Cardiac safety profile of etamicastat, a novel peripheral selective dopamine-ß-hydroxylase inhibitor in non-human primates, human young and elderly healthy volunteers and hypertensive patients

Abstract

The aim of this work was to evaluate the cardiac risk for etamicastat, a peripheral reversible dopamine-ß-hydroxylase inhibitor. Etamicastat blocked the hERG current amplitude with an IC50 value of 44 μg/ml. Etamicastat had no substantial effects on arterial blood pressure, heart rate and the PR interval in male Cynomolgus monkeys when administered orally up to 90 mg/kg. Administered orally at 15 and 45 mg/kg/day in female and male Cynomolgus monkey for 91 days, etamicastat had no effect on heart rate and the waveform or intervals of the electrocardiogram. At the highest dose level of 45 mg/kg, mean plasma concentrations of etamicastat ranged from 1875 to 3145 ng/ml on Day 1 and Day 91 of treatment, respectively. The effect of age on the tolerability and pharmacokinetics of etamicastat in elderly (≥ 65 years) and young adult (18–45 years) subjects showed that supine systolic (SBP) and diastolic (DBP) blood pressure, ECG heart rate, PR interval, QRS duration and QTcF interval were not affected following once-daily administration of 100 mg/day etamicastat for 7 days. In hypertensive patients the decrease of blood pressure tended to be more important in subjects who had received etamicastat (50, 100 and 200 mg) than in subjects who had received placebo. No clinically significant out-of-range values in vital signs or ECG parameters, ECG heart rate, PR interval, QRS duration and QTcF interval were observed in hypertensive subjects following once-daily administration of etamicastat for 10 days. In conclusion, etamicastat is not likely to prolong the QT interval at therapeutic doses.

Full text

 
Cardiac safety profile of etamicastat, a novel peripheral selective dopamine-
ß-hydroxylase inhibitor in non-human primates, human young and elderly
healthy volunteers and hypertensive patients
Manuel Vaz-da-Silva, Jos´e-Francisco Rocha, Pierre Lacroix, Am´ılcar
Falc˜ao, Lu´ıs Almeida, Patr´ıcio Soares-da-Silva
PII: S2214-7624(15)00013-4
DOI: doi: 10.1016/j.ijcme.2015.03.002
Reference: IJCME 68
To appear in: International Journal of Cardiology
Received date: 19 December 2014
Accepted date: 4 March 2015
Please cite this article as: Vaz-da-Silva Manuel, Rocha Jos´e-Francisco, Lacroix Pierre,
Falc˜ao Am´ılcar, Almeida Lu´ıs, Soares-da-Silva Patr´ıcio, Cardiac safety profile of etami-
castat, a novel peripheral selective dopamine-ß-hydroxylase inhibitor in non-human pri-
mates, human young and elderly healthy volunteers and hypertensive patients, Interna-
tional Journal of Cardiology (2015), doi: 10.1016/j.ijcme.2015.03.002
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Cardiac safety profile of etamicastat, a novel peripheral selective dopamine-ß-hydroxylase
inhibitor in non-human primates, human young and elderly healthy volunteers and hypertensive
patients
Manuel Vaz-da-Silva1,2, José-Francisco Rocha3, Pierre Lacroix4, Amílcar Falcão5, Luís Almeida52,6,7 &
Patrício Soares-da-Silva2,3,7
Affiliations:
1 Department of Cardiology, Faculty of Medicine, University of Porto, Portugal.
2 MedInUP - Center for Drug Discovery and Innovative Medicines, University of Porto, Porto,
Portugal.
3 Department of Research and Development, BIAL – Portela & Cª, S.A., 4745-457 S. Mamede do
Coronado, Portugal.
4 Pierre Lacroix Consultant, 3 rue Théodore Botrel, 35830 Betton, France.
5 Laboratory of Pharmacology, Faculty of Pharmacy, University of Coimbra, Portugal.
6 Health Sciences Department, University of Aveiro, Portugal.
7 Department of Pharmacology & Therapeutics, Faculty of Medicine, University of Porto, Portugal.
Correspondence:
P. Soares-da-Silva, Department of Research and development, BIAL – Portela & Cª, S.A., 4745-457 S.
Mamede do Coronado, Portugal, Tel – 351-229866100, Fax - 351-229866102, E-mail –
psoares.silva@bial.com

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Abstract
The aim of this work was to evaluate the cardiac risk for etamicastat, a peripheral reversible
dopamine-ß-hydroxylase inhibitor. Etamicastat blocked the hERG current amplitude with an IC50
value of 44 µg/ml. Etamicastat had no substantial effects on arterial blood pressure, heart rate and
the PR interval in male Cynomolgus monkeys when administered orally up to 90 mg/kg. Administered
orally at 15 and 45 mg/kg/day in female and male Cynomolgus monkey for 91 days, etamicastat had
no effect on heart rate and the waveform or intervals of the electrocardiogram. At the highest dose
level of 45 mg/kg, mean plasma concentrations of etamicastat ranged from 1875 to 3145 ng/ml on
Day 1 and Day 91 of treatment, respectively. The effect of age on the tolerability and
pharmacokinetics of etamicastat in elderly (>65 years) and young adult (18-45 years) subjects
showed that supine systolic (SBP) and diastolic (DBP) blood pressure, ECG heart rate, PR interval, QRS
duration and QTcF interval were not affected following once-daily administration of 100 mg/day
etamicastat for 7 days. In hypertensive patients the decrease of blood pressure tended to be more
important in subjects who had received etamicastat (50, 100 and 200 mg) than in subjects who had
received placebo. No clinically significant out-of-range values in vital signs or ECG parameters, ECG
heart rate, PR interval, QRS duration and QTcF interval were observed in hypertensive subjects
following once-daily administration of etamicastat for 10 days. In conclusion, etamicastat is not likely
to prolong the QT interval at therapeutic doses.
Key words: Etamicastat; cardiac risk assessment; QT prolongation.
Running title: Pre-clinical and clinical assessment of cardiac risk for etamicastat.

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Introduction
Iteest i the deelopet of ihiitos of dopaie β-hdolase DβH; EC ...;
dopaie β-monooxygenase) is centered on the hypothesis that inhibition of this enzyme may
provide significant clinical improvements in patients suffering from cardiovascular disorders such as
hypertension or congestive heart failure [1-8]. The atioale fo the use of DβH ihiitos is ased o
their capacity to inhibit the biosynthesis of noradrenaline, which is achieved via enzymatic
hydroxylation of dopamine [9-13].
“eeal DβH ihiitos hae ee epoted [4-6], but none achieved marketing approval due to
weak potency, poor DBH selectivity [14] and/or significant adverse effects [15]. Nepicastat, (Figure 1)
a 5-substituted imidazole-2-thione derivative, is a highly potent DβH inhibitor that, in beagle dogs,
produced a dose-dependent noradrenaline reduction and dopamine increase in renal artery, heart
left ventricle and cerebral cortex [7]. These data indicate that nepicastat crosses the blood brain
barrier (BBB) causing central as well as peripheral effects, a situation that could lead to undesired
and potentially serious CNS adverse effects. Etamicastat, also known as BIA 5-453, (Figure 1) is a
reversible DßH inhibitor that prevents the conversion of dopamine to noradrenaline in
sympathetically innervated tissues and reduces sympathetic nervous system activity [1, 2, 14]. As a
result of its reduced ability to cross the blood-brain barrier [1], etamicastat acts preferentially in the
periphery and is currently being developed for the treatment of cardiovascular diseases. In contrast
to the effects in peripheral tissues, etamicastat failed to affect dopamine and noradrenaline tissue
levels in the brain [1], which is unique among the DßH inhibitors previously tested for the treatment
of cardiovascular disorders [3, 8, 14].
As previously observed with other DH inhibitors that are endowed with potent antihypertensive
effects in the spontaneously hypertensive rat [6], etamicastat showed to reduce both systolic (SBP)
and diastolic (DBP) blood pressure, alone or in combination with other antihypertensive drugs, and
to decrease the urinary excretion of noradrenaline in spontaneously hypertensive rats with no
change in heart rate [16-19]. Recently, etamicastat demonstrated blood pressure lowering effects in
hypertensive patients [20]. In healthy subjects, etamicastat was well tolerated and showed
approximate linear pharmacokinetics following single oral doses [21] and multiple once-daily oral
doses [22], with no significant differences being observed in elderly versus young healthy subjects
[23].
The aim of the present work was to evaluate the effects of etamicastat for cardiac risk both in
vitro, testing on the hERG potassium channel of human embryonic kidney (HEK293) cells, and in vivo
in the Cynomolgus monkey monitored by telemetry (up to 90 mg/kg etamicastat). This evaluation
was complemented in male and female Cynomolgus monkey dosed daily with etamicastat (up to 45
mg/kg/day) for at least 91 days, and in three clinical studies in human healthy volunteers receiving
single doses of etamicastat (up to 1200 mg), young and elderly healthy subjects receiving etamicastat

