the mechanism of action of 
the ARB/NEPi will enable us to target its use appropriately and may lead to the development of 
new drugs for the treatment of HF. 
 
2.0      Rationale and Specific Aims 
 
HF affects approximately 5.7 million people in the United States, and the number is expected 
to grow to more than eight million by 2030.1 ACEis, ARBs, beta blockers, and MR antagonists 
reduce mortality in patients with HF with reduced EF.40-44  Still mortality from HF remains high, 
and approximately 50% of individuals with HF die within five years of diagnosis.1   
 
In 2015, the FDA approved LCZ696, a molecular complex of the ARB valsartan with an 
inhibitor of NEP (neutral endopeptidase-24.11) sacubitril,45 after the PARADIGM-HF trial 
demonstrated a mortality benefit over enalapril in patients with HF, reduced EF, and increased 
circulating brain natriuretic peptide (BNP) or N-terminal pro-BNP (NT-proBNP) concentrations.2, 
3 This combined ARB/NEPi targets at least two pathophysiological mechanisms underlying HF - 
activation of the renin-angiotensin-aldosterone system (RAAS) and decreased sensitivity to the 
natriuretic peptides. 
 
In addition to degrading natriuretic peptides, NEP degrades other vasoactive peptides including 
angiotensins (Ang) I and II, endothelins, bradykinin, and substance P (Figure 1). Indeed, 
observations that NEP inhibition increases endogenous Ang II and the pressor response to 
exogenous Ang II provided the rationale for combining NEPi with RAAS inhibitors.46, 47 Increased 
angioedema during combined ACE and NEP inhibition (vasopeptidase inhibition),33 presumably 
due to decreased degradation of bradykinin and substance P through two pathways,48 led to 
development of the ARB/NEPi. Studies in isolated resistance vessels from patients with coronary 
artery disease (CAD) and preserved left ventricular function suggest that NEP inhibition alone (or 
with ACE inhibition) potentiates vasodilator effects of bradykinin, however.19 NEP inhibition 
could potentiate bradykinin either by preventing its degradation or by enhancing effects at the B2 
receptor.49 Increased bradykinin can lead to decreased afterload, increased natriuresis, and 
enhanced fibrinolysis.27, 28, 50, 51 
 
Bradykinin could also contribute to angioedema in patients taking an ARB/NEPi. We have 
reported the association of a polymorphism in the gene encoding NEP (MME) with ACEi-
associated angioedema.37 In addition to increasing vascular permeability directly, bradykinin 
induces the release of the NEP substrate substance P from nerve terminals.52 Both bradykinin and 
substance P have been implicated in the pathogenesis of angioedema.53, 54 In the PARADIGM-HF 


                 6                                                                           2/11/19 
trial the incidence of angioedema was 0.45% during combined ARB/NEPi versus 0.23% during 
ACEi (p=0.13).2 While the incidence of angioedema was statistically comparable, the numerical 
differences raise concerns described below. 
 
This proposal tests the overarching hypothesis that bradykinin contributes to effects of 
combined ARB/NEP inhibition compared to ARB alone. Specifically, we will: 
 
Specific Aim 1: Test the hypothesis that ARB/NEP inhibition potentiates the effects of 
exogenous bradykinin, substance P, and BNP on forearm blood flow or endothelial t-PA 
release compared to ACE inhibition or ARB. We will compare LCZ696 to bioequivalent doses 
of valsartan to focus on the mechanistic effects of NEP inhibition in this aim. 
 
3.0      Animal Studies and Previous Human Studies 
 
Preliminary Studies 
 
Our group has experience studying the effect of peptidase inhibitors on responses to 
vasoactive peptides such as bradykinin, substance P, and BNP in the human forearm 
vasculature. For instance, we and others have shown that bradykinin causes vasodilation and 
stimulates t-PA release from the forearm vasculature,55 through the bradykinin B2 receptor,56 that 
risk factors for CAD are associated with diminished bradykinin- and substance P-stimulated 
endothelial t-PA release,57-59 and that forearm vascular t-PA release correlates with coronary t-PA 
release and predicts future coronary events.60, 61 We have also found that 1) blocking the B2 
receptor attenuates the BP response to ACE inhibition in patients with hypertension,27  2) that ACE 
inhibition increases endothelial t-PA release via the B2 receptor,28 3) that bradykinin contributes 
to hypotension following protamine in patients undergoing cardiopulmonary bypass,62 and 4) that 
bradykinin contributes to inflammation during hemodialysis.63 Relevant to the actions of LCZ696, 
bradykinin does not contribute to the BP response to valsartan alone.64   
 
More recently, we have studied the effects of the DPP4i sitagliptin on responses to vasoactive 
peptides. We have reported that there is no effect of DPP4 inhibition on vasodilator responses to 
bradykinin or substance P (Figure 3), whereas DPP4 inhibition reduces substance P-stimulated 
endothelial t-PA release and increases norepinephrine release (from 212.5±60.7 pg/mL during 
placebo to 331.9±228.5 pg/mL during combined inhibition, p=0.02).30 (Data are provided as mean 
± SD unless indicated.) We found no effect of DPP4 inhibition alone on the vasodilator response 
to intra-arterial BNP.21 
 
 


                 7                                                                           2/11/19 
 
 
 
 
 
 
 
 
 
 
 
4.0      Inclusion/Exclusion Criteria 
 
Inclusion criteria 
1) Patients with essential hypertension defined as having 
a) untreated, seated systolic BP (SBP) of 130 mmHg or greater on three separate 
occasions, or 
b) untreated, seated diastolic BP (DBP) of 80 or greater on three separate 
occasions, or 
c) taken anti-hypertensive agent(s) for a minimum of six months. 
 
2) For female subjects, the following conditions must be met: 
a) postmenopausal status for at least one year, or 
b) status post-surgical sterilization, or 
c) if of childbearing potential, utilization of adequate birth control and willingness 
to undergo urine beta-hCG testing prior to drug treatment and on every study 
day. 
 
3) Age 18 to 60 years of age 
 
 
Figure 3: Effect of angiotensin-converting enzyme inhibition (Enalaprilat , enal) and dipeptidyl peptidase 4 
inhibition (sitagliptin 200 mg) on forearm blood flow (FBF) and tissue-plasminogen activator (t-PA) release in 
response to exogenous intra-arterial bradykinin and substance P.30 *P<0.05 versus placebo, †P<0.05 versus 
sitagliptin alone. Data are mean ± SEM. 
0
50
75
100
25
0
2
4
6
8
10
12
14
16
18
20
*†
*†
*†
*
*
*
bradykinin (pmol/min)
FBF (mL/min/100mL)
0
25
50
75
100
0
25
50
75
100
125
150
175
200
*†
*†
*
*
*
bradykinin (pmol/min)
t-PA release (ng/min/100mL)
0
2
4
6
8
10
0
2
4
6
8
10
12
14
16
18
20
substance P (pmol/min)
FBF (mL/min/100mL)
0
2
4
6
8
10
0
20
40
60
80
100
120
*
†
†
substance  P (pmol/min)
t-PA release (ng/min/100mL)
Placebo
Placebo+Enal
Sitagliptin
Sitagliptin+Enal
ACEi withdrawan
chronic ACEi
M100240 2.5 mg/d
Placebo
Valsartan 160 mg/d
Ramipril 5 mg/d
0
50
100
150
200
250
bradykinin (fmol/mL)
ACEi withdrawn
chronic ACEi
M100240 2.5 mg/d
0
20
40
60
80
100
120
BK1-5 (fmol/mL)
Figure 4: Bradykinin and BK1-5 concentrations measured 
prior to cardiac surgery in patients whose chronic ACE 
inhibitor was stopped 48 hours prior to surgery (ACEi 
withdrawn, N=12) or continued (chronic ACEi, N=19),62  as 
well as in healthy volunteers treated daily with an ACE/NEPi 
for a week (M10024, N=8), and dialysis patients treated daily 
with placebo, valsartan, or ramipril for one week (N=13).69 
Mean ± SEM 


