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\begin{document}

\title{Effect of zamicastat on blood pressure and heart rate response to cold
pressor test: a double-blind, randomised, placebo-controlled study in
healthy subjects}



\author[1]{Marlene Fonseca}%
\author[2]{Cheila Ribeiro}%
\author[3]{Guillermo Fernández}%
\author[4]{Helena Gama}%
\author[2]{Luís Magalhães}%
\author[1]{Sara Carolina Henriques}%
\author[1]{Nuno Silva}%
\author[1]{Filipe Pinto}%
\author[1]{Luís Almeida}%
\author[5]{Patricio Soares-da-Silva}%
\affil[1]{BlueClinical Phase I}%
\affil[2]{Bial-Portela \& Cª S.A.}%
\affil[3]{BIAL R\&D Investments, S.A}%
\affil[4]{Bial-Portela \& Cª S.A}%
\affil[5]{Faculty of Medicine}%


\vspace{-1em}



  
  \date{June 11, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Aim: Inhibitors of dopamine-\selectlanguage{greek}β-\selectlanguage{english}hydroxylase (D\selectlanguage{greek}β\selectlanguage{english}H), such as zamicastat,
emerged as promising drugs for pulmonary arterial hypertension (PAH).
This study intended to validate the mechanism of action of zamicastat by
studying its effect on the overdrive of sympathetic nervous system
(SNS). Methods: This was a single-centre, prospective, double-blind,
randomised, placebo-controlled, crossover study, with 400 mg zamicastat,
in 22 healthy male subjects. Cold pressor test (CPT) was performed at
screening and each treatment period at day 1 and day 10. The
concentration of dopamine (DA), epinephrine (EPI), norepinephrine (NE)
in plasma and 24h-urine, and D\selectlanguage{greek}β\selectlanguage{english}H activity in plasma were measured.
Results: For zamicastat compared to placebo, the difference between cold
stimulus and rest phases on the change from baseline to day 10 of CPT
showed an estimated decrease of -4.62 mmHg for systolic blood pressure
(SBP; p=0.020). Zamicastat caused a decrease of -2.62 mmHg in mean
arterial pressure (MAP) response to cold stimulus during CPT (p=0.025).
At day 10, zamicastat elicited a statistically significant increase of
12.63 ng/L (p=0.040) and 19.22 ng/L (p=0.001) in plasma DA, before CPT
and after CPT, and a significant estimated increase in plasma EPI change
from baseline after CPT (p=0.040). Inhibition of plasma D\selectlanguage{greek}β\selectlanguage{english}H activity
ranged from 19.8\% to 25.0\%. At day 10, statistically significant
reductions in 24-hour urinary excretion of EPI (p=0.002) and NE
(p=0.001) were observed. Conclusions: Zamicastat decreased SBP and MAP
response to cold stimulus during CPT, evidencing its effect on the
overdrive sympathetic response to cold stimulus.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Despite advances in treatment options for pulmonary arterial
hypertension (PAH), the disease is still associated with worsening
symptoms and increased mortality, so research for novel treatments
persists.

The development of more effective therapeutic treatments has been
recently focused on the hyperactivation of neurohumoral systems --
essentially the sympathetic nervous system (SNS) -- involved in the
development and progression of hypertension and chronic heart failure.
In fact, hyperactivation of SNS has been implicated in the progression
of right heart failure and arrhythmia as premature ventricular
contractions, ventricular arrhythmia, and sudden cardiac death. This
involvement has been confirmed by both indirect (elevated plasma
concentrations of catecholamines) and direct evidence, such as muscle
sympathetic nerve activity, via microneurography of the peroneal nerve,
and heart rate (HR) and baroreceptor reflex variability.

In this context, the inhibition of the sympathetic nerve function with
adrenoceptor antagonists appeared to be a promising approach to manage
the hyperactivation of the SNS. However, most patients, particularly
those with heart failure, do not tolerate the immediate hemodynamic
deterioration that follows \selectlanguage{greek}β-\selectlanguage{english}blocker treatment. An alternative strategy
is to reduce the biosynthesis of norepinephrine (NE) through the
inhibition of the enzyme dopamine-\selectlanguage{greek}β-\selectlanguage{english}hydroxylase (D\selectlanguage{greek}β\selectlanguage{english}H). This approach has
several putative merits, such as gradual modulation, as opposed to
abrupt inhibition of the sympathetic system, and increased availability
of dopamine (DA), which can improve renal function. Therefore, it could
be anticipated that inhibitors of D\selectlanguage{greek}β\selectlanguage{english}H may provide significant clinical
advantages in PAH patients over conventional adrenoceptor antagonists.

In this setting, zamicastat (also known as BIA 5-1058), a reversible D\selectlanguage{greek}β\selectlanguage{english}H
inhibitor developed by Bial-Portela \& C\selectlanguage{ngerman}ª S.A., emerged as an orally
administered small molecule for the treatment of cardiovascular
disorders, including PAH. It is a potent peripheric selective inhibitor
of D\selectlanguage{greek}β\selectlanguage{english}H, with limited access to the brain, that converts DA to NE in
sympathetically innervated tissues, reducing the drive of the SNS. At
this point, the confirmation of the mechanism of action of zamicastat in
the SNS overdrive, through D\selectlanguage{greek}β\selectlanguage{english}H inhibition, would validate and strengthen
the efficacy and safety data collected so far.

Different methods for the evaluation of the autonomic nervous system
(ANS) have been described, including the cold pressor test (CPT) and
measurements of the levels of neurotransmitters. CPT is a simple,
non-invasive validated test of sympathetic activation. It consists of a
sympatho-excitatory stressor (cold) that activates afferent pain and
temperature neurons, resulting in a centrally mediated stimulation of
sympathetic efferent neurons. This induces vascular sympathetic
activation, causing arteriolar constriction, increased HR, and increased
cardiac contractility, with consequent increase in diastolic (DBP) and
systolic blood pressure (SBP).

