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J O U R N A L O F T H E AM E R I C A N C O L L E G E O F C A R D I O L O G Y V O L . 7 8 , N O . 1 0 , 2 0 2 1
ª 2 0 2 1 T H E A U T HO R S . P U B L I S H E D B Y E L S E V I E R O N B E H A L F O F T H E A M E R I C A N
C O L L E G E O F C A R D I O L O G Y F OU N D A T I O N . T H I S I S A N O P E N A C C E S S A R T I C L E U N D E R
T H E C C B Y - N C - N D L I C E N S E ( h t t p : / / c r e a t i v e c o mm o n s . o r g / l i c e n s e s / b y - n c - n d / 4 . 0 / ) .ORIGINAL INVESTIGATIONSMetoprolol in Critically Ill Patients
With COVID-19
Agustín Clemente-Moragón, BSC,a,* Juan Martínez-Milla, MD, PHD,a,b,* Eduardo Oliver, PHD,a,c
Arnoldo Santos, MD, PHD,d,e Javier Flandes, MD, PHD,f Iker Fernández, MD,f Lorena Rodríguez-González, TECH,g,h
Cristina Serrano del Castillo, MD,i Ana-María Ioan, MD,d María López-Álvarez, RN,b,c Sandra Gómez-Talavera, MD,a,b,c
Carlos Galán-Arriola, DVM, PHD,a,c Valentín Fuster, MD, PHD,a,j César Pérez-Calvo, MD, PHD,d
Borja Ibáñez, MD, PHDa,b,cABSTRACTISS
Fro
Jim
IIS
fDe
Fu
Ma
va
Mi
ChBACKGROUND Severe coronavirus disease-2019 (COVID-19) can progress to an acute respiratory distress syndrome
(ARDS), which involves alveolar infiltration by activated neutrophils. The beta-blocker metoprolol has been shown to
ameliorate exacerbated inflammation in the myocardial infarction setting.
OBJECTIVES The purpose of this study was to evaluate the effects of metoprolol on alveolar inflammation and on
respiratory function in patients with COVID-19–associated ARDS.
METHODS A total of 20 COVID-19 patients with ARDS on invasive mechanical ventilation were randomized to meto-
prolol (15 mg daily for 3 days) or control (no treatment). All patients underwent bronchoalveolar lavage (BAL) before and
after metoprolol/control. The safety of metoprolol administration was evaluated by invasive hemodynamic and elec-
trocardiogram monitoring and echocardiography.
RESULTS Metoprolol administration was without side effects. At baseline, neutrophil content in BAL did not differ
between groups. Conversely, patients randomized to metoprolol had significantly fewer neutrophils in BAL on day 4
(median: 14.3 neutrophils/ml [Q1, Q3: 4.63, 265 neutrophils/ml] vs median: 397 neutrophils/ml [Q1, Q3: 222, 1,346 neu-
trophils/ml] in the metoprolol and control groups, respectively; P ¼ 0.016). Metoprolol also reduced neutrophil extra-
cellular traps content and other markers of lung inflammation. Oxygenation (PaO2:FiO2) significantly improved after
3 days of metoprolol treatment (median: 130 [Q1, Q3: 110, 162] vs median: 267 [Q1, Q3: 199, 298] at baseline and day 4,
respectively; P ¼ 0.003), whereas it remained unchanged in control subjects. Metoprolol-treated patients spent fewer
days on invasive mechanical ventilation than those in the control group (15.5  7.6 vs 21.9  12.6 days; P ¼ 0.17).
CONCLUSIONS In this pilot trial, intravenous metoprolol administration to patients with COVID-19–associated
ARDS was safe, reduced exacerbated lung inflammation, and improved oxygenation. Repurposing metoprolol for
COVID-19–associated ARDS appears to be a safe and inexpensive strategy that can alleviate the burden of the COVID-19
pandemic. (J Am Coll Cardiol 2021;78:1001–1011) © 2021 The Authors. Published by Elsevier on behalf of the
American College of Cardiology Foundation. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).N 0735-1097 https://doi.org/10.1016/j.jacc.2021.07.003
m the aCentro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; bCardiology Department, IIS-Fundación
énez Díaz University Hospital, Madrid, Spain; cCIBER de Enfermedades Cardiovasculares, Madrid, Spain; dIntensive Care Unit,
-Fundación Jiménez Díaz University Hospital, Madrid, Spain; eCIBER de Enfermedades Respiratorias, Madrid, Spain;
partment of Pulmonary Medicine, IIS-Fundación Jiménez Díaz University Hospital, Madrid, Spain; gPathology Department, IIS-
ndación Jiménez Díaz University Hospital, Madrid, Spain; hBiobank Patform-PT20/00141, IIS-Fundación Jiménez Díaz Hospital,
drid, Spain; iFlow Citometry Laboratory, IIS-Fundación Jiménez Díaz University Hospital, Madrid, Spain; and the jCardio-
scular Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA. *Drs Clemente-Moragón and Martínez-
lla contributed equally to this work.
ristie Ballantyne, MD, served as Guest Editor-in-Chief for this paper.