                 8                                                                           2/11/19 
Exclusion criteria 
1) Presence of secondary form of hypertension 
2) Symptomatic hypertension and/or SBP>170 mmHg or DBP>110 mmHg, relevant to 
the washout period 
3) History of hypersensitivity or allergy to any of the study drugs, drugs of similar 
chemical classes, ACEi, ARBs, or NEPi, as well as known or suspected 
contraindications to the study drugs 
4) History of angioedema 
5) History of pancreatitis or known pancreatic lesions 
6) History of significant cardiovascular disease (other than essential hypertension and LV 
hypertrophy) 
7) Symptomatic hypotension and/or a SBP<100 mmHg at screening or <95 mmHg 
during the study 
8) Serum potassium >5.2 mmol/L at screening or >5.4 mmol/L during the study 
9) Individuals using oral contraceptives and smokers in order to reduce the risk of 
thrombosis following arterial line placement 
10) History of serious neurologic disease such as cerebral hemorrhage, stroke, seizure, or 
transient ischemic attack within six months 
11) Presence of significant pulmonary disorders 
12) Type 1 diabetes 
13) Poorly controlled type 2 diabetes mellitus (T2DM), defined as a HgbA1c >9% 
14) Hematocrit <35%  
15) Impaired renal function (eGFR of <30mL/min/1.73 m2) as determined by the four-
variable Modification of Diet in Renal Disease (MDRD) equation, where serum 
creatinine (Scr) is expressed in mg/dL and age in years: eGFR (mL/min/1.73m2)=175 
• Scr-1.154 • age-0.203 • (1.212 if Black) • (0.742 if female)  
16) Use of hormone-replacement therapy 
17) Breast feeding and pregnancy 
18) History or presence of immunological or hematological disorders 
19) History of malignancy other than non-melanoma skin cancer 
20) Diagnosis of asthma requiring use of inhaled beta agonist more than once a week 
21) Clinically significant gastrointestinal impairment that could interfere with drug 
absorption 
22) Impaired hepatic function [aspartate amino transaminase (AST) and/or alanine amino 
transaminase (ALT) >3.0 x upper limit of normal range] 
23) Any underlying or acute disease requiring regular medication which could possibly 
pose a threat to the subject or make implementation of the protocol or interpretation of 
the study results difficult, such as arthritis treated with non-steroidal anti-inflammatory 
drugs 
24) Treatment with chronic systemic glucocorticoid therapy within the last year 
25) Treatment with lithium salts 
26) History of alcohol or drug abuse 
27) Treatment with any investigational drug in the one month preceding the study 
28) Mental conditions rendering the subject unable to understand the nature, scope, and 
possible consequences of the study 


                 9                                                                           2/11/19 
29) Inability to comply with the protocol, e.g., uncooperative attitude, inability to return 
for follow-up visits, and unlikelihood of completing the study 
 
5.0      Enrollment/Randomization 
 
Written advertisements which have been approved by the Vanderbilt Institutional Review 
Board (IRB) and which give the name and phone number of a contact research nurse will also be 
placed on Vanderbilt clinic bulletin boards. A description of the study will also be placed on the 
Vanderbilt patient portal. Potential participants who call for information will be given a brief 
description of the study protocol and, if interested, will be invited to the Vanderbilt CRC for 
more information. In addition, the research nurse will make himself available to talk with 
patients at the Vanderbilt Heart and Vascular Institute and at other clinics where a high number 
of potential subjects have been identified using Subject Locator. During meetings with potential 
subjects, the research nurse or investigator will describe the study protocol in detail. Interested 
participants will be invited to read and sign an IRB-approved consent form and will be given a 
copy of the consent form to take with them. 
 
Assignment to treatment will be randomized and blinded to the investigators and subjects. 
Dr. Yu, study biostatistician, will provide an allocation schedule. The Vanderbilt Investigational 
Drug Service will be responsible for the storage, preparation, and labeling of all investigational 
agents and for maintaining accurate drug storage and dispensing logs. The Clinical Research 
Pharmacist will devise standard operating procedures for the pharmacy to follow with regard to 
preparing, labeling, blinding, and dispensing study drug.  
 
After subjects have been consented and screened, the investigator or research nurse will fax a 
copy of the consent form and a prescription containing check boxes for the inclusion and 
exclusion criteria to the Vanderbilt Investigational Drug Service. The pharmacist will confirm 
that consent was obtained and the subject met entry criteria, assign the subject a randomization 
number, and will provide the investigator with labeled study drug as indicated in the protocol. 
An extra label containing the randomization number will be attached to each study drug. The 
investigator will affix this extra label to the subject’s records. The Investigational Drug Service 
will retain a secure set of sealed envelopes containing the treatment assignment. These will be 
opened in the event of a clinical scenario which necessitates unblinding, as determined by the PI 
and the Data and Safety Monitoring Committee. Subjects randomized who do not complete the 
whole protocol for any reason will be replaced. 
 
6.0      Study Procedures 
 
Specific Aim 1: Test the hypothesis that ARB/NEP inhibition potentiates the effects of 
exogenous bradykinin, substance P, and BNP on forearm blood flow or endothelial t-PA 
release compared to ARB. 
 
Rationale 
NEP efficiently catalyzes the degradation of bradykinin and substance P (Kcat/Km 69 and 
158.7 min-1 µM-1, respectively).24 In small resistance arteries, NEP inhibition potentiates the 
vasodilator response to bradykinin.19 NEP hydrolyzes substance P in human vascular smooth 


                 10                                                                           2/11/19 
muscle cells.26 NEP inhibition has variable effects on the vasodilator response to exogenous 
natriuretic peptides in prior studies,12, 13, 19 but LCZ696 increases the ratio of BNP to NT-proBNP 
in HF patients.9, 10 Potentiation of bradykinin-, substance P-, or BNP-mediated vasodilation could 
contribute to beneficial effects of LCZ696 on BP and afterload reduction. Potentiation of t-PA 
release could reduce thrombotic events in patients taking LCZ696. Conversely potentiation of 
bradykinin or substance P could contribute to angioedema.66 
 
Thirty-five percent of the patients with HF and reduced EF in PARADIGM-HF had known 
diabetes, and another 13% had undiagnosed diabetes.2, 67 DPP4 inhibitors are often used to treat 
diabetes. Substance P and BNP are substrates common to NEP and DPP4 (Figure 2).22, 36 For 
this reason, we will also test the novel hypothesis that the LCZ696 and the DPP4i sitagliptin 
interact to potentiate effects of substance P and/or BNP. 
 
Subjects   
 
We will study patients with essential hypertension. Thirty-four non-smoking hypertensive 
subjects, age 18 to 60 years of age (50% Black, 50% female) will participate in this randomized, 
double-blind, placebo-controlled, crossover study. Subjects will be defined as hypertensive if 
they have an untreated, seated systolic blood pressure (SBP) of 140 mmHg or greater on three 
separate occasions; have an untreated, seated diastolic blood pressure (DBP) of 90 mmHg or 
greater on three separate occasions; or have taken anti-hypertensive agent(s) for a minimum of 
six months. Subjects with secondary forms of hypertension, significant cardiovascular disease 
(other than essential hypertension and LV hypertrophy), pulmonary and neurological disorders, 
diabetes, anemia, and renal insufficiency will be excluded. Pregnancy will be excluded in women 
of child-bearing potential by urine beta-hCG, and women will be studied during the follicular 
phase of the menstrual cycle.  
 
 
 
Protocol 
Figure 5 illustrates the study protocol. After informed consent is obtained, subjects will 
undergo a screening history and physical exam, and anti-hypertensive medications will be 
withdrawn. If appropriate, as in the case of beta-blockers and clonidine or other medications for 
which there may be rebound hypertension if they are discontinued suddenly, medications will be 
tapered. During this period, BP will be measured every one to three days. If at any time the 
seated SBP is >170 mmHg, or seated DBP >110 mmHg, or if a subject develops symptoms of 
high BP regardless of BP, the subject will be discontinued, and his or her anti-hypertensives 
restarted. In our experience, about three percent of subjects may be excluded during washout. 
Excluded subjects will be replaced. 
 