Measurements of plasma and urine catecholamines are a classic and
reliable method for assessing ANS function. The concentration of plasma
catecholamines has been shown to increase following various stressor
stimuli, being an excellent tool to evaluate the impact of drugs on the
SNS.

This study aimed to validate the proposed mechanism of action of
zamicastat by studying its effects on the SNS, through the evaluation of
the impact on blood pressure (BP) and HR response and assessment of
plasma and urinary concentration of catecholamines and their
metabolites, before and after CPT.

\textbf{Materials and Methods}

\textbf{Study population}

Study subjects were recruited from BlueClinical Phase I's pool of
healthy volunteers and underwent a screening procedure comprising
medical history, physical examination, vital signs, 12-lead
electrocardiogram (ECG), CPT, and clinical laboratory safety tests.

The main inclusion criteria were: healthy non-smoker or ex-smoker male
subject with age [?]18 and [?]55 years; absence of clinically relevant
diseases in medical history; resting seated SBP of 90-140 mmHg, DBP of
50-90 mmHg and pulse rate 45-100 bpm, inclusive; ability to tolerate the
3-minute cold stimulus phase of CPT; mean SBP increase of [?]7 mmHg during
the cold stimulus phase of CPT; absence of relevant abnormalities on
physical examination, 12-lead ECG, and clinical laboratory safety tests.
The main exclusion criteria were: previous exposure to zamicastat;
hypersensitivity and/or allergy reaction to the investigational
medicinal product (IMP) or any of the excipients; any medical or
surgical condition that may affect drug pharmacokinetics or subject
safety; clinically relevant history of fainting, syncope, orthostatic
hypotension, vasovagal reaction; history of alcoholism or drug abuse in
the past two years; history of arterial hypertension, diabetes mellitus
or Raynaud's phenomenon; orthostatic hypotension; QT interval corrected
with Fridericia's formula (QTcF) \textgreater{} 450 msec. Subjects who
met all the inclusion criteria and none of the exclusion criteria at the
screening visit were considered eligible for participation in the study.

Written informed consent was obtained from each prospective subject
prior to any study procedure, after the physician provided all the
information about the implications of participating in the study.
Subjects were assured that they might abandon the study at any time
without any prejudice.

SARS-CoV-2 testing was performed prior to admission to each study
period.

\textbf{Study design and treatments}

This was a single-centre, prospective, two-sequence, two-period
crossover study, in which healthy male subjects were randomised to
receive 400 mg zamicastat or placebo.

The study had a double-blind design and was placebo controlled to
reduced potential bias. Compared with a parallel-group study, the
crossover design reduces the influence of confounding covariates and
provides more precise estimates of effects with fewer participants.

The study consisted of a screening evaluation, two treatment periods,
and an end-of-study visit. In each treatment period, subjects were
administered a 400 mg once-daily oral dose of zamicastat or matching
placebo, for 10 days, in fed conditions. The two treatment periods were
separated by a washout of at least seven days. Subjects were screened
between days -28 to -3 (both inclusive) before the first IMP
administration to confirm that they met the eligibility criteria for the
study (Figure 1).

On both treatment periods, subjects were admitted to the clinical site
two days before (day -2) the first IMP administration. On both treatment
periods, the following procedures were completed on the day before (day
-1) the first IMP administration: measurement of vital signs (BP, HR,
and body temperature); 24-hour urine collection for catecholamines
assay; collection of blood for plasma catecholamines concentration, D\selectlanguage{greek}β\selectlanguage{english}H
activity determination, and zamicastat and metabolites (BIA 5-453 and
BIA 5-961) quantification; and CPT.

On the morning of days 1 to 10, subjects received 400 mg of zamicastat
or matching placebo orally, after a moderate breakfast. On days 3, 6, 8,
and 10, 24-hour urine was collected for catecholamines assay, and blood
samples were collected for D\selectlanguage{greek}β\selectlanguage{english}H activity determination and zamicastat and
metabolites quantification. On day 1 and day 10, CPT was performed, and
blood was collected for the determination of plasma catecholamines
concentration, before and after CPT.

During the treatment days, the following safety assessments were
performed: clinical laboratory safety test on day 5 (haematology and
biochemistry); ECG on days 1, 3, 5, 7, and 9; and vital signs from day 1
to 10.

\textbf{Cold pressure test}

Each subject performed CPT at screening, day -1, and day 10 of each
treatment period, approximately 3.5 hours after the start of a moderate
breakfast. CPT was conducted approximately at the same time of the day,
to reduce the impact of circadian variations of BP on the study
outcomes. BP and haemodynamic variables were collected during the CPT
using the Finapres\selectlanguage{ngerman}® NOVA monitor (Finapres Medical Systems, Amsterdam).
The CPT procedure was conducted using the Advanced Hemodynamics software
application (Finapres Medical Systems, Amsterdam) to allow for the
characterisation of further haemodynamic parameters. The following
parameters were collected: SBP, DBP, HR, mean arterial pressure (MAP),
cardiac output (CO), cardiac index (CIn), stroke volume (SV), SV index
(SVI), total peripheral resistance (TPR), cardiac contractility (dP/dt)
and left ventricular ejection time (LVET).

The test included three phases: i) rest phase (5 minutes), ii) cold
stimulus phase (3 minutes), and iii) recovery phase (5 minutes). During
the cold stimulus phase of CPT, subjects inserted their left hand up to
the wrist in a temperature-controlled (4±0.5 ºC) water bath, for three
minutes.

The pharmacodynamics analysis set (PD population) included all subjects
who received at least one dose of the IMP and had at least one valid
post-dose for the corresponding PD assessment. Those subjects who belong
to the PD set and did not deviate from the protocol in a way that might
affect the evaluation of the corresponding PD variable were included in
the per-protocol pharmacodynamics analysis set (PP-PD population).