ABBR EV I A T I ON S
AND ACRONYMS
ARDS = acute respiratory
distress syndrome
BAL = bronchoalveolar lavage
COVID-19 = coronavirus
disease-2019
ICU = intensive care unit
IMV = invasive mechanical
ventilation
NET = neutrophil extracellular
trap
The author
institutions
visit the Au
Manuscript
Clemente-Moragón et al. J A C C V O L . 7 8 , N O . 1 0 , 2 0 2 1
MADRID-COVID Pilot Trial S E P T E M B E R 7 , 2 0 2 1 : 1 0 0 1 – 1 0 1 1
1002C oronavirus disease-2019 (COVID-19),caused by severe acute respiratorysyndrome-coronavirus-2 (SARS-CoV-2)
infection, is an ongoing pandemic affecting
more than 145 million people worldwide
and responsible for more than 3 million
deaths to date. An estimated 6%-18% of
COVID-19 cases progress to an acute respira-
tory distress syndrome (ARDS) requiring
intensive care unit (ICU) admission and inva-
sive mechanical ventilation (IMV) (1). There
is currently a lack of specific therapies forCOVID-19–associated ARDS.SEE PAGE 1012In the early stages of SARS-CoV-2 infection, the
host immune system is activated to block disease
progression. However, in some cases rapid replication
of SARS-CoV-2 in the respiratory tract triggers an
exacerbated inflammatory response and a cytokine
storm (2). This situation leads to progression to ARDS
together with other clinical complications, such as
septic shock, microthrombi, coagulopathy, and mul-
tiple organ dysfunction (3).
ARDS of different etiologies (4), including SARS-
CoV-2 infection (5,6), is highly dependent on the ac-
tion of neutrophils. Activated neutrophils contribute
to alveolar injury by releasing prestored inflamma-
tory mediators (reactive oxygen species and myelo-
peroxidase [MPO]) and by interacting with other
cells, such as platelets, to induce microthrombi. In
addition, the formation of neutrophil extracellular
traps (NETs) and highly injurious histones activates
the inflammasome and triggers the release of pro-
inflammatory cytokines (7). NETs released from
alveolar-infiltrated activated neutrophils increase
pulmonary inflammation and serum levels of proin-
flammatory cytokines, leading to extensive lung
damage and microthrombotic events in COVID-19
patients (2,3,8,9).
Despite the massive worldwide impact of
COVID-19, there is a shortage of effective therapies to
prevent transition from moderate to severe disease
and to improve prognosis. Given the intense pressure
COVID-19 is placing on ICUs worldwide, there is an
urgent need to identify therapies to reduce the
number of days in the ICU. The most sought-after
interventions are those able to mitigate COVID-19–s attest they are in compliance with human studies committe
and Food and Drug Administration guidelines, including patien
thor Center.
received April 28, 2021; revised manuscript received June 17, 20associated immune dysregulation (10). An attractive
candidate approach is to use host-directed therapies,
which have emerged in recent years as an adjuvant
strategy to limit damage during infectious or sterile
exacerbated inflammation.
Beta-adrenergic receptor antagonists (b-blockers)
have been used for many decades to treat cardiovas-
cular conditions such as hypertension, arrhythmias,
and myocardial infarction (11). Observational retro-
spective studies have established a link between
b-blocker therapy and increased survival in critically
ill patients caused by different conditions, such as
sepsis (12-14), acute respiratory failure (15), severe
traumatic brain injury (16,17), and others (18,19).
Recent findings show that the b1-selective blocker
metoprolol has a direct effect on neutrophils, damp-
ening their deleterious effects during exacerbated
inflammation (20). In the context of ischemia/
reperfusion (acute myocardial infarction), metoprolol
targeting of neutrophils has been shown to have a
strong cardioprotective effect, both in animal models
and in patients (20-23). More recently, our group
demonstrated that metoprolol (but not other
clinically available intravenous b-blockers) abrogates
neutrophil-driven exacerbated inflammation,
neutrophil-platelet interaction, and NETs formation
in a mouse model of LPS-induced acute lung injury
(24). These experimental data prompted us to inves-
tigate whether treatment with intravenous (IV)
metoprolol could ameliorate lung inflammation—and
eventually improve prognosis—in patients with
COVID-19–associated ARDS.
METHODS
STUDY DESIGN AND POPULATION. The MADRID-
COVID (Intravenous Metoprolol in Respiratory
Distress Due to COVID-19) pilot trial was approved by
the Fundación Jiménez Díaz University Hospital
ethics committee (Eudract registry number 2020-
002310-41). All patients, or a close relative, gave
written consent to participate. Inclusion criteria were
age 18-80 years, rt-PCR–confirmed SARS-CoV-2
infection (in either nasal swab or bronchoalveolar
lavage), invasive mechanical ventilation #72 hours,
heart rate $60 beats/min, and invasive systolic blood
pressure $120 mm Hg. Exclusion criteria included
prolonged hospital admission (>5 days) beforees and animal welfare regulations of the authors’
t consent where appropriate. For more information,
21, accepted July 1, 2021.
J A C C V O L . 7 8 , N O . 1 0 , 2 0 2 1 Clemente-Moragón et al.