                 11                                                                           2/11/19 
 
 
 
 
 
After subjects have been off anti-hypertensive medications for three weeks (four for 
spironolactone), they will be randomized to four-week treatment with valsartan 160 mg bid (80 
mg bid for one week, then 160 mg bid) or LCZ696 200 bid (100 mg bid for one week, then 200 
mg bid) in a double-blind fashion. Valsartan bioavailability is enhanced when administered as 
LCZ696, and these doses of valsartan and LCZ696 give equivalent serum concentrations of 
valsartan.68 The doses of LCZ696 is that used in PARADIGM. On the morning of the 28th day 
of study drug, subjects will report to the Vanderbilt Clinical Research Center (CRC) after an 
overnight fast. Subjects will be studied in the supine position in a temperature-controlled room. 
They will be instrumented for intra-arterial infusions, as described below. Subjects will be given 
their last dose of study drug. One hour after drug administration, we will measure forearm blood 
flow (FBF) and give bradykinin, substance P, or BNP. The order of bradykinin and substance P 
will be randomized. BNP will be given third because it has a half-life of 18 minutes (versus 
seconds for the other peptides) and requires a longer washout period to avoid carryover effects.21 
Each peptide will be infused in three graded doses for five minutes. After administration of all 
three peptides, subjects will be allowed to rest for an hour. Then they will be given a single oral 
dose of sitagliptin 200 mg and be allowed to rest for 90 minutes. This dose of sitagliptin achieves 
complete inhibition of DPP4 comparable to 100 mg but with an earlier (one hour) and more 
sustained maximal response.69 We will repeat baseline measurements and the peptide infusions 
with an intervening rest period. The four-week study treatment and protocol will be repeated 
after a three-week washout, until subjects complete both arms. 
 
Anticipated Results 
We do not expect an effect of valsartan alone on the responses to bradykinin (or substance P 
or BNP) based on prior studies.64 We anticipate that LCZ696 will increase vasodilator and t-PA 
responses to bradykinin and may increase vasodilator and t-PA responses to substance P, as well 
as the vasodilator response to BNP compared to during valsartan. We will compare the effect of 
valsartan and LCZ696 on the responses to exogenous bradykinin, substance P, and BNP. We 
hypothesize that effects of LCZ696 on responses to BNP or substance P will be enhanced by 
concurrent inhibition of DPP4.  In the case of substance P, there may also be an effect on 
0
1
2
3
4
5
6
7
8
Instrumentation
Study Drug (LCZ696
or valsartan)
rest periods
rest
Sitagliptin
time (hours)
forearm blood flow
followed by a-v sampling
graded infusions bradykinin,
substance P and BNP
0
5
10
15 min
4.8
4.9
5.0
5.1
5.2
5.3
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
LCZ696 200 mg bid
or
Valsartan 160 mg bid
LCZ696 200 mg bid
or
Valsartan 160 mg bid
washout
4 weeks
4 weeks
3 weeks
randomization
Study Days
Figure 5: Study Protocol. Valsartan 160 mg and LCZ696 200 mg provide similar valsartan concentrations 
due to increased bioavailability of valsartan when administered as LCZ696.64 The LCZ696 dose is that used 
in PARADIGM-HF. 


                 12                                                                           2/11/19 
norepinephrine release, as we observed in an earlier study of combined ACE and DPP4 
inhibition.30 
 
Sample Size Calculation and Statistical Analysis 
 
The primary endpoints are FBF and net release of t-PA. Net norepinephrine release is a 
secondary endpoint. The primary analyses will focus on the LCZ696 versus valsartan 
comparisons on the primary and secondary endpoints. The secondary analysis will focus on the 
comparison of LCZ696 during sitagliptin versus LCZ696 alone on the primary and secondary 
endpoints. 
 
Sample Size and Power Calculation:  
In our study of the effect of ACE inhibition on responses to bradykinin, enalaprilat increased the 
FBF response to a 94.3 pmol/min dose of bradykinin from 10.57±3.24 (mean±SD) to 16.32±6.34 
mL/min/100 mL, with a SD of the difference of 4.07 mL/min/100 mL and a within-subject 
correlation of 0.85. Substance P increased FBF from 2.29±0.51 to 14.79±4.86 mL/min/100 mL 
at the highest dose proposed, and the within-subject correlation was 0.76. BNP increased FBF 
from 2.54±0.84 mL/min/100 mL at baseline to 6.15±1.95 mL/min/100 mL at the 720 pmol/min 
dose, and the within-subject correlation was 0.71. With 16 subjects in this crossover study we 
would have excellent (>90%) power to detect an increase in FBF similar in magnitude during 
LCZ696 versus valsartan or LCZ696/sitagliptin versus LCZ696 alone (Table 2). We will study 
32 subjects so we will have adequate power to detect this magnitude of difference within either 
racial or gender group alone. We also have power to stratify women on menopause status. If half 
of the 16 women are pre-menopausal, we would have >90% power to detect the difference 
observed with bradykinin. In our most recent study, enalaprilat increased the t-PA response to 
bradykinin (94.3 pmol/min) from 21.8±14.0 to 114.3±110.9 ng/mL/100mL, and the within-
subject correlation was 0.80. An N of 13 would be required to provide 85% power to detect this 
magnitude of change between LCZ696 and valsartan. 
Table 2: Expected FBF results.  For substance P and BNP, we conservatively projected LCZ696 
would increase the FBF response 30 percent.  
Infusion  
valsartan 
LCZ696, increase of the magnitude of enalapril 
 
 
 
Mean ± SD 
Correlation 
Increase±SD 
N for 85% 
power 
Power for 
N=16 
Bradykinin  
10.57±3.24 
16.32±6.34 
0.85 
5.75±3.97 
7 
>99% 
Substance P 
14.79±4.86 
19.23±6.32 
0.76 
4.44±4.46 
11 
96 
BNP  
6.15±1.95 
8.00±2.53 
0.71 
1.85±1.79 
11 
97 
 
 
Data Analysis Plan: 
We will use standard graphing and screening techniques to detect outliers and to ensure data 
accuracy. We will  use non-parametric analysis methods due to the relatively small sample size. 
We will provide summary statistics for both numerical and categorical variables by study arm. 
We will assess comparability among randomization groups.  
 


                 13                                                                           2/11/19 
We are using a two-by-two crossover design in this study. Although we have designed the 
study with a prolonged three-week washout to avoid carryover of any ARB or NEPi effect, we 
will take additional baseline measurements of key study endpoints right before study subjects 
take study medication (valsartan or LCZ696) in the two treatment periods. These data will allow 
us to estimate directly any residual carryover effect using within-subject comparisons or the 
mixed effects models. In the mixed-effects models with a random subject effect, we will code the 
appropriate first-order carryover effect. For example, if a subject receives treatments valsartan 
and LCZ696 in the two periods, then the model has carryover effect null, CV,  in the two 
periods. (CV denotes potential carryover effect due to valsartan in the prior period.) Since we are 
going to use the baseline measures for this evaluation, the main effect of the treatments will not 
be in the model.  If they are not significant, the carryover effects will not be included in the 
model when we estimate the main treatment effects in the following. We will use an 
autoregressive model of order 1 (AR1) or other plausible covariance structures for the error 
covariance. We will use a similar approach to evaluate for potential carryover of bradykinin, 
substance P, or BNP effects. 
 