\textbf{Plasma and urinary catecholamines concentration}

In each treatment period, for plasma catecholamines concentration
assessment, venous blood samples (10 mL) were drawn into
K\textsubscript{2}EDTA tubes, on day -1 and day 10, 30±5 minutes before
the rest phase of CPT and within 2 minutes after the recovery phase of
CPT. The determination of the plasma-free fractionated catecholamines
DA, epinephrine (EPI), and NE was performed by using liquid
chromatography with tandem mass spectrometry (LC-MS/MS). The lower limit
of quantification (LLOQ) and the upper limit of quantification (ULOQ) of
the method were 17.4 and 1116.8 ng/L, for DA, 17.6 and 1124.5 ng/L, for
EPI, and 63.0 and 3966.2 ng/L, for NE, respectively.

For the determination of the urinary creatinine and catecholamines
excretion profile, urine was collected for 24 hours, starting within 15
minutes before breakfast on days -1, 3, 6, 8, and 10, on each treatment
period. The determination of urinary concentrations of total
catecholamines and their metabolites was performed by high-performance
liquid chromatography (HPLC) with electrochemical detection.

The catecholamines metabolites determined were: i) normetanephrine; ii)
metanephrine; iii) 4-hydroxy-3-methoxy-phenylacetic acid, also named
homovanillic acid (HVA); and iv) 4-hydroxy-3-methoxy-mandelic acid
(HMMA), also named vanillylmandelic acid (VMA).

The 24-hour urinary catecholamines excretion profile was normalised by
using the 24-hour creatinine concentration.

\selectlanguage{greek}\textbf{Dopamine-β-hydroxylase (DβH) activity assay}\selectlanguage{english}

For the determination of D\selectlanguage{greek}β\selectlanguage{english}H activity in plasma, venous blood samples (6
mL) were drawn into lithium heparin tubes, 30 minutes after breakfast on
day -1 and at pre-dose (within 10 minutes) on days 3, 6, 8, and 10.

The D\selectlanguage{greek}β\selectlanguage{english}H activity assay relied on the conversion of tyramine to
octopamine by D\selectlanguage{greek}β\selectlanguage{english}H; octopamine was then determined in plasma, in
accordance with the applicable principles of Good Laboratory Practices
(GLP), using a previously validated LC-MS/MS analytical method. D\selectlanguage{greek}β\selectlanguage{english}H
activity was calculated as follows:\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

The LLOQ and the ULOQ of the method for octopamine were 1.00 and 29.98
\selectlanguage{greek}μ\selectlanguage{english}g/mL, respectively.

\textbf{Pharmacokinetic measurements}

In each treatment period, for the determination of plasma concentrations
of zamicastat and its metabolites, venous blood samples (6 mL) were
drawn into lithium heparin tubes, 30 minutes after breakfast on day -1
and at pre-dose (within 10 minutes) on days 3, 6, 8, and 10. Plasma
concentrations of zamicastat and its metabolites BIA 5-453 and BIA 5-961
were determined by SYNLAB Analytics \& Services Switzerland AG
(Sternenfeldstrasse 14 CH-4127 Birsfelden, Switzerland), in accordance
with the applicable principles of GLP, using a previously validated
LC-MS/MS analytical method.

The pharmacokinetics analysis set (PK population) included all subjects
who had received at least one dose of zamicastat without major protocol
deviation and had at least one valid evaluable post-dose plasma
zamicastat concentration.

\textbf{Safety and Tolerability Analysis}

Safety parameters were carefully monitored throughout the study.
Clinically significant abnormalities in physical examination, vital
signs, 12-lead ECG, laboratory safety tests, and any other relevant
safety variables were reported as adverse events (AEs), which were
tabulated and summarized using MedDRA 23.1.

Results of vital signs, clinical laboratory variables, and ECG
parameters {[}HR and the intervals: PR, QRS, QT, QT interval corrected
with Bazett's formula (QTcB), QT interval corrected with Fridericia's
formula (QTcF){]}, at each time point of measurement, were compared to
baseline.

The safety analysis set (safety population) included all subjects who
received at least one dose of the investigational product.

\textbf{Statistical Analysis}

Estimation of parameters and statistical analyses on PK and PD data were
conducted using non-compartmental analysis in Phoenix\selectlanguage{ngerman}® WinNonlin®
version 8.2 (Certara USA Inc, Princeton, NJ) and SAS® version 9.4 (SAS
Institute, Cary, NC). For the additional statistical analyses, SAS® 9.4
was also used.

Descriptive statistics were calculated according to the variable nature:
i) for continuous variables, number of subjects (n), arithmetic mean,
standard deviation (SD), standard error of the mean and range (minimum
and maximum); median, quartiles, coefficient of variation (CV\%),
geometric mean, geometric standard deviation, and geometric CV\% are
given, if pertinent; ii)~for categorical variables, number of subjects,
relative and absolute frequencies.

Actual p-values were reported, no multiplicity or adjustment of type I
error was implemented; therefore, an alpha of 0.05 (p\selectlanguage{english}[?]0.05) was set for
statistical significance. Hypotheses testing was defined to
statistically compare the main primary PD endpoints of the study with
zamicastat and placebo (SBP, DBP, and HR), with clinical margins of 5.5
mmHg, 3.7 mmHg and 4.1 bpm for mean SBP, DBP, and HR, respectively.
Hence the null hypotheses were as follows:

\selectlanguage{english}\textbf{SBP: ?0:??-??[?]-5.5}

\selectlanguage{english}\textbf{DBP: ?0:??-??[?]-3.7}

\selectlanguage{english}\textbf{HR: ?0:??-??[?]-4.1,}

where \selectlanguage{english}?\selectlanguage{english}? and \selectlanguage{english}?\selectlanguage{english}? represent the mean of each endpoint for zamicastat and
placebo, respectively.