S E P T E M B E R 7 , 2 0 2 1 : 1 0 0 1 – 1 0 1 1 MADRID-COVID Pilot Trial
1003enrollment, concomitant acute heart failure, left
ventricular ejection fraction <50%, right ventricular
systolic dysfunction, concomitant pulmonary embo-
lism, moderate-severe peripheral artery disease,
moderate-severe valvular heart disease, moderate-
severe COPD, or active treatment with b-blockers
before enrollment. A total of 20 patients with ARDS
secondary to SARS-CoV-2 infection under IMV were
enrolled and randomized to IV metoprolol tartrate
(Recordati) (3  5 mg boluses, 2 minutes apart, daily
for 3 days; n ¼ 12) or control (no treatment; n ¼ 8).
Two minutes after each bolus, blood pressure and
heart rate were measured, and if they were above the
limits set, the next bolus was injected.
Randomization was stratified by age (#59 years vs
>59 years), history of hypertension (yes/no), and
circulating neutrophil counts (<6,000 vs $6,000).
Bronchoalveolar lavage (BAL) fluid and blood samples
were obtained from patients at randomization (base-
line) and 24 hours after the third metoprolol dose/
control (day 4). The main study goal was to assess the
effect of metoprolol on inflammatory markers (mainly
neutrophil infiltration and NETs). The main secondary
goals were to assess the effect ofmetoprolol on days on
invasive mechanical ventilation and days in the ICU
after randomization, as well as pulmonary function.
The main safety outcome measure was hemodynamic
complications (cardiogenic shock, severe hypoten-
sion, or severe bradycardia/atrioventricular block).
Because this was a pilot trial, sample size was
calculated based on the capacity of identifying
changes in lung inflammation (neutrophil infiltra-
tion). Based on previous experimental studies, we
speculated that 20 patients would be enough to
detect a significant biological effect of metoprolol in
this context.
FLOW CYTOMETRY OF BAL SAMPLES. For flow
cytometry (FCM) studies, BAL samples (8 mL) were
previously inactivated with 2 mL of a cellular antigen
stabilization reagent containing formaldehyde
(TransFix, Cytomark Ltd). Samples were then centri-
fuged (5 minutes at 540g), the supernatant discarded,
and the cell pellet resuspended in 200 mL phosphate-
buffered saline. Afterwards, 100 mL of cell suspension
was stained for 15 minutes at room temperature with
the following color combination: antihuman CD15-
fluorescein isothiocyanate, CD33-phycoerythrin, and
CD3-V-450 and CD45-V-500 (Becton Dickinson Bio-
sciences). After staining, 2 mL of FACS lysing solution
(Becton/Dickinson Biosciences) was added, and after
5 minutes incubation, the sample was centrifuged
and resuspended in 100 mL phosphate-buffered sa-
line. Before acquisition, the fluorescent dye DRAQ5(Biostatus Limited) (25,26) and Perfect-COUNT
microspheres (Cytognos SL) (27) were added for the
selection of DNA-positive cells and cell count,
respectively. Samples were run on a FACSCanto II
flow cytometer (Becton Dickinson Biosciences)
equipped with FACSDiva software (Becton Dickinson
Biosciences), and information was acquired about all
events corresponding to nucleated cells present in
the stained sample aliquot.
Data were analyzed with INFINICYT software
(Cytognos SL). FCM analysis included a first-step
identification of nucleated cells by DRAQ5 staining.
Leukocyte populations were identified with a gating
strategy based on forward scatter, side scatter, and
CD45 expression. Neutrophils and macrophages were
identified from their relatively higher light-scattering
properties, their unique pattern of CD45 expression,
and the expression of CD15 (neutrophils) and CD33
(alveolar macrophages). Lymphocytes were also
identified according to their CD45 expression and
forward and side scatter properties. Neutrophil,
macrophage, and lymphocyte populations were
quantified as the percentage of total CD45 events.
CHEMOKINE ELISA ASSAYS. Samples were inacti-
vated by incubation in a final concentration of 0.2%
SDS per 0.1% Tween-20 and heat treatment at
60C for 15 minutes. Plasma and cell-free BAL sam-
ples were analyzed with human ELISA kits for von
Willebrand factor (RAB0556-1KT, Sigma) and the
chemokines monocyte chemoattractant protein
(MCP)-1 (orb315028, Biorbyt), interleukin (IL)-6
(orb219452, Biorbyt), and IL-8 (orb315028, Biorbyt).
NETosis MARKERS. A total of 3 NETosis biomarkers
were measured: citrullinated histone-3 (Cit-H3),
MPO-DNA complexes, and cell-free DNA. For Cit-H3
and MPO-DNA ELISA, samples were first inactivated
by suspension in 0.2% SDS per 0.1% Tween-20 and
heat treatment at 60C for 15 minutes. For cell-free
DNA measurement, samples were inactivated by
heat treatment at 60C for 1 hour.