We will use mixed-effects models with a random subject effect and with treatment factor one 
(valsartan or LCZ696) and treatment factor two (absence or presence of sitagliptin) as fixed 
effects. We will evaluate treatment effects and interactions using properly set up contrasts in the 
mixed-effects models. We will include sex and race as covariates and will conduct stratified 
analyses if appropriate. (We and others have observed racial differences in vasodilator responses 
to bradykinin.70, 71 ACE inhibition increases t-PA response to bradykinin to a greater extent in 
women than in men.72) In addition to evaluating treatment effects and interactions using 
regression analyses, we will calculate within-subject mean differences and 95% confidence 
intervals for specific effects of interest and test for treatment effects using  signed rank test. We 
will test all hypotheses at the level of α=0.05. We will use SPSS (version 23.0, SPSS, Chicago, 
IL) and the open-source statistical package R (version 3.1.0, R Core Team, 2014) for analyses. 
 
Subjects who drop out will be replaced. For this reason and based on prior experience, 
missing data will be unusual. Nevertheless, if data are missing for a particular time point, mixed-
effects models are robust in that subjects with missing data at some time points can be included 
to estimate effects of interest. In addition, we will conservatively impute missing data to perform 
corroborative analyses with and without missing data. 
 
Limitations and Future Directions 
We will study people with hypertension rather than HF in this aim because the study is 
invasive and to permit safe washout of vasoactive drugs. Because LCZ696 increases urine cGMP 
similarly in patients with hypertension and with HF,10, 73 we expect findings will be applicable 
across groups.  
 
We are studying effects of four-week treatment with LCZ696 and valsartan. Prior studies 
indicate that hemodynamic effects of valsartan and LCZ696 are at steady state by four weeks.73 
  
We have designed the study to avoid carryover effects. We know from many other studies 
that three weeks provides adequate washout for the effects of ARBs and ACEis,74, 75 and studies 
using LCZ696 suggest that this duration is adequate for NEP inhibition.2 As noted in Data 


                 14                                                                           2/11/19 
Analysis Plan, we will test for carryover and also use repeated baseline measurements to allow 
us to estimate any residual carryover effect.  
 
Bradykinin, substance P, and BNP are just three substrates of NEP that may have 
cardiovascular effects. Adrenomedullin is another candidate NEP substrate that causes 
vasodilation and natriuresis.76 NEP inhibition enhances the vasodilator response to intravenous 
adrenomedullin in sheep;77 omapatrilat (but not thiorphan) potentiates the vasodilator response to 
adrenomedullin in resistance arteries from patients with CAD.19 If we do not see an effect of 
NEP inhibition or NEP inhibition + DPP4 inhibition on the response to any one of the peptides, 
we will consider substituting adrenomedullin as one of the peptides. Likewise, it may be 
necessary to complete studies using ANP or CNP. 
 
7.0      Risks of Investigational Agents/Devices (side effects) 
1) Frequent blood draws can lead to anemia. 
2) Withholding anti-hypertensive medications may result in elevation in BP. We will 
monitor BP every one to three days after stopping or decreasing these medications. If 
the BP is too high or if there are symptoms of high BP during the washout period, the 
study will be stopped and prior medications will be restarted. 
3) LCZ696 may cause angioedema, leading to swelling of the tongue, lips, or pharynx 
that can result in airway compromise. DPP4i use has also been associated with 
angioedema and can increase the risk of ACEi-associated angioedema.78, 79 We will 
exclude patients with a history of angioedema, and the duration of LCZ696 is short in 
each study. Subjects in Aim 1 will receive sitagliptin during LCZ696 only one time. 
We have not observed angioedema in studies of short-term sitagliptin and ACEi 
exposure. 
4) Valsartan or LCZ696 can cause hypotension or orthostasis. We are giving the first 
dose in the CRC to monitor BP. We will start at a lower dose and escalate after one 
week. We will monitor BP after the first dose and throughout the study.  
5) Valsartan or LCZ696 could cause hyperkalemia in patients with renal dysfunction. 
We are excluding patients with an eGFR less than 30 mL/min/1.73 m2. We will 
monitor potassium during the study. If potassium is >5.4 mmol/L, we will repeat the 
blood sample. If potassium is persistently >5.4 mmol/L we will withdraw the subject 
from the study. 
6) Insertion of brachial artery and venous catheters may cause bleeding, bruising, or 
infection. Insertion of a brachial artery catheter may cause damage to the artery or 
thrombosis. This is potentially serious and could result in loss of blood supply to the 
arm, requiring intra-arterial administration of fibrinolytic agents and/or surgery. 
Complications arising from arterial line insertion are usually seen in the setting of low 
flow and prolonged arterial line placement, neither of which occur in these protocols. 
We have conducted studies involving the insertion of brachial artery catheters safely 
in over 400 subjects.  
7) We will use lidocaine to numb the skin prior to the insertion of the brachial artery 
catheter. Lidocaine can cause numbness and burning and/or local rash or irritation in 
subjects who are allergic to it.   
8) Bradykinin (U-1080, Clinalfa, Bachem AG), substance P (U-1180, Clinalfa, Bachem 
AG, IND 110,721), and BNP are vasoactive substances that will be infused into the 


                 15                                                                           2/11/19 
brachial artery at doses that have no detectable effect on systemic BP or heart rate. 
Given at higher doses bradykinin, substance P, and BNP can lower BP and cause a 
compensatory tachycardia. We have not observed an effect on BP at the doses 
proposed in prior studies.  
9) DPP4 inhibitors can cause hypoglycemia. The risk of fasting hypoglycemia is lower 
than with non-incretin-based anti-diabetic agents due to the mechanism of action. We 
have not observed hypoglycemia in our prior studies using the dose of sitagliptin 
proposed in this study. 
 
8.0  Reporting of Adverse Events or Unanticipated Problems involving Risk to Participants 
or Others 
 
All protocols will be reviewed and approved by the Vanderbilt IRB before any subject is 
enrolled. The Principal Investigator will closely oversee the protocol in conjunction with the 
dedicated research nurse.  
 
A DSMC will provide objective review of treatment results as they relate to human safety 
and data quality. The committee will be comprised of Marie Griffin, MD, Professor of Health 
Policy; Ashish S. Shah, MD, Professor of Cardiac Surgery and Surgical Director of Vanderbilt 
Heart Transplant and Mechanical Circulatory Support; and Alan B. Storrow, MD, Professor and 
Vice Chairman for Research and Academic Affairs for the Department of Emergency Medicine. 
Dr. Storrow will chair the committee. An important aspect of the membership of the DSMC is 
that all three members are senior faculty who hold a primary appointment outside the 
Department of Medicine.  
 
The DSMC will meet at least three times, after the first one-third of the subjects have been 
enrolled, to receive reports of the progress of the study. These reports will provide information 
regarding recruiting, safety reporting, and data quality. No early stopping is planned. The 
committee will assess safety data including hypotension or hypertension, angioedema, 
hyperkalemia, common adverse events, hospitalizations, and other serious adverse events. 
Interim data will be provided to the committee by Drs. Brown and Yu. The randomization will 
be blinded and presented on a coded basis unless the Committee votes to receive unblinded data. 
The Committee will have the authority to modify the protocols or to terminate the study if it 
deems such actions to be warranted. The DSMC will provide summary reports to the 
investigators that will be submitted to the IRB. 
 
The DSMC will also receive quarterly reports of enrollment, protocol adherence, data 
quality, and adverse events via e-mail. The DSMC will review all serious adverse events. Any 
serious adverse event will be reported to the DSMC, IRB, and NIH (and FDA if appropriate) as 
soon as possible, but not more than 7 business days from the PI's notification of the event. Any 
untoward medical event will be classified as an adverse event, regardless of its causal 
relationship with the study. An adverse event will be classified as serious if it a) results in death, 
b) is life-threatening, c) requires inpatient hospitalization or prolongation of existing 
hospitalization, d) results in persistent or significant disability or incapacity, e) is a congenital 
anomaly or birth defect. The DSMC may choose to become unblinded; however, it is expected 


                 16                                                                           2/11/19 
that such unblinding would not occur without reasonable concern related either to patient safety 
or to data validity.   
 