Missing data were not replaced, and no imputation was conducted;
descriptive statistics and statistical analysis were performed based on
the available data only.



\textbf{RESULTS}

\textbf{Study population}

A total of 57 male subjects were screened for participation however, 22
subjects were randomized and participated in the study.

Out of the total, 35 participants were not admitted to randomisation due
to the following reasons: 21 subjects did not meet one or more of the
selection criteria (16 at screening and 5 at admission), 7 subjects
decided to discontinue after the screening procedures, 3 subjects were
excluded due to an AE, 1 subject was lost to follow-up, 1 subject was
discontinued according to physician's decision, and 2 subjects served as
back-up.

Regarding the 22 subjects admitted to randomisation, their age ranged
between 20 and 53 years (mean +- SD: 34 +- 7.5 years) and the majority
were Caucasian (81.8\%). Weight varied between 57.8 kg and 93.4 kg
(76.4+-10.1kg) and height was in the range of 167 cm and 190 cm (179+-6.2
cm); mean body mass index (BMI) 23.8+-2.8 kg/m\textsuperscript{2}). All
these 22 subjects received at least one dose of IMP and had at least one
valid post-dose PD assessment (Safety population = 22 subjects; PD
population = 22 subjects). Of those, 21 subjects were included in the PK
set, completed both study periods without major protocol deviations, and
had at least one valid evaluable plasma zamicastat concentration (PP-PD
population = 21 subjects; PK population = 21 subjects). A total of 21
subjects completed the study, as one subject had a serious adverse event
and discontinued after first dosing, in period 1 of the study. This
subject was excluded from the PP-PD and PK populations since he was only
dosed with placebo. No concomitant medications administered to study
subjects were considered to have a relevant impact on PK, PD, and safety
analyses.

\textbf{Cold pressure test}

The arithmetic means of SBP, DBP, and HR in each of the three phases of
CPT, at day -1 and day 10, following administration of zamicastat and
placebo are presented in \textbf{Table 1} . The CPT was effective in
eliciting a BP and HR response. During the cold stimulus phase of the
CPT, SBP, DBP, and HR increased significantly in relation to the rest
phase, irrespective of study day (day -1 or day 10) and treatment
(zamicastat or placebo) (\textbf{Table 2} ).

In comparison to placebo, the difference between cold stimulus and rest
phases at day 10 adjusted to baseline (day -1), following administration
of zamicastat, showed an estimated decrease of -4.62 mmHg for SBP which
attained statistical significance (p=0.020). For DBP and HR, the
difference between zamicastat against placebo was -1.86 mmHg and 0.81
bpm respectively; however, they were not statistically significant
(p\textgreater{}0.05) (\textbf{Table 3} ).

Compared to placebo, the difference between cold stimulus and rest
phases at day 10, adjusted to baseline (day -1) following administration
of zamicastat showed a statistically significant decrease of -2.62 mmHg
in MAP response to cold stimulus during CPT (p=0.025), but not for the
other haemodynamic parameters assessed (\textbf{Table 4} ). Maximum
response achievable (E\textsubscript{max}) and time to occurrence of
E\textsubscript{max} (TE\textsubscript{max}) for SBP, DBP, and HR were
similar between zamicastat and placebo treatments, and the area under
the effect time curve (AUEC) was slightly decreased with zamicastat
treatment when compared to placebo. However, there were no statistically
significant differences between zamicastat and placebo in
E\textsubscript{max}, TE\textsubscript{max} and AUEC derived for SBP,
DBP, and HR, following 10 days of treatment.

At each CPT phase (rest, cold stimulus, and recovery), no differences
between zamicastat and placebo adjusted to baseline were observed for
SBP, DBP, HR, MAP and haemodynamic parameters (CO, CI, SV, SVI, TPR,
dP/dT and LVET)-..

\textbf{Plasma Catecholamines}

At day 10, zamicastat elicited a significant increase of 12.63 ng/L
(p=0.040) and 19.22 ng/L (p=0.001) in plasma DA before CPT and after
CPT. For day -1 and 10, following treatment with zamicastat and placebo,
no differences were observed for DA, EPI, and NE plasma concentrations,
when adjusted to baseline plasma concentrations, after CPT and before
CPT. After CPT, a significant difference between zamicastat and placebo
of 15.93 ng/L was observed (p=0.040) on the change from baseline EPI
plasma concentration. For DA and NE change from baseline plasma
concentrations, no statistically significant differences
(p\textgreater{}0.05) were observed (\textbf{Table 5} ).

\textbf{Twenty-four-hour urinary excretion profile of catecholamines and
catecholamine metabolites} \emph{} The observed 24-hour urinary
excretion of DA, EPI, and NE was generally lower with zamicastat than
with placebo from day 3 to day 10 (n=21). At day 10, zamicastat was
shown to have a statistically significant decrease in EPI (-3.96 \selectlanguage{greek}μ\selectlanguage{english}g/24h,
p=0.007) and NE (-7.54 \selectlanguage{greek}μ\selectlanguage{english}g/24h, p=0.017) on the change from baseline
24-hour urine excretion profile when compared to placebo. No difference
was observed for DA 24-hour urinary excretion profile, following 10 days
of treatment (\textbf{Figure 2} ). Compared to placebo, zamicastat was
associated to an increase in HVA urinary excretion of 1.75 mg/24 h
(p\textless{}0.001) and a decrease in VMA urinary excretion of 1.97
mg/24 h (p\textless{}0.001), which was observed from baseline up to day
10 (\textbf{Figure 2} ). No differences were observed for
normetanephrine or metanephrine.