Cit-H3 was measured with an ELISA kit (clone 11D3,
Cayman, 501620). Quantification of MPO-DNA com-
plexes was based on a previously described protocol
(28,29) that uses several reagents from the Cell Death
Detection ELISA Kit (Roche, 11544675001) but in-
cludes a high-binding EIA/RIA 96-well plate differ-
ently coated overnight at 4C with antihuman MPO
antibody (Bio-Rad, 0400-0002). Cell-free DNA was
measured using the Quant-iT PicoGreen dsDNA Assay
Kit (Invitrogen, Thermo Fisher Scientific, P11496).
NEUTROPHIL AND NET VISUALIZATION IN BAL.NETs
were visualized by Giemsa staining of BAL samples
Clemente-Moragón et al. J A C C V O L . 7 8 , N O . 1 0 , 2 0 2 1
MADRID-COVID Pilot Trial S E P T E M B E R 7 , 2 0 2 1 : 1 0 0 1 – 1 0 1 1
1004(30,31). BAL samples were centrifuged for 10 minutes
at 2,500 revolutions/min. The pellet was resuspended
and spread for staining with Giemsa solution. Sam-
ples were then inactivated and fixed for 10 minutes at
room temperature with an alcohol-based spray fixa-
tive for cyto-diagnosis (M-Fix spray fixative). For
image analysis, fixed samples were digitalized with a
scanner (Nanozoomer-RS C110730, Hamamatsu) and
analyzed using NDP view image analysis soft-
ware (Hamamatsu).
STATISTICAL ANALYSIS. Data were analyzed with
Graphpad Prism version 8.4 and RStudio. Due to the
small sample size, all distributions were considered
non-normal, and nonparametric tests were applied
for statistical analyses. Paired comparisons between
pretreatment and post-treatment samples (basal and
4 days) were by Wilcoxon matched pairs signed rank
test. Comparisons between treatment conditions
(vehicle vs metoprolol) at baseline or after treatment
were made by unpaired Mann-Whitney U test. For
hemodynamics and functional parameters during
metoprolol administration, differences at baseline or
pre-post boluses were calculated by the nonpara-
metric chi-square Friedman test with correction by
the Durbin-Conover test for pairwise comparisons.
For categorical data, percentages were compared by
exact methods. Differences were deemed statistically
significant at P values below 0.05.
RESULTS
PATIENT CHARACTERISTICS. Between October 19,
2020, and January 19, 2021, a total of 20 patients were
enrolled; 12 were randomized to metoprolol and 8 to
control. There were no between-group differences in
baseline characteristics (Table 1). All patients were
treated during ICU admission with corticosteroids
(dexamethasone 6 mg daily), anticoagulants, mela-
tonin, and acetylcysteine. Before enrollment in the
trial, all patients (except 1 in the metoprolol group)
were treated with bolus and maintenance dose of
corticosteroids (methylprednisolone and/or dexa-
methasone) in the ward before admission to the ICU
without differences between groups.
Of the patients randomized to metoprolol, 11
received all scheduled IV doses (15 mg daily for
3 days). The remaining patient received 15 mg of
metoprolol on the first 2 days but not the third
because the heart rate was <50 beats/min caused by
intensified sedation (initiation of propofol). BAL was
conducted without complications in all patients
before and 24 hours after treatment. Clinicallaboratory analyses at baseline and after treatment
are presented in Supplemental Table 1.
CARDIOVASCULAR SAFETY OF INTRAVENOUS
METOPROLOL ADMINISTRATION TO ARDS PATIENTS
ON MECHANICAL VENTILATION. Administration of
IV b-blockers has largely been proven to be
safe except for patients with acute pump failure.
Given the cardiovascular effects of metoprolol,
patients were monitored invasively and by echocar-
diography before and on every day after metoprolol
injection/control. As expected, metoprolol signifi-
cantly reduced heart rate (P < 0.01) and invasively
measured systolic blood pressure (P < 0.05),
although both remained within the physiological
range (Supplemental Table 2). Echocardiography
showed no deterioration of cardiac function parame-
ters after metoprolol treatment (Supplemental
Table 3). Overall, metoprolol intravenous adminis-
tration was shown to be safe and without side effects
in severe COVID-19 patients with ARDS on IMV.
METOPROLOL ADMINISTRATION ATTENUATES
NEUTROPHIL-DRIVEN LUNG EXACERBATED
INFLAMMATION. To assess the ability of metoprolol
to ameliorate neutrophil-mediated exacerbated lung
inflammation, we analyzed leukocyte populations in
BAL samples by flow cytometry at baseline and on
day 4. At baseline, the metoprolol and control groups
showed no differences in BAL neutrophil content
(Supplemental Figure 1). In contrast, on day 4 (after
3 days of metoprolol/control treatment), neutrophil
content was significantly lower in BAL from patients
in the metoprolol group than in those randomized to
control (median: 14.3 neutrophils/ml [Q1, Q3: 4.63, 265
neutrophils/ml] vs median: 397 neutrophils/ml [Q1, Q3:
222, 1,346 neutrophils/ml]; P ¼ 0.016). Day 4 BAL from
metoprolol-treated patients also had lower total
inflammatory-cell content and lower monocyte/
macrophage content, whereas lymphocytes did not
differ between groups (Figure 1A). We further
explored the impact of metoprolol on MCP-1 in BAL,
because this chemokine has been shown to promote
pulmonary fibrosis in late-stage ARDS (32,33). MCP-1
in cell-free BAL was significantly attenuated after
3 days of metoprolol treatment (median: 298 pg/mL
[Q1, Q3: 236, 350 pg/mL] vs median: 203 pg/mL [Q1,
Q3: 175, 258 pg/mL] for baseline and day 4, respec-
tively; P ¼ 0.009), whereas it remained unchanged in
control patients (Figure 1B). Conversely, changes in
IL-8 and -6 in cell-free BAL did not differ between
treatment groups (Supplemental Figure 2).