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(100 mg/day) for 7 days, and hypertensive patients administered once-daily with etamicastat (up to
200 mg) for up to 10 days.
Methods
1. In vitro studies
1.1. DβH activity
DβH atiit as easued  a odifiatio of the ethod of Nagatsu ad Udefied [24],
which is based on the enzymatic hydroxylation of tyramine into octopamine, in SK-N-SH cell
homogenates. SK-N-SH cells (ATCC HTB-11) obtained from LGC Standards (Tedington, UK) were
cultured in Eagle´s minimum essential medium supplemented with 25 mM Hepes, 100 U/ml penicillin
G, 0.25 µg/ml amphotericin B, 100 µg/ml streptomycin and 10% Gibco® fetal bovine serum. Cells
were grown in T162 cm flasks (Corning, NY) in a humidified atmosphere of 5% CO2-95% air at 37ºC.
For the preparation of homogenates, fetal bovine serum was removed from cell medium 4 h prior to
homogenate preparation. At the appropriate time media was removed and cell monolayers were
washed with 50 mM Tris-HCl pH 7.4. Cells were subsequently scrapped off the flasks and were
resuspended in 50 mM Tris pH 7.4. Cell suspensions were homogenized with SilentCrusher M
(Heidolph) for a short stroke and homogenates were aliquoted and were stored frozen at -80ºC.
Total protein in cell homogenates was determined with the BioRad Protein Assay (BioRad) using a
standard curve of BSA (50-250 µg/ml). The octopamine formed is subsequently oxidized to p-
hydroxybenzaldehyde and measured by spectrophotometry. Experimental assay conditions for the
cellular homogenates were previously optimized by evaluating time and protein dependency of the
enzymatic assay. In brief, reaction mixture (total volume 500 µl) contained: cellular homogenate (75
µg total protein) sodium acetate pH 5.0 (200 mM), NEM (30 mM), CuSO4 (5 µM), catalase aqueous
solution (0.5 mg/ml), pargyline-HCl (1 mM), sodium fumarate (10 mM), ascorbic acid (10 mM),
inhibitor or vehicle and tyramine (25 mM). After a 10 min pre-incubation period at 37ºC, the reaction
was initiated by the addition of tyramine. Reaction was carried out for 45 min at 37ºC before
termination with 50 µl PCA (2 M). Samples were centrifuged for 3 min at 16100 g and supernatants
were transferred to SPE cartridges ISOLUTE SCX-3 (100 mg, 1 ml) previously equilibrated with MilliQ
water. Columns were centrifuged at 150 g for 2 min. Eluate was discarded and matrix was washed
with 1 ml of MilliQ water after which octopamine was eluted with 2 x 0.25 ml ammonium hydroxide
(4 M). The oxidation of octopamine to p-hydroxybenzaldehyde was carried out for 6 min with 100 µl
sodium periodate (2 % ) and was stopped with 100 µl sodium metabisulfite (10 % ). Absorbance was
measured at 330 nm on a Spectramax microplate reader (Molecular Devices, Sunnyvale, CA). Under
the experimental conditions described in above, cellular homogenates were incubated with various
concentrations (1, 3, 10, 30, 100, 300, 1000, 3000 nM) of either etamicastat, or nepicastat.

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1.2. hERG K+ channel
The whole-cell patch-clamp technique was used to investigate the effects of etamicastat and
nepicastat on hERG potassium channels stably expressed in stably transfected human embryonic
kidney (HEK 293) ells. Both opouds ee tested at oetatios of . to . μg/l i ode
to determine their effects on the hERG mediated current. All solutions applied to cells including the
pipette solution were maintained at room temperature (19-30°C). A vehicle group (water for
injection 1% used as solvent for etamicastat and nepicastat) was included in the study for
comparison, and E-4031 (1 µM), which selectively blocks the rapid delayed rectifier potassium
current IKr, was used as reference substance.
HEK 293 cells stably expressing the hERG channel were incubated at 37°C in a humidified
atmosphere with 5% CO2. For electrophysiological measurements, HEK 293 cells were seeded onto
35 mm sterile culture dishes containing 2 ml culture medium without antibiotics. Tetracycline was
added to induce channel expression. Because responses in distant cells are not adequately voltage
clamped and because of uncertainties about the extent of coupling [25], cells were cultivated at a
density that enabled single cells (without visible connections to neighboring cells) to be measured.
The cells were continuously maintained in and passaged in sterile culture flasks containing a 1:1
mixture of Duleo’s odified eagle ediu ad utiet itueF-12 (D-MEM/F-12 1x, liquid, with
GlutaMax I, Gibco-BRL) supplemented with 9 % fetal bovine serum (Gibco-BRL) and 0.9 %
Penicillin/Streptomycin solution (Gibco-BRL). The complete medium as indicated above was
supplemented with 100 mg/ml Hygromycin B (Invitrogen) and 15 mg/ml Blasticidin (Invitrogen). The
final bath solution had the following composition (in mM): NaCl 137, KCl 4, CaCl2 1.8, MgCl2 1, HEPES
10, d-Glucose 10, pH (NaOH) 7.4. The pipette solution had the following composition (in mM): KCl
130, MgCl2 1, Mg-ATP 5, HEPES 10, EGTA 5, pH (KOH) 7.2. The 35 mm culture dishes upon which cells
were seeded at a density allowing single cells to be recorded were placed on the dish holder of the
microscope and continuously perfused (approximately 1 ml/min) with the bath solution. All solutions
applied to cells including the pipette solution were maintained at room temperature (19-30°C). After
formation of a Gigaohm seal between the patch electrodes and individual hERG stably transfected
HEK 293 cells (pipette resistance range: 2.0 MW - 7.0 MW; seal resistance range: > 1GW), the cell
membrane across the pipette tip was ruptured to assure electrical access to the cell interior (whole-
cell patch-configuration). As soon as a stable seal was established, hERG outward tail currents were
measured upon depolarization of the cell membrane to +20 mV for 2 s (activation of channels) from
a holding potential of -80 mV and upon subsequent repolarization to -40 mV for 3 s. This voltage
protocol was run at least 10 times at intervals of 10 s. If current density was judged to be too low for
measurement, another cell was recorded. Once control recordings had been accomplished, cells
were continuously perfused with a bath solution containing etamicastat at 3, 10, 30 and 100 µM.
During wash-in of the test item, the voltage protocol indicated above was run continuously again at

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10 s intervals until the steady-state level of block was reached. Complete cumulative concentration-
response analysis was accomplished per cell and the IC50 value was calculated. After measurement of
the control period, concentrations of etamicastat were applied to the perfusion bath. During wash-in
of etamicastat, the voltage protocol was run until the steady-state level of channel inhibition was
reached. Values (in pA/nA) of the peak amplitudes of outward tail currents were generated for each
voltage step. The recorded current amplitudes at the steady-state level of current inhibition were
compared to those from control conditions measured in the pre-treatment phase of the same cell.
The amount of current block was calculated as percentage of control. To determine whether any
observed current inhibition was due to etamicastat interaction with the hERG channel or due to
current rundown, these residual currents were compared to those measured in vehicle treated cells.
Data from 3 individual cells were collected and the corresponding mean values and standard errors
calculated. For the validation of the test system, the selective IKr blocker E-4031 was evaluated at 100
nM in 3 cells.
2. In vivo studies
2.1. Cynomolgus primates
For cardiac and pharmacokinetic assessments, male and female Cynomolgus monkeys, with
bodyweight ranging from 1.8-2.7 kg (females) and 2.2-3.1 kg (males), were obtained from Bioculture
Mauritius Ltd (Senneville, Riviere des Anguilles, Mauritius) or Guangxi Grand forest Scientific Primate
Company (Beijing, China). The animals were housed in group cages. Each cage contained all animals
of the same sex and treatment group. The animal house was maintained under a 12-h fluorescent
light / 12-h dark cycle at a controlled ambient temperature of 20-24°C and relative humidity ranged
from 40-70% . Animal diet consisted of pelleted standard Teklad diet 2055 and 2055C monkey diet
(150 g/day/animal) and a fresh piece of fruit four times a week. During the treatment period, the diet
was offered approximately one hour after completion of dosing. Any remaining diet was withdrawn
early on the following day. All animal interventions were performed in accordance with the European
Dietie ue /, ad the ules of the Guide fo the Cae ad Use of Laoato Aials,
7th edition, 1996, Institute for Laboratory Animal Research (ILAR), Washington, DC.
2.1.1. Telemetry monitored evaluations in Cynomolgus primates
The effects of etamicastat (15, 45 and 90 mg/kg) on arterial blood pressure, heart rate and the
main parameters of the electrocardiogram were evaluated following oral (p.o. capsule)
administration in the conscious male Cynomolgus primate monitored by telemetry. The doses of
etamicastat were based on the results from a maximum tolerated dose (MTD) study in Cynomolgus
monkeys in which the MTD was established as 120 mg/kg/day (BIAL data on file). There was 1
treatment group of 4 primates following a 4x4 Latin-square design. There was a washout period of 1
week between each treatment. Animals were dosed at approximately the same time each day.