\selectlanguage{greek}\textbf{Dopamine-β-Hydroxylase (DβH) activity}\selectlanguage{english}

Following multiple-dose administration of zamicastat 400 mg, the
pre-dose plasmatic inhibition of D\selectlanguage{greek}β\selectlanguage{english}H activity ranged from 19.8\% on day
3 to 25.0\% on day 10 (\textbf{Figure 3)} . The variability between
individuals was moderate, with coefficients of variation ranging from
32.6\% to 39.8\% during the 10 days of evaluation. With placebo, the
inhibition of D\selectlanguage{greek}β\selectlanguage{english}H activity ranged from -3.9\% (day 3) to -4.3\% (day
10). Compared to placebo, zamicastat was associated with a statistically
significant increase in the inhibition of plasma D\selectlanguage{greek}β\selectlanguage{english}H activity
(p\textless{}0.001) from day 3 up to day 10.

\textbf{Pharmacokinetic results}

Geometric mean trough plasma concentration \emph{versus} time profiles
of zamicastat, following administration of zamicastat 400 mg once daily
for 10 days, are displayed in \textbf{Figure 2} , in linear scale
(n=21). From visual inspection, steady-state plasma concentrations of
zamicastat and its metabolites appeared to be reached at day 6
approximately. The geometric mean of zamicastat concentration ranged
from 45.86\selectlanguage{ngerman}±1.46 ng/mL on day 3 to 58.64±1.52 ng/mL on day 10. The
inter-individual variability was moderate, with coefficients of
variation ranging from 32.6\% to 36.6\% during the 10 days of zamicastat
400 mg once daily administration.

\textbf{Safety results}

Sixteen subjects reported a total of 26 treatment-emergent adverse
events (TEAEs). Among the 22 subjects who received placebo, 6 (27\%)
reported a total of 10 TEAEs; 9 were considered drug-related. Among the
21 subjects who received zamicastat, 12 (57\%) reported a total of 16
TEAEs; of these, 13 were considered related to the treatment. The most
common drug-related TEAE was ``orthostatic hypotension'' of mild
intensity, which was reported by 4 (18\%) subjects with placebo and 5
(24\%) subjects with zamicastat. One TEAE (``acute cardiac event''),
which occurred with placebo, was considered of severe intensity. It was
considered a serious adverse event (SAE) due to hospitalization, leding
to subject withdrawal from the study. All remaining TEAEs were of mild
intensity. No deaths, further SAEs, or discontinuations due to TEAEs
were registered, and no clinically relevant change or clinically
significant abnormality in vital signs, physical examinations, and
clinical laboratory tests were observed. Vital signs and ECG parameters
did not show any overall trend.

\textbf{DISCUSSION}

Herein, we describe a single-centre, prospective, double-blind,
randomised, placebo-controlled, two-treatment, two-sequence, two-period
crossover study with 400 mg zamicastat/placebo, in healthy male
subjects. The cold stimulus caused a marked sympathetic response
evidenced by a significant increase in BP and HR during CPT, at baseline
(day -1) and after a 10-day treatment with zamicastat or placebo.

CPT was selected to explore the effect of oral zamicastat, at
steady-state conditions, on the overdrive of the sympathetic system and
consequent effects on the cardiovascular system. This test has been used
both clinically and experimentally to evaluate non-baroreflex-mediated
sympathetic neural control in humans, cardiovascular reactivity to
stress in normotensive and hypertensive subjects, and the efficacy of
lifestyle and pharmacological interventions on BP and vascular
reactivity.

Even though our results followed the tendencies presented in other
reports, showing increased BP and HR, the increment in HR was lower than
in other studies, in which the increase could be higher than 20 bpm,
after a 90-second immersion in cold water. However, in other studies CPT
showed to have lower or no effect on HR. On the other hand, the change
in BP was higher than in previous studies, reaching about 30 mm Hg.
These variations may be related to differences in the baseline BP values
of the participants as well as to other demographic and behavioural
patterns such as age, gender, weight, and exercise habits, which have
been shown to influence reactivity to cold-induced stress. Despite the
differences, our results on day 10 illustrate the absence of habituation
or learning from exposure to the cold stimulus. In fact, both BP and HR
kept the same increasing trend with similar magnitude, after 10 days.
This observation combined with the referred influence of demographic and
behavioural patterns on the reactivity to CPT can guide the definition
of future selection criteria that can guarantee the inclusion of
subjects with more robust baseline conditions to promote adaptation and
learning during cold exposure. This would enrich the cohort by reducing
selection bias. However, this endeavour requires a more detailed
analysis of the correlation of the baseline characteristics of our
participants with habituation to the cold stressor.

Women were not eligible for this study to avoid the potential influence
of fluctuation of sex hormones throughout the menstrual cycle with
neurohumoral variations with impact on the cardiovascular system.

Compared to placebo, zamicastat significantly decreased SBP and MAP
response to cold stimulus during CPT, following 10 days of treatment,
evidencing its effect on the overdrive sympathetic response to cold
stimulus. Zamicastat reduced SBP (-4.62 mmHg) and DBP (-1.86 mmHg) when
compared with placebo. The HR slightly increased (0.81 bpm) which may be
related to compensatory hemodynamic effects. The one-sided 95\%
confidence interval (-7.77 mmHg) was below the established clinical
margin for SBP (-5.5 mmHg) supported by a statistically significant
difference (p=0.020) between zamicastat and placebo.

The measurement of plasma and urine catecholamines proved to be a valid
tool to evaluate the impact of zamicastat on the SNS. These data showed
that zamicastat 400 mg, given once daily, inhibited D\selectlanguage{greek}β\selectlanguage{english}H plasmatic
activity, increased the 24-h urinary excretion of HVA, and significantly
decreased the 24-h urinary excretion of EPI, NE, and VMA, when compared
to placebo. The decrease of NE in urine 24h explains the reduction of
its metabolite VMA, which reflects a decrease in the total production of
catecholamines caused by zamicastat treatment. On the other hand, the
levels of DA in urine 24h were similar with zamicastat and placebo, but
its metabolite HVA increased, which may imply that a higher amount of DA
was metabolized. Our results confirm that the 24h urinary catecholamines
is a collector of plasmatic levels of catecholamines and, as such, is
more assertive as a potential biomarker of SNS modulation than the
sporadic assessment of catecholamines in plasma, which is more affected
by fluctuations and interferences than an integrated measurement over 24
h.