Excessive neutrophil activation in the lungs is
associated with NET formation and the release of
TABLE 1 Patient Characteristics at Randomization
All Metoprolol Control P Value
Age, y 60 (53.8, 68) 60 (57.8, 68.5) 58.5 (43.3, 65.8) 0.354
Male 13 (65.0) 8 (66.7) 5 (62.5) 1.000
BMI, kg/m2 27.1 (25.3, 31.1) 26.8 (25.1, 30.4) 27.1 (26.2, 31.5) 0.422
Comorbidities
Hypertension 6 (30.0) 4 (33.3) 2 (25.0) 1.000
Diabetes 2 (10.0) 2 (16.7) 0 (0.0) 0.648
Smokers 3 (15.0) 1 (8.3) 2 (25.0) 0.701
Dyslipidemia 6 (30.0) 4 (33.3) 2 (25.0) 1.000
Previous treatment
RAS inhibitors 5 (25.0) 3 (25.0) 2 (25.0) 1.000
Anticoagulants 0 (0.0) 0 (0.0) 0 (0.0) 1.000
Values are median (Q1, Q3) or n (%).
BMI ¼ body mass index; RAS ¼ renin-angiotensin system.
J A C C V O L . 7 8 , N O . 1 0 , 2 0 2 1 Clemente-Moragón et al.
S E P T E M B E R 7 , 2 0 2 1 : 1 0 0 1 – 1 0 1 1 MADRID-COVID Pilot Trial
1005reactive oxygen species and proteolytic enzymes,
which can drive severe epithelial and endothelial
injury (2,34). To study whether the inflammation-
disrupting effect of metoprolol reduced the produc-
tion of these neutrophil activation byproducts,
we measured the NETosis markers Cit-H3 and
MPO-DNA complexes. Levels of both markers were
decreased in day 4 BAL from metoprolol-treated
patients (P ¼ 0.005 and P ¼ 0.086 vs baseline,
respectively), whereas no changes were observed in
BAL from control patients (Figure 1C). Lower NET
formation and inflammatory content in the meto-
prolol group was confirmed by Giemsa staining
(Figure 1D). We found no differences in cell-free DNA
content (Supplemental Figure 3), probably reflecting
its nonspecific nature as a NETosis biomarker (3).
To determine if attenuated immune-cell infiltra-
tion in the lungs was associated with a systemic
effect, we assessed changes in circulating levels of
chemokines known to be markedly elevated in severe
COVID-19 patients (34). The 3-day treatment with
metoprolol was associated with a significant reduc-
tion in the circulating concentrations of the pro-
inflammatory cytokine IL-8 (median: 94.4 pg/mL
[Q1, Q3: 72.1, 168 pg/mL] vs median: 80.1 pg/mL
[Q1, Q3: 69.5, 85.2 pg/mL] for baseline and day 4,
respectively; P ¼ 0.003), whereas no changes were
observed in controls (Supplemental Figure 4). Meto-
prolol had no significant effect on circulating levels
of IL-6 (Supplemental Figure 4) or NETosis markers
(Supplemental Figure 5).
METOPROLOL TREATMENT IMPROVES RESPIRATORY
FUNCTION. Oxygenation was measured as the ratio
between arterial oxygen partial pressure and frac-
tional inspired oxygen (PaO2:FiO2). Baseline and post-
treatment oxygenation parameters are shown inTable 2. At baseline, oxygenation was worse in pa-
tients randomized to metoprolol than in the control
group, despite higher FiO2. After the 3-day metoprolol
treatment, PaO2 significantly improved (median:
87.5 mm Hg [Q1, Q3: 78.8, 110 mm Hg] vs median:
108 mm Hg [Q1, Q3: 98.3, 139 mm Hg] for baseline and
day 4, respectively; P ¼ 0.017). Metoprolol treatment
also significantly improved PaO2:FiO2 (median: 130
[Q1, Q3: 110, 162] vs median: 267 [Q1, Q3: 199, 298] at
baseline and day 4, respectively; P ¼ 0.007).
Conversely, in control subjects, PaO2 and PaO2:FiO2
both deteriorated, although the change did not reach
statistical significance (P ¼ 0.107 and P ¼ 0.363 vs
baseline, respectively) (Figures 2A and 2B).