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Venous blood samples (approximately 1 ml) for determination of etamicastat in plasma were taken
prior to dosing and at 2 and 4 h after the start of dose administration, or as close as was reasonably
practicable to these time points.
Four male Cynomolgus primates were surgically implanted with telemetry transducers, type
TL11M2-D70-PCT (Data Sciences International), for the measurement of arterial blood pressure and
lead II electrocardiogram (ECG). Briefly, using sterile techniques and with the animals anaesthetized
with isofluorane, the implant body was placed intraperitoneally in the lower quarters of the
abdominal cavity and the blood pressure catheter introduced into the femoral artery with the tip
estimated to be in the terminal abdominal aorta. The ECG electrodes were tunneled subcutaneously
and fixed in the thorax to give a lead II ECG waveform morphology. Approximately 3 weeks later, i.e.
after a recovery period during which visual inspection of waveform morphology of arterial blood
pressure and of the ECG was performed at times to confirm suitability of the animals, a telemetry
eeie as positioed ea eah aial’s hoe age to eod mean blood pressure (MBP), SBP
and DBP and heart rate (HR), which was derived from pulse blood pressure. The PR and the QT
iteals s ee also easued ad the QT iteal as alulated aodig to Fideiia’s
formula QTcF = QT (ms) / 3[60/HR(bpm), and to individual animal specific correction formula
QTcQ = QT (ms) + #(500-RR). Arterial blood pressure, heart rate and lead II ECG variables in all
groups were extracted at -0.5, 1, 2, 4, 6, 8, 10, 14, 18 and 22 h post-dose, where time 0 is the time at
the end of dosing. Etamicastat was examined at 3 ascending single doses, i.e. 15, 45 and 90 mg/kg,
administered p.o. (as powder using gelatine capsules). Each animal received the vehicle (empty
capsule), then etamicastat, with a washout period of at least 5 days between each treatment. The
animals were offered food between approximately 2 h post-dosing, on completion of the blood
sampling. Approximately 1 ml of venous blood was collected from a jugular vein into lithium-
heparinized glass vacuum tubes before and 2 and 4 h after administration of etamicastat or vehicle;
the tubes were stored in ice until centrifugation (approximately 1500 g at 4C for 15 minutes) and
until required for analysis.
2.1.2. Cardiac evaluation after repeated oral administration in Cynomolgus primates
Four animals of each sex were dosed daily by oral capsule at 0, 15 and 45 mg/kg/day for at least
91 days. On each day of treatment, the animals were housed individually immediately prior to and
after dosing to allow detection of any treatment-related clinical signs. Thereafter, the animals were
released into group cages to allow interaction within the dose group and sex. Animals were dosed
orally by gastric gavage and individual doses were adjusted weekly according to the most recently
recorded bodyweight. ECGs were recorded for all animals before the start of treatment and again
before dosing and 1 h after dosing on one day during Week 1 and Week 13 of the study.
Electrocardiograms were obtained using Einthoven (I, II and III) and Goldberger (aVR, aVL, aVF) leads.
The heart rate, P wave duration and amplitude, P-Q interval, QRS interval and Q-T intervals were

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measured manually using a representative section of the electrocardiogram from lead II, on the basis
of an average of at least 10 consecutive complexes data
Blood samples for determination of plasma concentrations of etamicastat were taken, by direct
vein puncture, into lithium-heparin tubes at pre-dose and then at 1, 2, 4, 8, 12 and 24 h after dosing.
After collection, blood samples were centrifuged at approximately 1500 g for 10 min at 4°C. The
resulting plasma was then separated into 2 aliquots of 250 µL and stored at -80°C until required for
analysis.
2.2. Human subjects
The healthy status of human volunteers was determined by an interview, medical history, physical
examination, vital signs, 12-lead ECG, and results of clinical laboratory safety tests (including
hematology, plasma biochemistry), urinalysis, drugs of abuse screen, and HIV and hepatitis B and C
serology considered clinically acceptable at screening and admission. At admission to the unit, the
medical history and physical examination were updated. Etamicastat was administered in the
morning, with 250 mL of water, after at least 8 hours of fasting. The doses were obtained as
combinations of etamicastat 1 mg, 10 mg and 50 mg capsules and placebo capsules identical in
appearance manufactured by BIAL (S. Mamede do Coronado, Portugal). Healthy subjects
participating in the single ascending dose study were requested to abstain from consuming
grapefruit or other citrus (e.g. orange) or their juice, and alcohol- or xanthine-containing beverages
or foods until 120 h after the last dose. Hypertensive patients participating in the hypertension study
were required to abstain from strenuous physical activity, smoking, consumption of grapefruit or
other citrus fruit (e.g. orange) or of their juice, alcohol and beverages containing xanthine derivatives
(i.e. no coffee, tea, chocolate or Coca-Cola like drinks) from 2 days before the first dose until 72 h
after the last dose. Concomitant medications were not permitted throughout the study, unless
required for treatment of adverse events (AEs). Blood samples (3 ml) for the determination of plasma
concentrations of etamicastat and its metabolite BIA 5-961 were collected in lithium-heparin
Vacuette® (Greiner Bio-One) tubes by means of an indwelling catheter or venipuncture at the
following time points: pre dose, and at 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36, 48, 72, 96, and 120 h post
dose in the young and elderly study, or at pre-dose and 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, 48 and
72 h post last dose (Day 10) in the hypertensive patient study. After collection, blood samples were
placed on ice and within 60 minutes after collection, the blood samples were centrifuged at
approximately 1500 g and at 4°C during 10 min. For the assay of etamicastat and metabolites, 4
aliuots of  μl eah of the esultig plasa were withdrawn and placed into 2-ml cryotubes which
were labeled, frozen and stored at <-70°C until analysis. Safety was evaluated from reported AEs,
physical examination, vital signs, digital 12-lead ECG, and clinical laboratory test results.