Non-serious, unexpected adverse events and incidences of noncompliance with the protocol 
will be reported to the IRB at the time of annual review. If warranted, appropriate changes will 
be made to the consent form. Adverse events will be graded as mild (no limitation of usual 
activities), moderate (some limitation), or severe (inability to carry out usual activities) and 
attributed according to the relationship to the study drug and/or procedures as Not related, 
Unlikely, Possible, Probable, or Definite. Any instance of non-compliance with the protocol will 
be reported at the time of annual review. Summary reports will be submitted to the IRB at least 
annually and will contain a) the number of adverse events and an explanation of how each event 
was handled, b) the number of complaints and how each complaint was handled, c) the number 
of subject withdrawals and an explanation of why the subject withdrew or was withdrawn, and d) 
the number of instances of non-compliance with the protocol and how each was handled. 
 
9.0      Study Withdrawal/Discontinuation 
 
If at any time during the study, a subject develops any symptoms related to study participation 
that subject will be withdrawn from the study. If, in the opinion of the investigator, a subject is 
non-compliant, that subject will be withdrawn from the study. Subjects who are withdrawn will 
be followed until symptoms have resolved.  
 
10.0 
Statistical Considerations 
 
See above under protocol. 
 
11.0 
Privacy/Confidentiality Issues 
 
A unique identification case number will be used to protect the confidentiality of the study 
participants. Only case numbers will be included in spreadsheets used for the statistical analysis.   
 
We will use the web-based Vanderbilt Research Electronic Data Capture (REDCap) system to 
design electronic data collection forms in all Aims. These forms will be pilot tested before use. 
Data will be input into a protected, web-based case report form (which can be readily 
downloaded into SAS, STATA, R, or SPSS). The form allows for direct data entry by 
investigators and is designed to minimize errors and erroneous values. Results from the 
Vanderbilt clinical laboratory can also be directly imported to REDCap, which further reduces 
typographical data entry errors. Expected ranges are pre-specified to prevent errors such as the 
shifting of decimal points. The program includes a computerized audit trail so that the identity of 
individuals entering or changing data and, in the case of changes, both original and revised data 
are saved. Data are backed up daily. Clinical data, including clinical laboratory, will be entered 
by the research nurse. Research laboratory data will be entered by a predoctoral student in the 
laboratory. 
 
12.0 
Follow-up and Record Retention 
 


                 17                                                                           2/11/19 
All records will be retained for 7 years following publication of the data. After that time, records 
may be archived for an additional 5 years and then shredded. 
 
13.0 
Standard Techniques 
 
BP Measurements: During screening, washout, and active treatment outpatient BP, HR, and MAP 
will be measured with an aneroid sphygmomanometer (Welch Allyn, Skaneateles Falls, NY), 
using the appearance and complete disappearance of the Korotokoff sounds (K1 and K5) as SBP 
and DBP. The mean of three supine measurements will be used. During study days, BP will be 
measured before and using an automated oscillometric recording device (Dinamap, Critikon, 
Carlsbad, CA). 
 
Instrumentation for intra-arterial infusions: An intravenous catheter will be placed in the 
antecubital vein of the nondominant arm.  After subdermal 1% lidocaine, an 18-gauge 
polyurethane catheter (Cook Inc., Bloomington, IN) will be inserted in the brachial artery of the 
same arm. Prior to the infusion of vasoactive peptides, arterial catheter patency will be maintained 
by infusion of 5% dextrose at rate of 1 mL/minute.  After study drug, a one-hour rest period, and 
measurement of basal FBF and blood sampling, peptides will be infused. We will infuse 
bradykinin (U-1080, Clinalfa, Bachem AG) at 23.6, 47.2, and 94.3 pmol/min. The highest dose 
increases FBF to 10.57±3.24 mL/min/100 mL forearm volume in the absence of ACE inhibition. 
We will give substance P (U-1180, Clinalfa, Bachem AG), at 2, 4, and 8 pmol/min to achieve 
similar vasodilation (Figure 3). We will give BNP (Nesiritide, Scios Inc., Mountain View, CA) at 
240, 480, and 720 pmol/min. These doses cause vasodilation comparable to the lower doses of the 
other agonists.21 Each dose will be infused for five minutes, and FBF will be measured during the 
last two minutes.  Drug concentrations in the infusate will be adjusted to maintain infusion volumes 
at one mL/minute. FBF will be measured using mercury-in-silastic strain-gauge plethysmography, 
as detailed elsewhere.28 
 
Forearm blood flow measurements: FBF will be measured using mercury-in-silastic strain-gauge 
plethysmography. The wrist will be supported to raise the forearm above the level of the atrium, 
and a strain gauge placed on the widest part of the forearm. The strain gauge will be connected to 
a plethysmograph (Model EC-4, D.E. Hokanson, Issaquah, WA), calibrated to measure percent 
change in volume and connected to a chart recorder to record flow measurements. For each 
measurement, a cuff around the upper arm will be inflated to 40 mmHg with a rapid cuff inflator 
(Model E-10, Hokanson) to occlude venous outflow from the extremity. The hand will be occluded 
from measurement of FBF by inflation of a pediatric sphygmomanometer cuff to 200 mmHg 
around the wrist. Flow measurements will be recorded for approximately 7 sec out of 15 sec, and 
the slope will be derived from the first 3-4 pulses; 7 readings will be obtained for each mean. 
 
Blood Sampling and Calculations: Following measurement of FBF, simultaneous arterial and 
venous samples will be obtained from the infused arm prior to and at the end of each dose of study 
drug for measurement of peptide concentrations, cGMP, t-PA (bradykinin and substance P), and 
norepinephrine.  We will also measure venous NEP activity and DPP4 activity at baseline. 
Infusions will be interrupted during arterial sampling. All samples will be obtained after the first 
three mL of blood are discarded. Blood samples will be collected on ice and centrifuged 
immediately. Arterio-venous concentration gradients will be calculated by subtracting the plasma 
level measured in simultaneously collected venous (CV) and arterial (CA) blood.  Forearm plasma 
flow (FPF) will be calculated from the FBF and arterial hematocrit corrected for 1% trapped 
plasma.  Thus, individual net release or uptake rates at each time point will be calculated: Net 
release = (CV-CA) x [FBF x ((101-hematocrit)/100)]. 
 


                 18                                                                           2/11/19 
 
References 
 
 
1. 
Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, de Ferranti S, 
Despres JP, Fullerton HJ, Howard VJ, Huffman MD, Judd SE, Kissela BM, Lackland DT, 
Lichtman JH, Lisabeth LD, Liu S, Mackey RH, Matchar DB, McGuire DK, Mohler ER, 3rd, 
Moy CS, Muntner P, Mussolino ME, Nasir K, Neumar RW, Nichol G, Palaniappan L, Pandey 
DK, Reeves MJ, Rodriguez CJ, Sorlie PD, Stein J, Towfighi A, Turan TN, Virani SS, Willey JZ, 
Woo D, Yeh RW and Turner MB. Heart disease and stroke statistics--2015 update: a report from 
the American Heart Association. Circulation. 2015;131:e29-322. 
 
2. 
McMurray JJ, Packer M, Desai AS, Gong J, Lefkowitz MP, Rizkala AR, Rouleau JL, Shi 
VC, Solomon SD, Swedberg K and Zile MR. Angiotensin-neprilysin inhibition versus enalapril 
in heart failure. The New England journal of medicine. 2014;371:993-1004. 
 
3. 
Claggett B, Packer M, McMurray JJ, Swedberg K, Rouleau J, Zile MR, Jhund P, 
Lefkowitz M, Shi V and Solomon SD. Estimating the Long-Term Treatment Benefits of 
Sacubitril-Valsartan. The New England journal of medicine. 2015;373:2289-90. 
 
4. 
Hubers SA and Brown NJ. Combined Angiotensin Receptor Antagonism and Neprilysin 
Inhibition. Circulation. 2016;133:1115-24. 
 
5. 
Jessup M. Neprilysin inhibition--a novel therapy for heart failure. The New England 
journal of medicine. 2014;371:1062-4. 
 