Overall, the profile of AEs showed that placebo and zamicastat
treatments were similarly well tolerated.

Our results suggest that mechanism of action mediated by zamicastat can
be a promising therapeutic option for morbidities characterized by
excessive activation of the SNS, such as PAH, ischemic disease, among
others.

In conclusion, our study confirmed the effect of zamicastat on the SNS
through the decrease of SBP and MAP response to cold stimulus during CPT
and inhibition of D\selectlanguage{greek}β\selectlanguage{english}H plasmatic activity. In addition, the effect of
zamicastat in 24 h urine catecholamines and their metabolites proved to
be clinically relevant, thereby confirming its potential as SNS
modulator.

\textbf{Funding statement}

This study was sponsored by Bial-Portela \& C\selectlanguage{ngerman}ª S.A.

\textbf{Conflict of interest disclosure}

CR, GCF, HG, and LM are or were employees of Bial-Portela \& Cª S.A.

MF, SCH, FP, and LA are or were employees of BlueClinical.

NS is a Consultant of BlueClinical.

\textbf{Data Availability Statement}

The data that support the findings of this study are available on
request from the corresponding author. The data are not publicly
available due to privacy or ethical restrictions.

\textbf{Ethics approval statement}

The study was implemented and conducted according to the principles of
the Declaration of Helsinki and of the ICH Good Clinical Practice. The
protocol (BIA-51058-123, EudraCT No 2020-003262-40) was approved by the
National Ethics Committee for Clinical Research (CEIC).

\textbf{Patient consent statement}

Written informed consent was obtained from each prospective subject
prior to any study procedure, after the physician in charge had the
conviction that the subject was aware of the implications of
participating in the study and the subject confirmed his willingness to
participate. Subjects were assured that they might abandon the study at
any time without any prejudice.

\textbf{References}

\textbf{Table Captions}

\textbf{Table 1} . Arithmetic means (SD) of SBP, DPP, and HR measured
during CPT, at day -1 and day 10, following administration of zamicastat
and placebo (PP-PD population, n=21).

\textbf{Table 2.} Arithmetic means (SD) of the differences between cold
stimulus and rest phases for SBP, DPP, and HR measured during CPT, at
day -1 and day 10, following administration of zamicastat and placebo
(PP-PD population, n=21).

\textbf{Table 3.} Comparison of zamicastat against placebo for cold
stimulus and rest phases (at day 10 adjusted to baseline) (PP-PD
population, n=21).

\textbf{Table 4.} Mixed effect model results of mean arterial pressure
and hemodynamic parameters during cold pressor test -- Least square
means differences between zamicastat and placebo for the difference
between cold stimulus and rest phases adjusted to baseline (PP-PD
population, day -1, n=21).

\textbf{Table 5.} Mixed effect model results of plasma catecholamines
concentration -- differences between zamicastat and placebo -- Least
square means before and after CPT at day 10, adjusted to baseline (PP-PD
population, n=21).

\textbf{Figure Captions}

\textbf{Figure 1.} Study design diagram

\textbf{Figure 2.} Least square means and corresponding standard error
of dopamine (A), epinephrine (B), norepinephrine (C), normetanephrine
(D), homovanillic acid (E), and vanillylmandelic acid (F) 24-hour urine
excretion profiles

\textbf{Figure 3.} Percentage (arithmetic mean) of plasmatic inhibition
of D\selectlanguage{greek}β\selectlanguage{english}H activity at pre-dose (trough) following administration of
zamicastat 400 mg (once daily) and placebo (PP-PD population, n=21).
(D\selectlanguage{greek}β\selectlanguage{english}H: dopamine-\selectlanguage{greek}β-\selectlanguage{english}hydroxylase; PP-PD: per-protocol pharmacodynamics)