Patients randomized to metoprolol spent fewer
days on mechanical ventilation, although this differ-
ence did not reach statistical significance (15.5 
7.6 days vs 21.9  12.6 days in the metoprolol and
control groups, respectively; P ¼ 0.17). A similar trend
was observed for days of ICU admission after enroll-
ment (14.5  7.2 days vs 21.4  13.4 days in the
metoprolol and control groups, respectively; P ¼ 0.15)
(Figure 2C). All patients were discharged from the ICU,
and 1 patient in each group died before hospital
discharge.
DISCUSSION
The COVID-19 pandemic and associated ARDS is
placing an immense burden on health care systems.
In addition to high mortality, COVID-19–associated
ARDS results in prolonged ICU admission, contrib-
uting to morbidity among survivors and high hospital
expenditure. The current approach with these
patients is mainly based on protective IMV (35,36),
which ensures sufficient gas exchange while causing
minimal alveolar damage. With the exception of
FIGURE 1 Metoprolol Disrupts COVID-19–Associated Exacerbated Lung Inflammation
0
1,000
No
. o
f C
el
ls
/
l
2,000
3,000
4,000
CellsA
B
C
D
Neutrophils Macrophages
MCP-1 Control
Cit-H3 MPO-DNA
Metoprolol
Lymphocytes
0
Control Metoprolol
100
200
300
400
M
CP
-1
 (p
g/
m
l)
500
0
5
10
15
20
25
30
P = 0.086
Control Metoprolol
Ci
t-
H3
 (n
g/
m
l)
35
Control Metoprolol
0
5
10
15
20
25
30
M
PO-DNA
Com
plexes ( g/m
l)
35
Con
tro
l
Me
top
rolo
l
Con
tro
l
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top
rolo
l
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tro
l
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tro
l
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top
rolo
l
P = 0.05
P = 0.05
*
**
P = 0.15
**
Baseline Day-4
Baseline Day-4
(A) Day 4 inflammatory cell populations in BAL from control and metoprolol-treated severe COVID-19 patients. Dots represent individuals and bars and error bars show
mean values (boxes)  SD (error bars). *P < 0.05 by unpaired Student’s t-test. (B) Attenuation of MCP-1 in cell-free BAL from metoprolol-treated patients. (C)
Attenuation of neutrophil hyperactivation biomarkers (Cit-H3 and MPO-DNA complexes) in cell-free BAL from metoprolol-treated patients. Data are presented as
individuals’ (dots) paired data between days 1 and 4. **P< 0.01 by paired Student’s t-test. (D) Representative images of Giemsa-stained BAL samples from control and
metoprolol-treated patients at day 4. Scale bar, 50 mm. Control, n ¼ 8; metoprolol, n ¼ 12. BAL ¼ bronchoalveolar lavage; Cit-H3 ¼ citrullinated histone-3;
COVID-19 ¼ coronavirus disease-2019; MCP ¼ monocyte chemoattractant protein; MPO ¼ myeloperoxidase.
Clemente-Moragón et al. J A C C V O L . 7 8 , N O . 1 0 , 2 0 2 1
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1006
TABLE 2 Baseline and Post-Treatment Ventilation Parameters
Baseline Day 4
Metoprolol Control P Value Metoprolol Control P Value
PaO2, mm Hg 87.5 (78.8, 110.0) 104.0 (93.0, 122) 0.105 108.0 (98.3, 139.0) 83.5 (77.3, 92.5) 0.004
PaCO2, mm Hg 48.5 (43.8, 52.5) 47 (41.5, 48.8) 0.562 51.0 (46.5, 53.3) 47.0 (45.3, 50.5) 0.353
PEEP, cm H2O 12.0 (10.0, 12.5) 13.0 (10.0, 14.0) 0.625 10.0 (9.00, 12.0) 11.0 (10.0, 12.0) 0.666
FiO2 0.60 (0.5, 0.75) 0.48 (0.44, 0.60) 0.241 0.40 (0.39, 0.53) 0.43 (0.40, 0.57) 0.634
PaO2/FiO2, 130 (110, 162) 223 (188, 242) 0.076 267 (199, 298) 163 (145, 209) 0.037
Lactic acid, mmol/L 1.3 (1.2, 1.8) 1.2 (0.98, 2.00) 0.785 1.4 (1.20, 1.73) 1.9 (1.50, 2.05) 0.094
pH 7.41 (7.38, 7.42) 7.42 (7.37, 7.45) 0.485 7.43 (7.40, 7.46) 7.41 (7.38, 7.44) 0.461
Values are median (Q1, Q3). Bold indicates statistical significance.
FiO2 ¼ fraction of inspired oxygen; PaCO2 ¼ partial pressure of carbon dioxide; PaO2 ¼ partial pressure of oxygen; PEEP ¼ positive end-expiratory pressure.
J A C C V O L . 7 8 , N O . 1 0 , 2 0 2 1 Clemente-Moragón et al.
S E P T E M B E R 7 , 2 0 2 1 : 1 0 0 1 – 1 0 1 1 MADRID-COVID Pilot Trial
1007dexamethasone, which showed promising results in
an early trial (37), there are no therapies specifically
targeting exacerbated inflammation in ARDS (38).