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2.2.1. Cardiac evaluation after single ascending administration in young healthy humans
This was a single-center, entry-into-man, phase 1, double-blind, randomized, placebo-controlled
study (trial registration EudraCT No. 2007-001181-33) in 10 sequential groups of 8 healthy male
subjects each. In compliance with a randomization list generated by using computerized techniques,
within each group 2 subjects were randomized to receive placebo and the remaining 6 to receive
etamicastat. Etamicastat was administered as single oral doses of 2, 10, 20, 50, 100, 200, 400, 600,
900 or 1200 mg. Eligible volunteers were admitted to the unit 2 days prior (Day -2) to receiving the
study medication (Day 1) and remained in the unit under clinical supervision for at least 72 h after
dosing (Day 4). During admission, SBP and DBP and HR were recorded in supine position (after
resting for at least 10 minutes) using a Dinamap® (GE Healthcare) monitor at the following times: Day
-1 (the day prior to dosing) – time 0 (24 h before dosing) and 1, 2, 3, 4, 5, 6, 8, 10, 12 and 16 h after;
Day 1 (dosing day) – pre-dose and 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, 48, and 72 h post-dose. Orthostatic
blood pressure and HR were recorded in standing position (after standing for approximately 2
minutes) at the following times: Day -1 – time 0 and 2, 4, 8, and 12 h after; Day 1 – pre-dose and 2, 4,
8, 12, and 24 h post-dose. 12-lead digital ECG were recorded on Day -1 at time 0 and 1, 2, 3, 4, 6, 8,
10, 12 and 16 h after, and on Day 1 at pre-dose and 1, 2, 3, 4, 6, 8, 10, 12, 16, 24, 48, and 72 h post-
dose. Digital ECGs were recorded after 10 minutes of rest, in triplicate (with an interval of 5 minutes,
with a difference of at least 1 minute between each of the 3 recordings). A general safety assessment
and detailed pharmacokinetic evaluation was previously made available [21].
2.2.2. Cardiac evaluation after repeated oral administration in young and elderly healthy volunteers
This was an open-label, single-center, parallel-group study (trial registration EudraCT No. 2008-
002127-82) in a group of 12 healthy young male subjects and a group of 12 healthy elderly male
subjects. Participants were drawn from the study center’s pool of olutees. Volutees eligile fo
participation were healthy male volunteers between 18 and 45 years (young group) or 65 years or
more (elderly group), non-smokers or smokers of less than 10 cigarettes per day. Subjects were
considered ineligible for participation if they: had been administered any investigational drug within
90 days, prescription drug within 30 days or over-the-counter drugs within 7 days before admission;
showed to be prone to orthostatic hypotension, defined by a difference between supine SBP and
stadig “BP  Hg o a diffeee etee supie DBP ad stadig DBP  Hg; o
peseted a ECG QT iteal eadig  s oug sujets o  s eldel sujets. The
trial consisted of a 100 mg etamicastat multiple-dose period during which participants received 100
mg etamicastat once-daily, for 7 days. A general safety assessment and detailed pharmacokinetic
evaluation was previously made available [23].
2.2.3. Cardiac evaluation after repeated oral administration in hypertensive patients
This was a Phase IIa, double-blind, randomized, placebo-controlled study (trial registration
EudraCT No. 2008-002789-69) investigating three dosage regimens of etamicastat (50, 100 or 200

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mg, once-daily, for 10 days) in 3 groups of 8 hypertensive male patients aged between 18 and 45
years. Within each group, it was planned to randomize 2 subjects to receive placebo and the
remaining 6 subjects to receive etamicastat. Randomization was performed by means of
computerized techniques. Doses of 50, 100 and 200 mg of etamicastat were investigated in
ascending order. The decision to proceed to the next dose depended on the tolerability assessment
of the previous dose level. Each patient participated in the study for approximately 8 weeks.
Participation included several screening evaluations within 4 weeks before admission to an inpatient
period (Day -1 to Day 11), two ambulatory visits (Days 12 and 13) and a follow-up visit 7 to 10 days
after the last administration. During the screening period, subjects received placebo once daily, in
the morning, in a single-blind manner (subjects did not know they were receiving placebo, but the
investigator was aware). Patients were admitted to the research facility in the morning of Day -1 (day
before the first dose of investigational product) and remained inpatient until at least 24 h after the
last dose, on Day 11. Then patients were discharged and instructed to return for the 48 h (Day 12)
and 72 h (Day 13) post last dose assessments. During the inpatient period, from Day 1 to Day 10,
participants received in a double-blind fashion either etamicastat 50, 100 or 200 mg or placebo, once
daily, in the morning, with 250 mL water, under fasting conditions. Patients remained fasted until 4 h
post-dose on Days 1 and 10, and until 1 h post-dose on Days 2 to 9. A general safety assessment and
detailed pharmacokinetic evaluation was previously made available [20].
Safety was assessed from reported AEs, physical examination, vital signs, digital 12-lead ECG, and
clinical laboratory test results. HIV and hepatitis B and C serology, and drugs of abuse screen were
performed at screening; alcohol test was performed at screening and Day -1. Laboratory safety tests
(hematology, blood chemistry and urinalysis) were performed at screening, Day -1, pre-dose on Days
5 and 10, Day 13 and follow-up visit.
Digital 12-lead ECG recordings were taken in supine position, after at least a 10-minute rest, at
the screening visit and then at the following times: Day -1: time 0 (24 h before first dosing), and 1, 2,
3, 4, 6, 8, 10, 12 and 16 h after; Day 1 (day of first dosing): pre-dose, and 1, 2, 3, 4, 6, 8, 10, 12 and 16
h post-dose; from Day 2 to Day 9: pre-dose and 2 h post-dose; Day 10 (day of last dosing): pre-dose,
and 1, 2, 3, 4, 6, 8, 10, 12, 16, 24, 48 and 72 h post-dose. The reported 12-lead ECG parameters
included HR, PR interval, QRS interval duration and axis deviation, and QT interval. QT interval was
corrected by the Frideiia’s QTF foula. The primary method of correction was QTcF. A manual
reading of the digital ECGs was used to assess QT/QTc interval prolongation.
Safety standing blood pressure and HR measurements taken after the patient had been standing
for 2 minutes were performed on Day -1, and at pre-dose and 2, 12 and 24 h post first (Day 1) and
last dose (Day 10).

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3. Assay of etamicastat and metabolites in plasma
Plasma concentrations of etamicastat and BIA 5-961 were determined using a validated method
consisting of reversed phase liquid chromatography coupled with triple-stage quadrupole mass-
spectrometric detection (LC/MS-MS), as previously described [26]. In brief, for the preparation of
calibration samples, etamicastat and BIA 5-961 were dissolved in methanol to a final concentration of
 μg/l (plasma assay). For the quality control (QC) samples, a second set of stock solutions was
prepared. For calibration and QC samples, working solutions in methanol were added to plasma
using a ratio of 2/98 (v/v). For the preparation of the internal standard (ISTD) solution for the plasma
assay, reference standard (BIA 5-1058, molecular formula C21H21F2N3OS) was dissolved in methanol
to a oetatio of  μg/l and then diluted in methanol to 5000 ng/ml; further dilutions to a
final concentration of 50 ng/ml were done using acetonitrile/ethanol (50/50, v/v). For the
preparation of the ISTD solution for the urine assays, reference standard was dissolved in methanol
to a final concentration of 50 ng/ml. Plasma samples were vortexed and centrifuged for 20 min at
approximately 3362 g and approximately 8ºC after unassisted thawing at room temperature. To an
aliquot of 100 l of plasa,  μl of acetonitrile/ethanol (50/50, v/v) containing 50 ng/ml of ISTD
were added. After protein precipitation at room temperature, plasma samples were filtrated using a
Captia filte plate ad a aliuot of  μl of the filtrate was injected onto the analytical column. To an
aliquot of 20 L of uie,  μl lithium-hepai plasa ee added ad ee peipitated   μL
of methanol containing the ISTD. After protein precipitation at room temperature, urine samples
were centrifuged for 20 min at 2773 g and 8ºC. A aliuot of  μl of the supernatant was
transferred into an ultrafiltration filter plate and centrifuged for about 2 h at 4000 rpm (2773 g) and
appoiatel ºC. A aliuot of  μl was injected onto the analytical column. The samples were
stored in the autosampler tray at approximately 8°C±5°C.
The analytical equipment consisted of a Rheos 2000 pump (Flux Instruments, Basel, Switzerland),
a SpeedROD, RP18e, 50-4.6 mm analytical column (Merck, Darmstadt, Germany), a ultra-low volume
precolumn filter,  μ Uphuh “ietifi I, Oak Hao, WA, U“A, a T“Q Quatu ass
spectrometer (Thermo Fisher Scientific, San Jose, CA, USA), and a HTS PAL autosampler (CTC
Analytics AG, Zurich, Switzerland). The MS detector was operated in positive ion mode with mass
transitions for etamicastat, BIA 5-961 and the ISTD of respectively 283.0 amu, 127.0 amu and 120.0
amu. Column temperature was 50ºC. The mobiles phases used water containing 0.5% formic acid
(phase A), water containing 1.0% formic acid (phase B), acetonitrile containing 1.0% formic acid
(phase C) and acetonitrile containing 0.01% formic acid (phase D). Calibration curves over the
nominal concentration range 5-5000 ng/ml for plasma assays and a set of quality control (QC)
samples were analyzed with each batch of study samples. The QC samples were prepared in
duplicates at three concentration levels (low, medium and high). The analytical method was
demonstrated to be precise and accurate. The descriptive statistics of the QC samples showed that