6. 
Schmitt M, Gunaruwan P, Payne N, Taylor J, Lee L, Broadley AJ, Nightingale AK, 
Cockcroft JR, Struthers AD, Tyberg JV and Frenneaux MP. Effects of exogenous and 
endogenous natriuretic peptides on forearm vascular function in chronic heart failure. 
Arteriosclerosis, thrombosis, and vascular biology. 2004;24:911-7. 
 
7. 
Forfia PR, Lee M, Tunin RS, Mahmud M, Champion HC and Kass DA. Acute 
phosphodiesterase 5 inhibition mimics hemodynamic effects of B-type natriuretic peptide and 
potentiates B-type natriuretic peptide effects in failing but not normal canine heart. Journal of 
the American College of Cardiology. 2007;49:1079-88. 
 
8. 
Chen HH. Heart failure: a state of brain natriuretic peptide deficiency or resistance or 
both! Journal of the American College of Cardiology. 2007;49:1089-91. 
 
9. 
Solomon SD, Zile M, Pieske B, Voors A, Shah A, Kraigher-Krainer E, Shi V, Bransford 
T, Takeuchi M, Gong J, Lefkowitz M, Packer M and McMurray JJ. The angiotensin receptor 
neprilysin inhibitor LCZ696 in heart failure with preserved ejection fraction: a phase 2 double-
blind randomised controlled trial. Lancet (London, England). 2012;380:1387-95. 
 
10. 
Packer M, McMurray JJ, Desai AS, Gong J, Lefkowitz MP, Rizkala AR, Rouleau JL, Shi 
VC, Solomon SD, Swedberg K, Zile M, Andersen K, Arango JL, Arnold JM, Belohlavek J, 


                 19                                                                           2/11/19 
Bohm M, Boytsov S, Burgess LJ, Cabrera W, Calvo C, Chen CH, Dukat A, Duarte YC, Erglis A, 
Fu M, Gomez E, Gonzalez-Medina A, Hagege AA, Huang J, Katova T, Kiatchoosakun S, Kim 
KS, Kozan O, Llamas EB, Martinez F, Merkely B, Mendoza I, Mosterd A, Negrusz-Kawecka M, 
Peuhkurinen K, Ramires FJ, Refsgaard J, Rosenthal A, Senni M, Sibulo AS, Jr., Silva-Cardoso J, 
Squire IB, Starling RC, Teerlink JR, Vanhaecke J, Vinereanu D and Wong RC. Angiotensin 
receptor neprilysin inhibition compared with enalapril on the risk of clinical progression in 
surviving patients with heart failure. Circulation. 2015;131:54-61. 
 
11. 
Miller WL, Phelps MA, Wood CM, Schellenberger U, Van Le A, Perichon R and Jaffe 
AS. Comparison of mass spectrometry and clinical assay measurements of circulating fragments 
of B-type natriuretic peptide in patients with chronic heart failure. Circulation Heart failure. 
2011;4:355-60. 
 
12. 
Cruden NL, Fox KA, Ludlam CA, Johnston NR and Newby DE. Neutral endopeptidase 
inhibition augments vascular actions of bradykinin in patients treated with angiotensin-
converting enzyme inhibition. Hypertension. 2004;44:913-8. 
 
13. 
Lainchbury JG, Richards AM, Nicholls MG, Espiner EA and Yandle TG. Brain 
natriuretic peptide and neutral endopeptidase inhibition in left ventricular impairment. The 
Journal of clinical endocrinology and metabolism. 1999;84:723-9. 
 
14. 
Barletta G, Lazzeri C, Vecchiarino S, Del Bene R, Messeri G, Dello Sbarba A, Mannelli 
M and La Villa G. Low-dose C-type natriuretic peptide does not affect cardiac and renal function 
in humans. Hypertension. 1998;31:802-8. 
 
15. 
Pham I, Sediame S, Maistre G, Roudot-Thoraval F, Chabrier PE, Carayon A and Adnot 
S. Renal and vascular effects of C-type and atrial natriuretic peptides in humans. The American 
journal of physiology. 1997;273:R1457-64. 
 
16. 
Nakamura M, Arakawa N, Yoshida H, Makita S and Hiramori K. Vasodilatory effects of 
C-type natriuretic peptide on forearm resistance vessels are distinct from those of atrial 
natriuretic peptide in chronic heart failure. Circulation. 1994;90:1210-4. 
 
17. 
Hunt PJ, Richards AM, Espiner EA, Nicholls MG and Yandle TG. Bioactivity and 
metabolism of C-type natriuretic peptide in normal man. The Journal of clinical endocrinology 
and metabolism. 1994;78:1428-35. 
 
18. 
Ichiki T, Huntley BK, Sangaralingham SJ and Burnett JC, Jr. Pro-Atrial Natriuretic 
Peptide: A Novel Guanylyl Cyclase-A Receptor Activator That Goes Beyond Atrial and B-Type 
Natriuretic Peptides. JACC Heart failure. 2015;3:715-23. 
 
19. 
Dalzell JR, Seed A, Berry C, Whelan CJ, Petrie MC, Padmanabhan N, Clarke A, 
Biggerstaff F, Hillier C and McMurray JJ. Effects of neutral endopeptidase (neprilysin) 
inhibition on the response to other vasoactive peptides in small human resistance arteries: studies 
with thiorphan and omapatrilat. Cardiovascular therapeutics. 2014;32:13-8. 
 


                 20                                                                           2/11/19 
20. 
Honing ML, Smits P, Morrison PJ, Burnett JC, Jr. and Rabelink TJ. C-type natriuretic 
peptide-induced vasodilation is dependent on hyperpolarization in human forearm resistance 
vessels. Hypertension. 2001;37:1179-83. 
 
21. 
Devin JK, Pretorius M, Nian H, Yu C, Billings FTt and Brown NJ. Dipeptidyl-peptidase 
4 inhibition and the vascular effects of glucagon-like peptide-1 and brain natriuretic peptide in 
the human forearm. Journal of the American Heart Association. 2014;3. 
 
22. 
Brandt I, Lambeir AM, Ketelslegers JM, Vanderheyden M, Scharpe S and De Meester I. 
Dipeptidyl-peptidase IV converts intact B-type natriuretic peptide into its des-SerPro form. 
Clinical chemistry. 2006;52:82-7. 
 
23. 
Rawlings ND, Barrett AJ and Bateman A. Using the MEROPS Database for Proteolytic 
Enzymes and Their Inhibitors and Substrates. Current protocols in bioinformatics. 
2014;48:1.25.1-33. 
 
24. 
Matsas R, Kenny AJ and Turner AJ. The metabolism of neuropeptides. The hydrolysis of 
peptides, including enkephalins, tachykinins and their analogues, by endopeptidase-24.11. The 
Biochemical journal. 1984;223:433-40. 
 
25. 
Watanabe Y, Nakajima K, Shimamori Y and Fujimoto Y. Comparison of the hydrolysis 
of the three types of natriuretic peptides by human kidney neutral endopeptidase 24.11. 
Biochemical and molecular medicine. 1997;61:47-51. 
 
26. 
Wang L, Sadoun E, Stephens RE and Ward PE. Metabolism of substance P and 
neurokinin A by human vascular endothelium and smooth muscle. Peptides. 1994;15:497-503. 
 
27. 
Gainer JV, Morrow JD, Loveland A, King DJ and Brown NJ. Effect of bradykinin-
receptor blockade on the response to angiotensin-converting-enzyme inhibitor in normotensive 
and hypertensive subjects. The New England journal of medicine. 1998;339:1285-92. 
 
28. 
Pretorius M, Rosenbaum D, Vaughan DE and Brown NJ. Angiotensin-converting 
enzyme inhibition increases human vascular tissue-type plasminogen activator release through 
endogenous bradykinin. Circulation. 2003;107:579-85. 
 