\textbf{Tables}

\textbf{Table 1.} Arithmetic means (\selectlanguage{ngerman}±SD) of SBP, DPP, and HR measured
during CPT, at day -1 and day 10, following administration of zamicastat
and placebo (PP-PD population, n=21).\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Treatment Period Day}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Treatment Period Day}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Treatment Period Day}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Treatment Period Day}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Treatment Period Day}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Treatment Period Day}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Day -1}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Day -1}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Day -1}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Day 10}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Day 10}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Day 10}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Rest}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Cold Stimulus}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Recovery}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Rest}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Cold Stimulus}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Recovery}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Systolic~Blood~Pressure~(mmHg)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Systolic~Blood~Pressure~(mmHg)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Systolic~Blood~Pressure~(mmHg)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Systolic~Blood~Pressure~(mmHg)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Systolic~Blood~Pressure~(mmHg)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Systolic~Blood~Pressure~(mmHg)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Systolic~Blood~Pressure~(mmHg)}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Zamicastat\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
119\selectlanguage{ngerman}±9\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
149\selectlanguage{ngerman}±15\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
125\selectlanguage{ngerman}±10\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
124\selectlanguage{ngerman}±11\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
156\selectlanguage{ngerman}±15\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
130\selectlanguage{ngerman}±11\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Placebo\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
121\selectlanguage{ngerman}±13\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
150\selectlanguage{ngerman}±18\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
128\selectlanguage{ngerman}±14\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
122\selectlanguage{ngerman}±14\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
158\selectlanguage{ngerman}±19\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
131\selectlanguage{ngerman}±13\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Diastolic~Blood~Pressure~(mmHg)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Diastolic~Blood~Pressure~(mmHg)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Diastolic~Blood~Pressure~(mmHg)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Diastolic~Blood~Pressure~(mmHg)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Diastolic~Blood~Pressure~(mmHg)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Diastolic~Blood~Pressure~(mmHg)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Diastolic~Blood~Pressure~(mmHg)}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Zamicastat\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
59\selectlanguage{ngerman}±6\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
78\selectlanguage{ngerman}±10\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
63\selectlanguage{ngerman}±7\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
61\selectlanguage{ngerman}±4\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
82\selectlanguage{ngerman}±10\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
65\selectlanguage{ngerman}±7\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Placebo\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
59\selectlanguage{ngerman}±8\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
78\selectlanguage{ngerman}±13\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
63\selectlanguage{ngerman}±8\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
60\selectlanguage{ngerman}±5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
82\selectlanguage{ngerman}±11\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
65\selectlanguage{ngerman}±7\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Heart~Rate~(bpm)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Heart~Rate~(bpm)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Heart~Rate~(bpm)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Heart~Rate~(bpm)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Heart~Rate~(bpm)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Heart~Rate~(bpm)}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Heart~Rate~(bpm)}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Zamicastat\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
63\selectlanguage{ngerman}±8\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
70\selectlanguage{ngerman}±9\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
59\selectlanguage{ngerman}±6\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
63\selectlanguage{ngerman}±9\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
73\selectlanguage{ngerman}±8\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
60\selectlanguage{ngerman}±9\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Placebo\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
62\selectlanguage{ngerman}±7\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
71\selectlanguage{ngerman}±9\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
59\selectlanguage{ngerman}±6\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
62\selectlanguage{ngerman}±6\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
72\selectlanguage{ngerman}±9\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
59\selectlanguage{ngerman}±6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
CPT: cold pressure test; DPP: diastolic blood pressure; HR: heart rate;
n: number of subjects; PP-PD: per-protocol pharmacodynamics; SBP:
systolic blood pressure.\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
CPT: cold pressure test; DPP: diastolic blood pressure; HR: heart rate;
n: number of subjects; PP-PD: per-protocol pharmacodynamics; SBP:
systolic blood pressure.\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
CPT: cold pressure test; DPP: diastolic blood pressure; HR: heart rate;
n: number of subjects; PP-PD: per-protocol pharmacodynamics; SBP:
systolic blood pressure.\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
CPT: cold pressure test; DPP: diastolic blood pressure; HR: heart rate;
n: number of subjects; PP-PD: per-protocol pharmacodynamics; SBP:
systolic blood pressure.\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
CPT: cold pressure test; DPP: diastolic blood pressure; HR: heart rate;
n: number of subjects; PP-PD: per-protocol pharmacodynamics; SBP:
systolic blood pressure.\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
CPT: cold pressure test; DPP: diastolic blood pressure; HR: heart rate;
n: number of subjects; PP-PD: per-protocol pharmacodynamics; SBP:
systolic blood pressure.\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
CPT: cold pressure test; DPP: diastolic blood pressure; HR: heart rate;
n: number of subjects; PP-PD: per-protocol pharmacodynamics; SBP:
systolic blood pressure.\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2.} Arithmetic means (\selectlanguage{ngerman}±SD) of the differences between cold
stimulus and rest phases for SBP, DPP, and HR measured during CPT, at
day -1 and day 10, following administration of zamicastat and placebo