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the overall imprecision of the method, measured by the inter-ath oeffiiet of aiatio, as 
.% fo etaiastat ad .% fo BIA -961. The inter-batch accuracy ranged from 101.5% to
105.3% for etamicastat and 101.0% to 105.3% for BIA 5-961. The lower limit of quantification of the
assay (LLOQ) was 5 ng/ml in plasma, for both compounds.
4. Statistical analysis
Reported values are expressed as means ± standard error of the mean (SEM) or means with
confidence intervals (CI). Statistical analysis was performed using the Dynamic Microsystem software
GB-Stat version 6.5. For in vitro (hERG) study, ultisaple aalsis ANOVA folloed  Duett’s
test) was performed to test statistical significance of all concentrations tested. For in vivo (telemetry)
studies, intra-group comparison was performed using an one-way analysis of variance (time) with
epeated easues at eah tie, folloed  Duett’s t test i ase of sigifiat tie effet, to
compare each time value with the T0 values (i.e. basal value before each treatment). Inter-group
statistical analysis was also performed using a two-way analysis of variance (group, time) with
repeated measures at each time, followed by a one-way analysis of variance (group) at each time in
ase of sigifiat goup  tie iteatio. The aalsis as opleted  Duett’s t tests hee
the group effect was significant. In case of data considered invalid or missing data, the retained value
was taken 5 min before or after the theoretical time, or was represented by the mean of data taken 5
or 10 min before and 5 or 10 min after the theoretical time.
5. Drugs
Etamicastat, BIA 5-961 and reference standard (BIA 5-1058) were supplied by BIAL (Laboratory of
Chemistry, S. Mamede do Coronado, Portugal).
Results
6. In vitro studies
6.1. DβH activity
The experimental conditions, for evaluating the conversion of tyramine into octopamine by
human SK-N-SH cell homogenates, were previously optimized with time and protein dependency
experiments (data not shown). The formation of octopamine was dependent on the incubation time
up to 1 h (r2=0.998) and on protein amount up to 125 µg total protein per assay (r2=0.995). With 75
µg total protein and 45 min incubation time, octopamine was formed by SK-N-SH cell homogenates
with a Km value of 9 (CI, 6; 13) mM for tyramine and a Vmax of 1725±76 nmol/mg protein/h. Under
these optimized conditions, etamicastat and nepicastat inhibited SK-N-“H DβH activity in a
concentration dependent manner (Figure 2) with IC50 values (in ng/ml) of 37.2 (CI, 30.7; 44.9) and
13.3 (CI, 11.6; 15.4), respectively.

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6.2. hERG K+ channel
Etamicastat and nepicastat produced reductions of hERG current amplitude (Figure 3) with IC50
values μg/l of 44.0 (CI, 34.8; 55.7) and 5.6 (CI, 4.7; 6.5), respectively. In comparison, the positive
control substance E-4031 at 100 nM markedly blocked the hERG tail current (8.88 ± 1.65% relative
tail current). The observed inhibition of hERG tail currents by E-4031 was in line with its known
pharmacological profile [27].
7. In vivo studies
7.1. Cynomolgus primates monitored by telemetry
The effects of etamicastat (15, 45 and 90 mg/kg) on arterial blood pressure, heart rate and the
main parameters of the electrocardiogram were evaluated following oral (p.o. capsule)
administration in the conscious male Cynomolgus primate monitored by telemetry (Table 1 and
Figure 4). The doses of etamicastat were based on the results from a maximum tolerated dose (MTD)
study in Cynomolgus monkeys in which the MTD was established as 120 mg/kg/day (BIAL data on
file). There was 1 treatment group of 4 primates following a 4x4 latin square design. There was a
washout period of 1 week between each treatment. Animals were dosed at approximately the same
time each day. Venous blood samples (approximately 1 ml) for determination of test substance in
plasma were taken prior to dosing and at 2 and 4 h after the start of dose administration, or as close
as was reasonably practicable to these time points. Loose faeces were noted in the cage of 1 animal
following the administration of 90 mg/kg etamicastat. Although this was an isolated incidence in a
single animal, it was observed following the highest dose of test substance and therefore may be
related to etamicastat administration.
Arterial blood pressure (SBP, DBP and MBP) was generally unaffected following the administration
of etamicastat at any of the doses examined with the exception of a non statistically significant
increase 2 h following administration of 15, 45 and 90 mg/kg etamicastat (Table 1 and Figure 4).
However, the magnitude of these changes were small, reflecting increases from pre-dose baseline in
MBP of 12.1±8.3% , 10.2±5.2% and 2.3±4.0% following 15, 45 and 90 mg/kg etamicastat
administration, respectively, in comparison to a decrease of 2.0±1.5% at this time following placebo
treatment and were mainly due to an increase in the arterial blood pressure of a single animal (Table
1). Furthermore, no dose-dependency was evident, consequently, the observed increases were not
considered to be etamicastat related. In addition, a decrease in arterial blood pressure was evident
22 h following administration of 90 mg/kg etamicastat (p<0.05 for DBP and MBP) (Figure 4B). At this
time, arterial blood pressure was maintained at similar values to those observed throughout the dark
phase of the aials’ light / dak le, as ithi the age of alues oseed folloig plaeo
treatment and as this decrease was noted some considerable time following an oral dose, it was not
considered to be related to the administration of etamicastat.

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HR was noted to be slightly elevated, although not significantly, in comparison to placebo at 2 h
following administration of 15, 45 and 90 mg/kg etamicastat (Figure 4C). However, when compared
to pre-dose baseline values, this increase was no longer evident. HR was otherwise unaffected
following administration of etamicastat with the exception of a tendency for this parameter to be
decreased 14 h following the administration of 45 mg/kg etamicastat. As this was an isolated
incidence, not observed following the highest dose of etamicastat and was noted some time
following an oral administration, it was not considered to be etamicastat related. RR interval was
generally unaffected following administration of etamicastat with the exception of changes
concomitant with those observed in HR (Table 1). Similarly, and for reasons discussed for HR, these
changes were not considered to be related to the administration of etamicastat. PR interval and QRS
duration were unaffected following the administration of etamicastat at any of the doses examined
(Figure 4E).
There was no marked effect on QT interval following administration of 15, 45 or 90 mg/kg
etamicastat with the exception of a prolongation 14 h following administration of 45 mg/kg
etamicastat, corresponding with the changes observed in heart rate and RR interval at this time.
When the QT interval was corrected for changes in HR / RR interval by calculation of the QTcF
interval (Figure 4F), this prolongation, although not significant, was still observed; however, when
corrected using the individual animal specific correction formula, QTcQ, it was no longer apparent
(Table 1). A slight shortening of the QTcQ interval was noted at 14 h following administration of
15 mg/kg etamicastat when compared to placebo (Table 1). However, values remained unchanged
when compared to pre-dose baseline; therefore, this was not considered to be a drug-related effect.
Mean plasma concentrations of etamicastat at 2 and 4 h were 610160 and 64668, 771304 and
1968356, and 105576.5 and 1926592 ng/ml at the dose levels of 15, 45 and 90 mg/kg,
respectively.
7.2. Cardiac evaluation after repeated oral administration in Cynomolgus primates
In this set of experiments, cumulative effects of etamicastat were assessed in the female and
male Cynomolgus monkey following oral administration by gastric gavage once daily for at least 91
days. Animals received etamicastat at 0, 15 or 45 mg/kg/day; the control group only received distilled
water as vehicle. Diarrhea and soft feces were frequently observed in groups receiving 15 or 45
mg/kg/day etamicastat. In all groups, no treatment-related ophthalmoscopic alteration was
recorded. No effects resulting from treatment with etamicastat were observed on hematology,
biochemistry or urinalysis. There were no treatment-related findings in the electrocardiograms (heart
rate, P amplitude, P duration, P-Q interval, QRS interval and Q-T interval) (Table 2). Occasional
incidences of incomplete right bundle branch block were observed in three vehicle-treated animals
and sinus arrhythmia was observed in two vehicle-treated and one animal receiving 45 mg/kg/day