29. 
Witherow FN, Helmy A, Webb DJ, Fox KA and Newby DE. Bradykinin contributes to 
the vasodilator effects of chronic angiotensin-converting enzyme inhibition in patients with heart 
failure. Circulation. 2001;104:2177-81. 
 
30. 
Devin JK, Pretorius M, Nian H, Yu C, Billings FTt and Brown NJ. Substance P increases 
sympathetic activity during combined angiotensin-converting enzyme and dipeptidyl peptidase-4 
inhibition. Hypertension. 2014;63:951-7. 
 
31. 
Newby DE, Sciberras DG, Ferro CJ, Gertz BJ, Sommerville D, Majumdar A, Lowry RC 
and Webb DJ. Substance P-induced vasodilatation is mediated by the neurokinin type 1 receptor 


                 21                                                                           2/11/19 
but does not contribute to basal vascular tone in man. British journal of clinical pharmacology. 
1999;48:336-44. 
 
32. 
Latini R, Masson S, Anand I, Salio M, Hester A, Judd D, Barlera S, Maggioni AP, 
Tognoni G and Cohn JN. The comparative prognostic value of plasma neurohormones at 
baseline in patients with heart failure enrolled in Val-HeFT. European heart journal. 
2004;25:292-9. 
 
33. 
Kostis JB, Packer M, Black HR, Schmieder R, Henry D and Levy E. Omapatrilat and 
enalapril in patients with hypertension: the Omapatrilat Cardiovascular Treatment vs. Enalapril 
(OCTAVE) trial. American journal of hypertension. 2004;17:103-11. 
 
34. 
Byrd JB, Adam A and Brown NJ. Angiotensin-converting enzyme inhibitor-associated 
angioedema. Immunology and allergy clinics of North America. 2006;26:725-37. 
 
35. 
Ward PE, Chow A and Drapeau G. Metabolism of bradykinin agonists and antagonists by 
plasma aminopeptidase P. Biochemical pharmacology. 1991;42:721-7. 
 
36. 
Wang LH, Ahmad S, Benter IF, Chow A, Mizutani S and Ward PE. Differential 
processing of substance P and neurokinin A by plasma dipeptidyl(amino)peptidase IV, 
aminopeptidase M and angiotensin converting enzyme. Peptides. 1991;12:1357-64. 
 
37. 
Pare G, Kubo M, Byrd JB, McCarty CA, Woodard-Grice A, Teo KK, Anand SS, Zuvich 
RL, Bradford Y, Ross S, Nakamura Y, Ritchie M and Brown NJ. Genetic variants associated 
with angiotensin-converting enzyme inhibitor-associated angioedema. Pharmacogenetics and 
genomics. 2013;23:470-8. 
 
38. 
Brown NJ, Ray WA, Snowden M and Griffin MR. Black Americans have an increased 
rate of angiotensin converting enzyme inhibitor-associated angioedema. Clinical pharmacology 
and therapeutics. 1996;60:8-13. 
 
39. 
Miller DR, Oliveria SA, Berlowitz DR, Fincke BG, Stang P and Lillienfeld DE. 
Angioedema incidence in US veterans initiating angiotensin-converting enzyme inhibitors. 
Hypertension. 2008;51:1624-30. 
 
40. 
Effects of enalapril on mortality in severe congestive heart failure. Results of the 
Cooperative North Scandinavian Enalapril Survival Study (CONSENSUS). The CONSENSUS 
Trial Study Group. The New England journal of medicine. 1987;316:1429-35. 
 
41. 
Effect of enalapril on survival in patients with reduced left ventricular ejection fractions 
and congestive heart failure. The SOLVD Investigators. The New England journal of medicine. 
1991;325:293-302. 
 
42. 
Cohn JN and Tognoni G. A randomized trial of the angiotensin-receptor blocker 
valsartan in chronic heart failure. The New England journal of medicine. 2001;345:1667-75. 


                 22                                                                           2/11/19 
43. 
Effect of metoprolol CR/XL in chronic heart failure: Metoprolol CR/XL Randomised 
Intervention Trial in Congestive Heart Failure (MERIT-HF). Lancet (London, England). 
1999;353:2001-7. 
 
44. 
Pitt B, Zannad F, Remme WJ, Cody R, Castaigne A, Perez A, Palensky J and Wittes J. 
The effect of spironolactone on morbidity and mortality in patients with severe heart failure. 
Randomized Aldactone Evaluation Study Investigators. The New England journal of medicine. 
1999;341:709-17. 
 
45. 
Feng L, Karpinski PH, Sutton P, Liu Y, Hook DF, Hu B, Blacklock TJ, Fanwick PE, 
Prashad M, Godtfredsen S and Ziltener C. LCZ696: a dual-acting sodium supramolecular 
complex. Tetrahedron Letters. 2012;53:275-276. 
 
46. 
Richards AM, Wittert GA, Espiner EA, Yandle TG, Ikram H and Frampton C. Effect of 
inhibition of endopeptidase 24.11 on responses to angiotensin II in human volunteers. 
Circulation research. 1992;71:1501-7. 
 
47. 
Richards AM, Wittert GA, Crozier IG, Espiner EA, Yandle TG, Ikram H and Frampton 
C. Chronic inhibition of endopeptidase 24.11 in essential hypertension: evidence for enhanced 
atrial natriuretic peptide and angiotensin II. Journal of hypertension. 1993;11:407-16. 
 
48. 
Lu B, Figini M, Emanueli C, Geppetti P, Grady EF, Gerard NP, Ansell J, Payan DG, 
Gerard C and Bunnett N. The control of microvascular permeability and blood pressure by 
neutral endopeptidase. Nature medicine. 1997;3:904-7. 
 
49. 
Deddish PA, Marcic BM, Tan F, Jackman HL, Chen Z and Erdos EG. Neprilysin 
inhibitors potentiate effects of bradykinin on b2 receptor. Hypertension. 2002;39:619-23. 
 
50. 
Witherow FN, Dawson P, Ludlam CA, Fox KA and Newby DE. Marked bradykinin-
induced tissue plasminogen activator release in patients with heart failure maintained on long-
term angiotensin-converting enzyme inhibitor therapy. Journal of the American College of 
Cardiology. 2002;40:961-6. 
 
51. 
Cruden NL, Witherow FN, Webb DJ, Fox KA and Newby DE. Bradykinin contributes to 
the systemic hemodynamic effects of chronic angiotensin-converting enzyme inhibition in 
patients with heart failure. Arteriosclerosis, thrombosis, and vascular biology. 2004;24:1043-8. 
 
52. 
Seyedi N, Maruyama R and Levi R. Bradykinin activates a cross-signaling pathway 
between sensory and adrenergic nerve endings in the heart: a novel mechanism of ischemic 
norepinephrine release? The Journal of pharmacology and experimental therapeutics. 
1999;290:656-63. 
 
53. 
Emanueli C, Grady EF, Madeddu P, Figini M, Bunnett NW, Parisi D, Regoli D and 
Geppetti P. Acute ACE inhibition causes plasma extravasation in mice that is mediated by 
bradykinin and substance P. Hypertension. 1998;31:1299-304. 
 


                 23                                                                           2/11/19 
54. 
Sulpizio AC, Pullen MA, Edwards RM and Brooks DP. The effect of acute angiotensin-
converting enzyme and neutral endopeptidase 24.11 inhibition on plasma extravasation in the rat. 
The Journal of pharmacology and experimental therapeutics. 2004;309:1141-7. 
 
55. 
Brown NJ, Gainer JV, Stein CM and Vaughan DE. Bradykinin stimulates tissue 
plasminogen activator release in human vasculature. Hypertension. 1999;33:1431-5. 
 
56. 
Brown NJ, Gainer JV, Murphey LJ and Vaughan DE. Bradykinin stimulates tissue 
plasminogen activator release from human forearm vasculature through B(2) receptor-dependent, 
NO synthase-independent, and cyclooxygenase-independent pathway. Circulation. 
2000;102:2190-6. 
 