In this study, we present the effects of 3-day
intravenous metoprolol administration on lung
inflammation in COVID-19 patients with ARDS. The
MADRID-COVID pilot trial shows the following: 1) IV
administration of the clinically approved b-blocker
metoprolol tartrate is safe in this clinical context; 2)
metoprolol treatment abrogates the exacerbated lung
inflammation associated with the disease; and 3) the
disruptive effect on exacerbated inflammation is
associated with better oxygenation and, conse-
quently, fewer days on IMV and in the ICU (Central
Illustration). These data suggest that metoprolol
repurposing for the treatment of ARDS in COVID-19
patients is a safe and inexpensive strategy with the
potential to improve outcomes.
The present study stems from our extensive
experience in the field of myocardial ischemia/
reperfusion injury. We previously demonstrated that
metoprolol protects the heart during ongoing
myocardial infarction by stunning neutrophils and
abrogating exacerbated inflammation (20,24). The
identification of this cardioprotective mechanism
created an opportunity to repurpose metoprolol for
other acute conditions in which exacerbated inflam-
mation plays a role, as is the case for COVID-19–
associated ARDS. The present study highlights the
importance of knowing the mechanism of action of
long-established drugs to identify other potential
indications.
Patients with severe COVID-19 present with bilat-
eral pneumonia that can lead to respiratory distress
requiring IMV. COVID-19–associated ARDS is charac-
terized by active neutrophil infiltration into the
alveolar space, which perpetuates exacerbated
inflammation, leading to a cytokine storm andhypoxemia (8,34). Neutrophil infiltration is thus a
major contributing factor to the poor prognosis of
these patients. Mitigation of immune dysregulation is
therefore a major therapeutic avenue for the treat-
ment and prevention of severe COVID-19.
Several studies have tested the potential benefits of
b-blockers in sepsis/septic shock. Retrospective
observational data have suggested that patients
admitted with septic shock and previously on main-
tenance b-blocker therapy have a better vital prog-
nosis than those who were not on b-blockers before
admission (13). In addition, small prospective clinical
trials have tested the benefits of IV b-blockers in sepsis
patients (12,39-41). The conclusion of most of these
trials is that b-blockers seem to offer a clinical benefit.
In an analysis of diverse experimental models of
exacerbated inflammation, we very recently showed
that not all b1-selective blockers exert the same ef-
fects on neutrophil biology. Of all tested b-blockers,
only metoprolol significantly attenuated exacerbated
inflammation and reduced neutrophil infiltration and
interaction with other cell types (24). Those results
position metoprolol as the b-blocker of choice in the
context of exacerbated inflammation.
The present study shows that 3-day treatment with
IV metoprolol reduces exacerbated inflammation in
critically ill COVID-19 patients with associated ARDS.
This was evidenced by the attenuation of infiltration
by immune cells, especially neutrophils, and reduced
levels of their related pro-inflammatory and NETosis
byproducts (Figure 1), which are potential drivers of
severe epithelial and endothelial injury. Lower
neutrophil infiltration in metoprolol-treated patients
was accompanied by a significant reduction in circu-
lating levels of the pro-inflammatory IL8, which ex-
erts chemotactic and activating functions on
neutrophils, suggesting a systemic anti-inflammatory
effect of this treatment (Supplemental Figure 4).
FIGURE 2 Metoprolol Rescues Pulmonary Function in ICU Patients With Severe COVID-19
A
C
PaO2
0
100
200
Control Metoprolol
METOPROLOL
CONTROL
Enrollment
P = 0.152
Intensive care unit admission
2.5 [1.8-4.0]
1.5 [0.0-2.3]
Data are expressed as days, median [25th-75th percentile]
3.2 [2.0-3.3]
2.5
[1.8-3.0]
11.5 [9.5-18.8]
17.0 [11.3-29.3]
pO
2 (
m
m
 H
g)
300
**
B PaO2:Fio2
0
Control Metoprolol
PA
FI
 In
de
x 
(p
O 2
:F
iO
2)
800
600
400
200
Baseline Day-4 Baseline Day-4
*
METOPROLOL
CONTROL
Invasive
mechanical
ventilation
P = 0.17512.0 [10.0-19.8]
18.5 [11.8-27.5]
Hospital admission (ward)
(A and B) Improved oxygenation (PaO2 and PaO2:FiO2) in patients receiving metoprolol, but not those in the control group. Data are presented as individuals’ (dots)
paired data between days 1 and 4. *P < 0.05, **P < 0.01 by paired Student’s t-test. (C) Days spent by severe COVID-19 patients on IMV and in the ICU according to
allocation to IV metoprolol or control (no treatment). Control, n ¼ 8; metoprolol, n ¼ 12. FiO2 ¼ fraction of inspired oxygen; ICU ¼ intensive care unit; IMV ¼ invasive
mechanical ventilation; PAFI ¼ PaO2:FiO2 ratio; other abbreviations as in Figure 1.