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etamicastat. These were considered not related to treatment with etamicastat as they were seen at
similar incidences in both groups and also observed during acclimatization.
Plasma samples were obtained on Day 1 and in Day 91 of the treatment period at 0 (pre-dose) 1,
2, 4, 8, 12 and 24 h after administration of etamicastat. All samples were analyzed for concentrations
of etamicastat. All plasma concentrations of etamicastat in male and female monkeys receiving only
vehicle were below the limit of quantification (5 ng/ml). All animals in the treated dose groups were
consistently exposed to etamicastat after single and repeated oral (gavage) administration. The
number of quantifiable samples was sufficient to calculate AUC0-t, tmax, Cmax, and t½,z, from the
concentration versus time profiles (Figure 5) and to investigate the effects of dose, gender and single
versus repeated administration on exposure. Maximum plasma concentrations (Cmax) of etamicastat
in female and male monkeys in all groups were reached 1 to 8 h after administration (Table 3). After
tmax, plasma concentrations declined rapidly (Figure 5) with half-lives ranging between 5.0 to 8.4 h
(Table 3). Throughout all groups, mean half-lives seemed to be dose, gender or time independent.
After single administration of etamicastat from 15 to 45 mg/kg/day with 3.0-fold dose increase,
exposure to etamicastat in males increased less than expected the given dose increase: AUC0-t
increased by a factor of 2.00.6. A similar increase was observed for Cmax (ratio of 2.00.5). In
females, from 15 to 45 mg/kg/day AUC0-t and Cmax increased more than dose-proportionally (ratios of
4.11.6 and 3.61.5, respectively). After 13 weeks of repeated treatment, from 15 to 45 mg/kg/day
with 3.0-fold dose increase, exposure to etamicastat in both gender increased more than dose-
proportionally: AUC0-t and Cmax increased by a factor of 3.91.2 and 3.61.1 for females and 3.41.2
to 3.51.1 for males, respectively. Throughout all groups, half-lives seemed to be dose-independent
(half-lives ratios of 0.90.2 to 1.00.1).
7.3. Cardiac evaluation after single ascending administration in young healthy humans
A total of 128 male subjects were screened in order to include 80 subjects in 10 successive groups
of 8 subjects each. All 80 included subjects were randomized and completed the study. There was no
subject withdrawal or premature discontinuation. No relevant differences in demographic
characteristics were found between the treatment groups (Table 4). Figure 6 displays the maximum
change from baseline of supine SBP, DBP, HR and QTcF interval in young healthy subjects after once-
daily single administration of placebo or etamicastat (2 to 1200 mg). There was no clinically
significant change in SBP, DBP, HR and QTcF interval, though important variability was observed. A
total of 18 AEs were reported in 13 subjects. There was no serious AE and no TEAE required the
withdrawal of a subject. All TEAEs were mild to moderate in intensity. Etamicastat was well tolerated
at all the investigated dose levels. Mean Cmax and area under the plasma concentrations versus time
curve from time 0 to infinity (AUC0-∞) etamicastat plasma levels following single doses of 2, 10, 20,
50, 100, 200, 400, 600, 900 and 1200 mg of etamicastat are displayed in Figure 6. Etamicastat plasma
concentrations could not be detected at the 2 mg dose level and were detected in only 3 subjects

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and at very few sampling time-points at the 10 mg dose level. BIA 5-961 plasma concentrations could
only be detected in two subjects at the 2 mg dose, and therefore mean pharmacokinetic parameters
are only depicted for etamicastat 10 mg and above doses (Figure 6). The corresponding
pharmacokinetic parameters are presented in Table 5. Although systemic exposure appeared to
increase close to dose-proportionally to the administered dose (Figure 6), dose proportionality could
not be demonstrated using an exponential regression model for Cmax or AUC0-∞ of both etamicastat
and BIA 5-961.
7.4. Cardiac evaluation after repeated administration in young and elderly healthy humans
In total, 25 subjects (13 young and 12 elderly) were enrolled. Their demographic and main
baseline characteristics are summarized in Table 4. All subjects were Caucasian but 1 Black in the
elderly group. One young subject was prematurely discontinued due to a serious AE and was
replaced. Twenty-four subjects (12 in each age group) completed the study. Figure 7 displays the
mean change from baseline of supine SBP, DBP, HR, PR interval, QRS duration and QTcF interval in
young and elderly healthy subjects after once-daily administrations of etamicastat for 7 days. There
was no clinically significant change in supine SBP and DBP in young subjects. However, in elderly
subjects, there was approximately 17 mmHg and 11 mmHg decreases in SBP and DBP respectively,
peaking 6 to 8 h after the last dosing. No clinically significant out-of-range value in vital signs or ECG
parameters, HR, PR interval, QRS duration and QTcF interval were observed in young and elderly
healthy subjects after once-daily administration of etamicastat for 7 days. In the elderly group, a
total of 3 AEs were reported (sciatica, asthenia, and back pain). One serious AE (myopericarditis)
occurred in the young group. Paracetamol was administered for the treatment of 2 AEs (sciatica and
back pain). All AEs were mild to moderate in intensity but the myopericarditis was severe and led to
the sujet’s discontinuation of the study. Myopericarditis occurred after 2 days of repeated dosing
and the subject recovered after 7 days.
The mean plasma concentration-time profiles of etamicastat and BIA 5-961 following the last dose
of a 100 mg once-daily regimen of etamicastat for 7 days in elderly and young subjects are displayed
in figure 7. The corresponding pharmacokinetic parameters are presented in Table 5. The systemic
exposure to etamicastat, as assessed by Cmax, AUC during the dosing interval (AUC0-24), AUC0-∞ and
minimum observed concentration (Cmin), was not significantly different in elderly and young subjects
both following repeated doses of etamicastat. Following multiple doses of etamicastat, BIA 5-961
AUC0-∞ and Cmin were significantly higher in elderly as compared with young subjects.
7.5. Cardiac evaluation after repeated administration in hypertensive patients
A total of 23 male volunteers, with ages between 49 and 64 years, were randomized and
constituted the safety and pharmacokinetic population. All subjects were Caucasian except one
Black. One subject administered 50 mg of etamicastat withdrew on Day 1 due to the occurrence of