(PP-PD population, n=21).\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Difference cold stimulus -- rest}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Difference cold stimulus -- rest}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Day -1}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Day 10}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Systolic~Blood~Pressure (mmHg)}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Systolic~Blood~Pressure (mmHg)}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Systolic~Blood~Pressure (mmHg)}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Zamicastat\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
30\selectlanguage{ngerman}±11\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
32\selectlanguage{ngerman}±11\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Placebo\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
29\selectlanguage{ngerman}±10\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
36\selectlanguage{ngerman}±9\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Diastolic~Blood~Pressure~(mmHg)}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Diastolic~Blood~Pressure~(mmHg)}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Diastolic~Blood~Pressure~(mmHg)}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Zamicastat\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
19\selectlanguage{ngerman}±6\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
21\selectlanguage{ngerman}±7\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Placebo\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
19\selectlanguage{ngerman}±8\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
22\selectlanguage{ngerman}±8\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Heart~Rate~(bpm)}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Heart~Rate~(bpm)}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Heart~Rate~(bpm)}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Zamicastat\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
8\selectlanguage{ngerman}±6\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
9\selectlanguage{ngerman}±7\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Placebo\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
10\selectlanguage{ngerman}±6\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
11\selectlanguage{ngerman}±6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
CPT: cold pressure test; DPP: diastolic blood pressure; HR: heart rate;
n: number of subjects; PP-PD: per-protocol pharmacodynamics; SBP:
systolic blood pressure; SD: standard deviation\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
CPT: cold pressure test; DPP: diastolic blood pressure; HR: heart rate;
n: number of subjects; PP-PD: per-protocol pharmacodynamics; SBP:
systolic blood pressure; SD: standard deviation\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
CPT: cold pressure test; DPP: diastolic blood pressure; HR: heart rate;
n: number of subjects; PP-PD: per-protocol pharmacodynamics; SBP:
systolic blood pressure; SD: standard deviation\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 3.} Comparison of zamicastat against placebo for cold
stimulus -- rest phases (at day 10 adjusted to baseline) (PP-PD
population, n = 21)\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& \textbf{Estimate*} & \textbf{One-Sided 95\% CI} &
\emph{p}\textbf{-value}\tabularnewline
\midrule
\endhead
\textbf{SBP (mmHg)} & -4.62 & -7.77 & \textbf{0.020}\tabularnewline
\textbf{DBP (mmHg)} & -1.86 & -3.55 & 0.073\tabularnewline
\textbf{HR (bpm)} & 0.81 & -0.66 & 0.354\tabularnewline
*Estimates correspond to the difference between zamicastat and placebo
for cold stimulus -- rest phases on the change from baseline; treatment
was used as fixed effect. DBP, Diastolic Blood Pressure; HR, Heart Rate;
PP-PD: per-protocol pharmacodynamics; SBP, Systolic Blood Pressure. &
*Estimates correspond to the difference between zamicastat and placebo
for cold stimulus -- rest phases on the change from baseline; treatment
was used as fixed effect. DBP, Diastolic Blood Pressure; HR, Heart Rate;
PP-PD: per-protocol pharmacodynamics; SBP, Systolic Blood Pressure. &
*Estimates correspond to the difference between zamicastat and placebo
for cold stimulus -- rest phases on the change from baseline; treatment
was used as fixed effect. DBP, Diastolic Blood Pressure; HR, Heart Rate;
PP-PD: per-protocol pharmacodynamics; SBP, Systolic Blood Pressure. &
*Estimates correspond to the difference between zamicastat and placebo
for cold stimulus -- rest phases on the change from baseline; treatment
was used as fixed effect. DBP, Diastolic Blood Pressure; HR, Heart Rate;
PP-PD: per-protocol pharmacodynamics; SBP, Systolic Blood
Pressure.\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 4.} Mixed effect model results of mean arterial pressure
and hemodynamic parameters during cold pressor test -- Least square
means differences between zamicastat and placebo for the difference
between cold stimulus and rest phases adjusted to baseline (day -1,
PP-PD population, n=21).\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& \textbf{Cold Stimulus -- Rest Phases} & \textbf{Cold Stimulus -- Rest
Phases} & \textbf{Cold Stimulus -- Rest Phases}\tabularnewline
\midrule
\endhead
& \textbf{Estimate*} & \textbf{One-sided 95\% CI} &
\emph{p}\textbf{-value}\tabularnewline
\textbf{Mean~Arterial~Pressure~(mmHg)} & -2.62 & -4.48 &
\textbf{0.025}\tabularnewline
\textbf{Cardiac Output (L/min)} & 0.02 & -0.26 & 0.890\tabularnewline
\textbf{Cardiac Index (L/min/m\textsuperscript{2})} & 0.02 & -0.13 &
0.827\tabularnewline
\textbf{Stroke~Volume~(mL)} & -0.05 & -3.31 & 0.980\tabularnewline
\textbf{Stroke~Volume~Index~(mL/m\textsuperscript{2})} & -0.03 & -1.63 &
0.976\tabularnewline
\textbf{Total~Peripheral~Resistance~(mmHg.s/mL)} & -0.03 & -0.07 &
0.304\tabularnewline
\textbf{Cardiac~Contractility~(mmHg/s)} & -43.43 & -96.70 &
0.175\tabularnewline
\textbf{Left~Ventricular~Ejection~Time (ms)} & -2.33 & -5.63 &
0.236\tabularnewline
*Estimates correspond to the difference between zamicastat and placebo
for cold stimulus -- rest phases on the change from baseline. n: number
of subjects; PP-PD: per-protocol pharmacodynamics. & *Estimates
correspond to the difference between zamicastat and placebo for cold
stimulus -- rest phases on the change from baseline. n: number of
subjects; PP-PD: per-protocol pharmacodynamics. & *Estimates correspond
to the difference between zamicastat and placebo for cold stimulus --
rest phases on the change from baseline. n: number of subjects; PP-PD:
per-protocol pharmacodynamics. & *Estimates correspond to the difference
between zamicastat and placebo for cold stimulus -- rest phases on the
change from baseline. n: number of subjects; PP-PD: per-protocol
pharmacodynamics.\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 5} . Mixed effect model results of plasma catecholamines
concentration -- differences between zamicastat and placebo -- Least
square means before and after CPT at day 10 adjusted to baseline (PP-PD
population, n = 21)\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& \textbf{Before CPT} & \textbf{Before CPT} & \textbf{Before CPT} &
\textbf{After CPT} & \textbf{After CPT} & \textbf{After
CPT}\tabularnewline
\textbf{Variable} & \textbf{Estimate*} & \textbf{One-sided 95\% CI} &
\emph{p}\textbf{-value} & \textbf{Estimate*} & \textbf{One-sided 95\%
CI} & \emph{p}\textbf{-value}\tabularnewline
\textbf{Dopamine (ng/L)} & 20.70 & -6.43 & 0.204 & 18.72 & -5.67 &
0.201\tabularnewline
\textbf{Epinephrine (ng/L)} & 13.86 & -0.93 & 0.122 & 15.93 & 3.30 &
\textbf{0.040}\tabularnewline
\textbf{Norepinephrine (ng/L)} & -27.74 & -102.86 & 0.541 & -18.16 &
-93.28 & 0.689\tabularnewline
\bottomrule
\end{longtable}

*Estimates correspond to the difference between zamicastat and placebo
on the change from baseline.

CI: confidence interval; CPT: cold pressure test; PP-PD: per-protocol
pharmacodynamics.

\textbf{Figures}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 1.} Study design diagram

(EOS: end of study)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 2.} Least square means plots of dopamine (A), epinephrine
(B), norepinephrine (C), normetanephrine (D), homovanillic acid (E), and
vanillylmandelic acid (F) 24-hour urine excretion profiles.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 3} . Percentage of plasmatic inhibition of D\selectlanguage{greek}β\selectlanguage{english}H activity
at pre-dose (trough) following administration of zamicastat 400 mg (once
daily) and placebo (PP-PD population, n = 21).

(D\selectlanguage{greek}β\selectlanguage{english}H: dopamine-\selectlanguage{greek}β-\selectlanguage{english}hydroxylase; PP-PD: per-protocol pharmacodynamics).

\selectlanguage{english}
\FloatBarrier
\end{document}