Clemente-Moragón et al. J A C C V O L . 7 8 , N O . 1 0 , 2 0 2 1
MADRID-COVID Pilot Trial S E P T E M B E R 7 , 2 0 2 1 : 1 0 0 1 – 1 0 1 1
1008Systemic markers of NETosis were unaffected at day
4; however, an effect over a longer time window after
metoprolol treatment cannot be discarded
(Supplemental Figure 5). The ameliorative effect of
metoprolol on pulmonary inflammation of COVID-19
patients with ARDS was associated with strong in-
dicators of clinical benefit, demonstrated by a sig-
nificant improvement in oxygenation (PaO2:FiO2) not
seen in control patients (Figure 2). These results are
very encouraging, but further large-scale trials are
needed to validate the clinical benefits of metoprolol
in this context. Given that neutrophils play a majorrole in the pathophysiology of ARDS of many causes
(not only COVID-19 related), further large validation
studies might include a wide spectrum of patients
with this condition.
The MADRID-COVID pilot trial has demonstrated
that IV administration of the clinically approved
b-blocker metoprolol to critically ill patients with
ARDS caused by COVID-19 is safe and disrupts the
exacerbated lung inflammation associated with the
disease. The beneficial effects on exacerbated
inflammation were associated with better oxygena-
tion and a nonsignificant reduction in the number of
CENTRAL ILLUSTRATION Metoprolol Repurposing for Treating ARDS in Critically Ill COVID-19 Patients
Clemente-Moragón, A. et al. J Am Coll Cardiol. 2021;78(10):1001–1011.
Reduced lung inflammation was associated with a significant improvement in oxygenation and fewer days on mechanical ventilation and of intensive care unit
admission. Repurposing metoprolol for the treatment of acute respiratory distress syndrome associated with coronavirus disease-2019 (COVID-19) appears to be a
safe and inexpensive strategy that can alleviate the burden of the COVID-19 pandemic.
J A C C V O L . 7 8 , N O . 1 0 , 2 0 2 1 Clemente-Moragón et al.
S E P T E M B E R 7 , 2 0 2 1 : 1 0 0 1 – 1 0 1 1 MADRID-COVID Pilot Trial
1009
PERSPECTIVES
COMPETENCY IN PATIENT CARE AND
PROCEDURAL OUTCOMES: In critically ill patients
with COVID-19 on mechanical ventilatory support,
intravenous administration of metoprolol upon
admission to the ICU is safe and improves pulmonary
function and clinical outcome.
TRANSLATIONAL OUTLOOK: Future studies with
larger sample sizes are needed to confirm the benefit
of metoprolol in critically ill patients with COVID-19
and potentially other inflammatory etiologies of
ARDS.
Clemente-Moragón et al. J A C C V O L . 7 8 , N O . 1 0 , 2 0 2 1
MADRID-COVID Pilot Trial S E P T E M B E R 7 , 2 0 2 1 : 1 0 0 1 – 1 0 1 1
1010days on mechanical ventilation and in the ICU.
Intravenous metoprolol appears as a promising
intervention that could improve the prognosis of
critically ill COVID-19 patients. Although these data
need to be corroborated in a larger sample, meto-
prolol is a clinically available and cheap drug (daily
treatment costs <2V) that can improve outcomes in
patients with severe COVID-19.
STUDY LIMITATIONS. The main limitation of this
study is the small sample size. The study was pow-
ered to detect differences in lung inflammation and
not clinical events. Another limitation is the single-
center nature of the study. This was an open-label
study, and treating physicians were not blinded to
treatment allocation. Finally, we cannot rule out a
selection bias resulting in patients with very poor
condition according to physicians not considered
for inclusion.
CONCLUSIONS
Our results show that IV administration of metoprolol
to patients with severe COVID-19–associated ARDS is
safe and abrogates the exacerbated lung inflamma-
tion associated with the disease. Reduced lung
inflammation was associated with a significant
improvement in oxygenation and with a trend toward
fewer days on mechanical ventilation and of ICU
admission. Metoprolol repurposing for the treatment
of ARDS associated with COVID-19 is a safe and cheap
intervention that can help to alleviate the massive
personal and health care burden associated with the
pandemic.
ACKNOWLEDGMENTS The authors thank the following
for their important support during this study: Noemí
Escalera, Rocío Escudero, and Antonio de Molina-
Iracheta at the CNIC; and Luis Nieto, Ana María
Venegas, Jose Tuñón, Ignacio Cornejo, Sandra Zazo,and Federico Rojo at the Fundación Jiménez Díaz.
Simon Bartlett provided English editing.
FUNDING SUPPORT AND AUTHOR DISCLOSURES
Mr Clemente-Moragón is supported by a fellowship from the Minis-
terio de Ciencia e Innovación (FPU2017/01932). The CNIC is supported
by the ISCIII, the Ministerio de Ciencia e Innovación, and the Pro
CNIC Foundation. Dr Ibáñez is supported by the European Commis-
sion (ERC-CoG grant No 819775) and by the Spanish Ministry of Sci-
ence and Innovation (MCN; “RETOS 2019” grant No PID2019-
107332RB-I00). Dr Oliver is supported by funds from the Comunidad
de Madrid Programa de Atracción de Talento (2017-T1/BMD-5185). All
other authors have reported that they have no relationships relevant
to the contents of this paper to disclose.
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Translational Laboratory for Cardiovascular Imaging
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APPENDIX For supplemental tables and
figures, please see the online version of this
paper.