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an AE (ECG repolarization abnormality), and 22 completed the study and constituted the
pharmacodynamic population. The demographic and other baseline characteristics of the study
population by treatment group are summarized in Table 4. No relevant between-group differences
were found. Figure 8 displays the mean change from baseline of supine SBP, DBP, HR, PR interval,
QRS duration and QTcF interval in hypertensive patients subjects after once-daily administrations of
etamicastat for 10 days. After 10 days of treatment, the decrease of SBP at day time and night time
tended to be more important in subjects who had received 50 mg of etamicastat (-7.6 and -7.4
mmHg, respectively), 100 mg (-9.1 and -10.2 mmHg, respectively) and 200 mg (-9.7 and -9.2 mmHg,
respectively) than in subjects who had received placebo (-2.4 and +3.8 mmHg). After 10 days of
treatment, the decrease of DBP at day time and night time also tended to be more marked in
subjects who had received 50 mg of etamicastat (-5.0 and -3.6 mmHg, respectively), 100 mg (-5.2 and
-6.8 mmHg, respectively) and 200 mg (-4.7 and -5.2 mmHg, respectively) than in subjects who had
received placebo (0.0 and +1.2 mmHg). After 10 days of treatment, no clinically relevant changes of
HR at day time and night time were observed in subjects who had received 50 mg of etamicastat (-
3.8 and -0.4 bpm, respectively), 100 mg (2.3 and 4.8 bpm, respectively) and 200 mg (+0.3 and -2.5
bpm, respectively) compared to subjects who had received placebo (+0.8 and +0.4 bpm,
respectively). No clinically significant out-of-range value in vital signs or ECG parameters, HR, PR
interval, QRS duration and QTcF interval were observed in hypertensive subjects after once-daily
administrations of etamicastat, for 10 days. No clinically relevant changes were observed in ECG
intervals. In particular, no subject had a change from baseline in QTcF of more than 60 ms and there
was no significant prolongation of QTcF interval > 480 msec.
Ten patients reported a total of 12 TEAEs: 3 AEs (gamma-glutamyl transferase increase, gout, and
diarrhea) in 3 patients with placebo, 1 AE (ECG repolarization abnormality) that led to study
discontinuation on Day 1 in a patient with etamicastat 50 mg, 4 AEs (generalized maculopapular rash,
pain in a extremity, headache, and vasovagal syncope) in 3 patients with etamicastat 100 mg, and 4
AEs (sciatica, pruritus, eczema, and dry skin) in 3 patients with etamicastat 200 mg. The
maculopapular rash was moderate and emerged at Day 10. There were no serious AEs. All AEs were
mild (7 cases) or moderate (5 cases) in intensity and recovered without sequellae.
The mean plasma concentration-time profiles of etamicastat and BIA 5-961 following the last dose
of a once-daily regimen of etamicastat for 10 days in hypertensive patients are displayed in figure 8.
The corresponding pharmacokinetic parameters are presented in Table 5. Etamicastat Cmax was
reached 1 h post-dose and declined thereafter with a mean t1/2 of 19 to 28 h following repeated
administration. Systemic exposure to etamicastat and BIA 5-961 increased less than dose-
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Discussion
Both nepicastat and etamicastat markedly inhibited the activity of native DβH expressed in SK-N-
SH human neuroblastoma cells, though the former was more potent than the later with IC50s of 13.3
and 37.2 ng/ml, respectively. On the other hand, the IC50 value for reduction of hERG current
amplitude by nepicastat (. μg/l was 7.9-fold that observed for etamicastat (44.0 μg/ml).
Assuming the ratio of IC50 values against hERG and DBH as a safety window, then etamicastat (IC50
hERG/DβH = 1183) is expected to offer a significant advantage over nepicastat (IC50 hERG/DβH =
421). This IC50 alue of etaiastat . μg/l agaist hERG is approximately 28 times higher than
the plasma concentration (1.6 µg/ml) that was found in healthy volunteers after single
administration of 1200 mg, a high well-tolerated dose that produced ca. 80% inhibition of DßB
activity [21].
In conscious telemetered Cynomolgus monkeys, etamicastat had no substantial effects on arterial
blood pressure, HR and the PR interval when administered orally at 15, 45 and 90 mg/kg with the
exception of a QT prolongation 14 h following administration of 45 mg/kg etamicastat,
corresponding with the changes observed in HR and RR interval at this time. When the QT interval
was corrected for changes in HR / RR interval by calculation of the QTcF interval, this prolongation
was still evident; however, when corrected using the individual animal specific correction formula,
QTcQ, it was no longer apparent. A slight shortening of the QTcQ interval was noted at 14 h following
administration of 15 mg/kg etamicastat when compared to vehicle, but values remained unchanged
when compared to pre-dose baseline. Therefore, this was not considered to be a drug-related effect.
No arrhythmia or other changes in the morphology of the electrocardiogram were observed at any
dose level of etamicastat. Mean plasma concentrations of etamicastat at 2 and 4 h were 610 and
646, 771 and 1968, and 1055 and 1926 ng/ml at the dose levels of 15, 45 and 90 mg/kg, respectively.
Administered orally at 15 and 45 mg/kg/day in female and male Cynomolgus monkey for 91 days,
etamicastat had no effect on HR and the waveform or intervals of the electrocardiogram. At the
highest dose level of 45 mg/kg/day, mean plasma concentrations of etamicastat were 1875 (males)
or 2497 (females) and 2895 (males) or 3145 ng/ml (females) on Day 1 and Day 91 of treatment,
respectively. Although male animal species are classically used for safety pharmacology studies, it is
not unusual to observe higher plasma concentrations in females than in males. It is also known that
females are more susceptible to drug-induced long QT interval and cardiac arrhythmias than males
[28, 29]. The plasma concentrations measured in monkeys at doses which did not show any
deleterious effects, including ECG disturbance, were at least 2 times greater than those measured in
humans at the highest cardiovascular active doses (1200 mg) or 10 to 15 times greater than those
measured in humans at therapeutic doses (100 to 200 mg). This finding is in agreement with that
observed in the hERG study: at 1 µg/ml, no effect was found on the hERG mediated current. Only

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concentrations 3 to 10 times higher were necessary to affect the delayed rectifier potassium
channels.
In the single ascending study in which young healthy volunteers were given once-daily oral doses
of placebo or etamicastat from 2 to 1200 mg, the extent of systemic exposure to etamicastat
increased in a close but not complete dose-proportional manner. Etamicastat Cmax occurred quickly
after administration, at approximately 1 to 3 hours after dosing. Etamicastat elimination appeared to
be slower for the highest doses, with elimination half-lives increasing when the dose increased, from
6.6 h at the 20 mg dose to 18.2 h at the 1200 mg dose. There was no clinically significant change in
supine SBP, DBP, HR and QTcF interval, though important variability and no clear trend was
observed. A total of 18 TEAEs were reported in 13 subjects. There was no serious AE and no AE
required the withdrawal of a subject. All AEs were mild to moderate in intensity.
The study that investigated the effect of age on the tolerability and pharmacokinetics of
etamicastat in elderly (65 years or older) and young adult (18-45 years) subjects followed a standard
design for a phase I study in healthy subjects aiming to investigate the effect of age on the drug
pharmacokinetics. This is useful at an early phase of drug development to define the dosage regimes
to be tested in elderly subjects enrolled in later therapeutic studies. The cardiovascular safety
evaluation comprised changes from baseline of supine SBP, DBP, HR, PR interval, QRS duration and
QTcF interval after once-daily administration of etamicastat for 7 days. There was no clinically
significant change in supine SBP and DBP in young subjects. However, in elderly subjects, there was
approximately 17 mmHg and 11 mmHg decreases in supine SBP and DBP, respectively, peaking 6 to 8
h after the last dosing. No clinically significant out-of-range value in vital signs or ECG parameters,
HR, PR interval, QRS duration and QTcF interval were observed in young and elderly healthy subjects.
The mean plasma concentration-time profiles of etamicastat and BIA 5-961 following the last dose of
a 100 mg once-daily regimen of etamicastat for 7 days indicated that the systemic exposure to
etamicastat, as assessed by Cmax, AUC0-24, AUC0-∞ and Cmin, was not significantly different in elderly
and young subjects both following repeated doses of etamicastat. However, the systemic exposure
to BIA 5-961, as assessed by AUC0-∞ and Cm in, were significantly higher in elderly as compared with
young subjects.
In hypertensive patients the decrease of SBP at day time and night time tended to be more
marked in subjects who received etamicastat (50, 100 and 200 mg) than in subjects who received
placebo. After 10 days of treatment, the decrease of DBP at day time and night time also tended to
be more marked in subjects who received etamicastat than in subjects who received placebo. This
was accompanied by no clinically relevant changes of HR at day time and night time in subjects who
received etamicastat (50, 100 and 200 mg) compared to subjects who received placebo. However, it
should be underlined that after the last dose of 100 mg etamicastat there was approximately 17
mmHg decrease in SBP and 11 mmHg decrease in DBP, peaking 3 to 6 h after the last dosing. No

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clinically significant out-of-range value in vital signs or ECG parameters, HR, PR interval, QRS duration
and QTcF interval were observed in hypertensive subjects after once-daily administrations of
etamicastat for 10 days. No clinically relevant changes were observed in ECG intervals. In particular,
no subject had a change from baseline in QTcF of more than 60 ms and there was no significant
prolongation of QTcF interval > 480 msec.
At 100 mg etamicastat, an effective therapeutic dose (ETPC), after repeated administration (trials
in elderly / young healthy subjects and in hypertensive patients), Cmax (0.124, 0.105 and 0.168 µg/ml,
respectively) was reached 1 h post-dose and declined, thereafter, with a mean half-life (t1/2) of 23 h
following the last dose. A 30-fold margin between Cmax and hERG IC50 may suffice for drugs currently
undergoing clinical evaluation, but this margin should be increased, particularly for drugs aimed at
non-debilitating diseases [30, 31]. Binding of [14C]-etamicastat to the human plasma proteins was
moderate with a mean of 73.9 % (Bial data on file). As such, the ratio between the 44.0 µg/ml IC50 for
hERG and the 0.035 µg/ml unbound ETPC (44.0/0.044=1000) is 33 times higher the 30-fold safety
margin. The no-effect concentration of 1.0 µg/mL is 23-fold higher than the unbound clinical Cmax
(0.044 µg/ml). As such, the observed in vitro effect in the hERG assay is highly unlikely to have any
clinical impact and this is supported by the review of Redfern et al [30], which concluded that a
greater than 30-fold margin between hERG IC50 and unbound clinical Cmax was sufficiently reassuring.
In conclusion, the blockade of hERG current amplitude by etamicastat together with the QTc
interval prolongation observed in conscious telemetered Cynomolgus monkeys can be considered as
modest with respect to the plasma concentrations found for these effects and to those expected for
beneficial cardiovascular activity in humans. These findings and the results of clinical trials in humans
suggest that etamicastat is not likely to prolong the QT interval at therapeutic doses.