57. 
Pretorius M, Rosenbaum DA, Lefebvre J, Vaughan DE and Brown NJ. Smoking impairs 
bradykinin-stimulated t-PA release. Hypertension. 2002;39:767-71. 
 
58. 
Newby DE, Wright RA, Labinjoh C, Ludlam CA, Fox KA, Boon NA and Webb DJ. 
Endothelial dysfunction, impaired endogenous fibrinolysis, and cigarette smoking: a mechanism 
for arterial thrombosis and myocardial infarction. Circulation. 1999;99:1411-5. 
 
59. 
Ridderstrale W, Ulfhammer E, Jern S and Hrafnkelsdottir T. Impaired capacity for 
stimulated fibrinolysis in primary hypertension is restored by antihypertensive therapy. 
Hypertension. 2006;47:686-91. 
 
60. 
Minai K, Matsumoto T, Horie H, Ohira N, Takashima H, Yokohama H and Kinoshita M. 
Bradykinin stimulates the release of tissue plasminogen activator in human coronary circulation: 
effects of angiotensin-converting enzyme inhibitors. Journal of the American College of 
Cardiology. 2001;37:1565-70. 
 
61. 
Robinson SD, Ludlam CA, Boon NA and Newby DE. Endothelial fibrinolytic capacity 
predicts future adverse cardiovascular events in patients with coronary heart disease. 
Arteriosclerosis, thrombosis, and vascular biology. 2007;27:1651-6. 
 
62. 
Pretorius M, Scholl FG, McFarlane JA, Murphey LJ and Brown NJ. A pilot study 
indicating that bradykinin B2 receptor antagonism attenuates protamine-related hypotension after 
cardiopulmonary bypass. Clinical pharmacology and therapeutics. 2005;78:477-85. 
 
63. 
Marney AM, Ma J, Luther JM, Ikizler TA and Brown NJ. Endogenous bradykinin 
contributes to increased plasminogen activator inhibitor 1 antigen following hemodialysis. 
Journal of the American Society of Nephrology : JASN. 2009;20:2246-52. 
 
64. 
LeFebvre J, Shintani A, Gebretsadik T, Petro JR, Murphey LJ and Brown NJ. Bradykinin 
B(2) receptor does not contribute to blood pressure lowering during AT(1) receptor blockade. 
The Journal of pharmacology and experimental therapeutics. 2007;320:1261-7. 
 


                 24                                                                           2/11/19 
65. 
Pretorius M, McFarlane JA, Vaughan DE, Brown NJ and Murphey LJ. Angiotensin-
converting enzyme inhibition and smoking potentiate the kinin response to cardiopulmonary 
bypass. Clinical pharmacology and therapeutics. 2004;76:379-87. 
 
66. 
Bas M, Greve J, Strassen U, Khosravani F, Hoffmann TK and Kojda G. Angioedema 
induced by cardiovascular drugs: new players join old friends. Allergy. 2015;70:1196-200. 
 
67. 
Kristensen SL, Preiss D, Jhund PS, Squire I, Cardoso JS, Merkely B, Martinez F, Starling 
RC, Desai AS, Lefkowitz MP, Rizkala AR, Rouleau JL, Shi VC, Solomon SD, Swedberg K, Zile 
MR, McMurray JJ and Packer M. Risk Related to Pre-Diabetes Mellitus and Diabetes Mellitus in 
Heart Failure With Reduced Ejection Fraction: Insights From Prospective Comparison of ARNI 
With ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure Trial. 
Circulation Heart failure. 2016;9. 
 
68. 
Gu J, Noe A, Chandra P, Al-Fayoumi S, Ligueros-Saylan M, Sarangapani R, Maahs S, 
Ksander G, Rigel DF, Jeng AY, Lin TH, Zheng W and Dole WP. Pharmacokinetics and 
pharmacodynamics of LCZ696, a novel dual-acting angiotensin receptor-neprilysin inhibitor 
(ARNi). Journal of clinical pharmacology. 2010;50:401-14. 
 
69. 
Bergman AJ, Stevens C, Zhou Y, Yi B, Laethem M, De Smet M, Snyder K, Hilliard D, 
Tanaka W, Zeng W, Tanen M, Wang AQ, Chen L, Winchell G, Davies MJ, Ramael S, Wagner 
JA and Herman GA. Pharmacokinetic and pharmacodynamic properties of multiple oral doses of 
sitagliptin, a dipeptidyl peptidase-IV inhibitor: a double-blind, randomized, placebo-controlled 
study in healthy male volunteers. Clinical therapeutics. 2006;28:55-72. 
 
70. 
Pretorius MM, Gainer JV, Van Guilder GP, Coelho EB, Luther JM, Fong P, Rosenbaum 
DD, Malave HA, Yu C, Ritchie MD, Vaughan DE and Brown NJ. The bradykinin type 2 
receptor BE1 polymorphism and ethnicity influence systolic blood pressure and vascular 
resistance. Clinical pharmacology and therapeutics. 2008;83:122-9. 
 
71. 
Ozkor MA, Rahman AM, Murrow JR, Kavtaradze N, Lin J, Manatunga A, Hayek S and 
Quyyumi AA. Differences in vascular nitric oxide and endothelium-derived hyperpolarizing 
factor bioavailability in blacks and whites. Arteriosclerosis, thrombosis, and vascular biology. 
2014;34:1320-7. 
 
72. 
Pretorius M, Luther JM, Murphey LJ, Vaughan DE and Brown NJ. Angiotensin-
converting enzyme inhibition increases basal vascular tissue plasminogen activator release in 
women but not in men. Arteriosclerosis, thrombosis, and vascular biology. 2005;25:2435-40. 
 
73. 
Ruilope LM, Dukat A, Bohm M, Lacourciere Y, Gong J and Lefkowitz MP. Blood-
pressure reduction with LCZ696, a novel dual-acting inhibitor of the angiotensin II receptor and 
neprilysin: a randomised, double-blind, placebo-controlled, active comparator study. Lancet 
(London, England). 2010;375:1255-66. 
 


                 25                                                                           2/11/19 
74. 
Brown NJ, Agirbasli M and Vaughan DE. Comparative effect of angiotensin-converting 
enzyme inhibition and angiotensin II type 1 receptor antagonism on plasma fibrinolytic balance 
in humans. Hypertension. 1999;34:285-90. 
 
75. 
Gamboa JL, Pretorius M, Todd-Tzanetos DR, Luther JM, Yu C, Ikizler TA and Brown 
NJ. Comparative effects of angiotensin-converting enzyme inhibition and angiotensin-receptor 
blockade on inflammation during hemodialysis. Journal of the American Society of Nephrology : 
JASN. 2012;23:334-42. 
 
76. 
Oya H, Nagaya N, Furuichi S, Nishikimi T, Ueno K, Nakanishi N, Yamagishi M, 
Kangawa K and Miyatake K. Comparison of intravenous adrenomedullin with atrial natriuretic 
peptide in patients with congestive heart failure. The American journal of cardiology. 
2000;86:94-8. 
 
77. 
Lisy O, Jougasaki M, Schirger JA, Chen HH, Barclay PT and Burnett JC, Jr. Neutral 
endopeptidase inhibition potentiates the natriuretic actions of adrenomedullin. The American 
journal of physiology. 1998;275:F410-4. 
 
78. 
Brown NJ, Byiers S, Carr D, Maldonado M and Warner BA. Dipeptidyl peptidase-IV 
inhibitor use associated with increased risk of ACE inhibitor-associated angioedema. 
Hypertension. 2009;54:516-23. 
 
79. 
Scirica BM, Bhatt DL, Braunwald E, Steg PG, Davidson J, Hirshberg B, Ohman P, 
Frederich R, Wiviott SD, Hoffman EB, Cavender MA, Udell JA, Desai NR, Mosenzon O, 
McGuire DK, Ray KK, Leiter LA and Raz I. Saxagliptin and cardiovascular outcomes in patients 
with type 2 diabetes mellitus. The New England journal of medicine. 2013;369:1317-26.