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Legends to figures
Figure 1. Structural formulae of etamicastat and nepicastat.
Figure 2. Inhibition curve for inhibition of dopamine-β-hydroxylase activity by etamicastat and
nepicatsat. Values are means ± SEM (n = 6).
Figure 3. Inhibition curve for blockade of hERG relative tail current by etamicastat. Values are means
± SEM (n = 3).
Figure 4. Effects of placebo and etamicastat on arterial blood pressure, heart rate, and the main
parameters of the electrocardiogram (PR interval, QRS duration and QTcF interval) following oral
(p.o. capsule) administration in the conscious male Cynomolgus primate monitored by telemetry.
Symbols represent mean ± SEM values of values of 4 animals per group.
Figure 5. Plasma concentration-time profiles of etamicastat after single and 91 once daily
administrations by oral gavage of 15 and 45 mg/kg/day etamicastat in male and female Cynomolgus
monkeys. Symbols represent mean ± SEM values of values of 4 animals per group.
Figure 6. Mean change from baseline of supine systolic blood pressure, diastolic blood pressure,
heart rate, PR interval, QTcF interval, Cmax and AUC0-∞ etamicastat and BIA 5-961 in young healthy
subjects after single once-daily administration of etamicastat. Symbols represent mean ± SEM values
of 6 subjects per group. Significantly different from corresponding values before dosing (# P<0.05)
following Dunnett test.
Figure 7. Mean change from baseline of supine systolic blood pressure, diastolic blood pressure,
heart rate, PR interval, QRS duration and QTcF interval, and concentration time profiles of
etamicastat and BIA 5-961 in young and elderly healthy subjects after once-daily administration of
etamicastat (100 mg/day) for 7 days. Symbols represent mean ± SEM values of 12 and 13 subjects
per group. Significantly different from corresponding values before dosing (* P<0.05) following
Dunnett test.
Figure 8. Mean change from baseline of supine systolic blood pressure, diastolic blood pressure,
heart rate, PR interval, QRS duration and QTcF interval, and concentration time profiles of
etamicastat and BIA 5-961 in hypertensive subjects after once-daily administration of placebo or
etamicastat (50, 100 and 200 mg/day) for 10 days. Symbols represent mean ± SEM values of 12 and
13 subjects per group.

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Figure 1
Etamicastat
Nepicastat
HCl
N
NH
F
F
S
O
(R)
NH2
(Z)
NH
N
S
H2N
(S)
F
F
(Z )
HCl

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Figure 2
-4 -3 -2 -1 0 1
0
25
50
75
100
Log [compound] (mg/ml)
Octopamine
(% control)
Etamicastat
Nepicastat

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Figure 3
-2 -1 0 1 2 3
0
25
50
75
100
Log [compound] (mg/ml)
Relative current
(%)
Etamicastat
Nepicastat

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Figure 4
A
0 4 8 12 16 20 24
50
75
100
125
Time (h )
Systolic arterial blood pressure (mm Hg)
Placebo
Etam (15 mg/kg)
Etam (45 mg/kg)
Etam (90 mg/kg)
C
0 4 8 12 16 20 24
75
100
125
150
175
200
Time (h )
Heart rate (bpm)
Placebo
Etam (15 mg/kg)
Etam (45 mg/kg)
Etam (90 mg/kg)
E
0 4 8 12 16 20 24
20
30
40
50
60
Time ( h)
QRS duration (ms)
Placebo
Etam (15 mg/kg)
Etam (45 mg/kg)
Etam (90 mg/kg)
B
0 4 8 12 16 20 24
25
50
75
100
Time ( h)
Diastolic arterial blood pressure (mm Hg)
Etam (45 mg/kg)
Placebo
Etam (15 mg/kg)
Etam (90 mg/kg)
*
D
0 4 8 12 16 20 24
60
70
80
90
100
110
Time (h)
PR interval (ms)
Placebo
Etam (15 mg/kg)
Etam (45 mg/kg)
Etam (90 mg/kg)
F
0 4 8 12 16 20 24
200
250
300
350
400
Time (h )
QTcF interval (ms)
Placebo
Etam (15 mg/kg)
Etam (45 mg/kg)
Etam (90 mg/kg)

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Figure 5
A
0 4 8 12 16 20 24
0
1000
2000
3000
4000
Time (h)
Etamicastat (ng/ml)
Vehicle - Male
Etami (15) - Male
Etami (45) - Male
Vehicle - Female
Etami (15) - Female
Etami (45) - Female
B
0 4 8 12 16 20 24
0
1000
2000
3000
4000
Time (h)
Etamicastat (ng/ml)
Vehicle - Male
Etami (15) - Male
Etami (45) - Male
Vehicle - Female
Etami (15) - Female
Etami (45) - Female

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Figure 6
A
0 2 10 20 50 100 200 400 600 900 1200
-25
-20
-15
-10
-5
0
5
10
15
20
25
Etamicastat (mg)
Maximum change of
SBP (mm Hg)
C
0 2 10 20 50 100 200 400 600 900 1200
-15
-10
-5
0
5
10
Etamicastat (mg)
Maximum change of
ECG HR (bpm)
E
0 2 10 20 50 100 200 400 600 900 1200
0
500
1000
1500
2000
Etamicastat (mg)
Cmax Etamicastat (ng/ml)
G
0 2 10 20 50 100 200 400 600 900 1200
0
1000
2000
3000
4000
Etamicastat (mg)
Cmax BIA 5-961(ng/ml)
B
0 2 10 20 50 100 200 400 600 900 1200
-25
-20
-15
-10
-5
0
5
10
15
Etamicastat (mg)
Maximum change of
DBP (mm Hg)
D
0 2 10 20 50 100 200 400 600 900 1200
-30
-20
-10
0
10
20
30
Etamicastat (mg)
Maximum change of
ECG QTcF interval (ms)
F
0 2 10 20 50 100 200 400 600 9001200
0
5000
10000
15000
20000
Etamicastat (mg)
AUC0-¥
Etamicastat (ng.h/ml)
H
0 2 10 20 50 100 200 400 600 9001200
0
5000
10000
15000
20000
25000
30000
Etamicastat (mg)
AUC0-¥
BIA 5-961 (ng.h/ml)

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Figure 7
A
0 4 8 12 16 20 24 30 60 90 120
-20
-15
-10
-5
0
5
10
Time (h)
Change from baseline of
SBP (mm Hg)
Elderly
Young
**
***
C
04812 16 20 24 30 60 90 120
-15
-10
-5
0
5
10
15
Time ( h)
Change from baseline of
ECG heart rate (bpm)
Elderly
Young
E
04812 16 20 24 30 60 90 120
-6
-4
-2
0
2
4
6
Time ( h)
Change from baseline of
ECG QRS interval (ms)
Elderly
Young
G
0 4 8 12 16 20 24 30 60 90 120
0
50
100
150
Time (h)
Etamicastat (ng/ml)
Elderly
Young
B
0 4 8 12 16 20 24 30 60 90 120
-15
-10
-5
0
5
Time ( h)
Change from baseline of
DBP (mm Hg)
Elderly
Young
*
*
*
D
0 4 8 12 16 20 24 30 60 90 120
-15
-10
-5
0
5
10
15
Time ( h)
Change from baseline of
ECG PR interval (ms)
Elderly
Young
F
0 4 8 12 16 20 24 30 60 90 120
-20
-15
-10
-5
0
5
10
Time ( h)
Change from baseline of
ECG QTcF interval (ms)
Elderly
Young
H
0 4 8 12 16 20 24 30 60 90 120
0
100
200
300
400
Time (h)
BIA 5-961 (ng/ml)
Elderly
Young

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Figure 8
A
0 4 8 12 16 24 72 120 168
-25
-15
-5
5
15
25
Time ( h)
Change from baseline of
SBP (mm Hg)
50 mg
100 mg
200 mg
Placebo
C
0 4 8 12 16 24 72 120 168
-15
-10
-5
0
5
10
15
Time ( h)
Change from baseline of
ECG heart rate (bpm)
50 mg
100 mg
200 mg
Placebo
E
0 4 8 12 16 24 72 120 168
-15
-10
-5
0
5
10
15
Time ( h)
Change from baseline of
ECG QRS interval (ms)
50 mg
100 mg
200 mg
Placebo
G
0 4 8 12 16 24 96 168
0
100
200
300
Time ( h)
Etamicastat (ng/ml)
50 mg
100 mg
200 mg
B
0 4 8 12 16 24 72 120 168
-20
-10
0
10
20
Time ( h)
Change from baseline of
DBP (mm Hg)
50 mg
100 mg
200 mg
Placeb o
D
0 4 8 12 16 24 72 120 168
-20
-10
0
10
20
Time (h )
Change from baseline of
ECG PR interval (ms)
50 mg
100 mg
200 mg
Placebo
F
0 4 8 12 16 24 96 168
-25
-15
-5
5
15
25
Time (h )
Change from baseline of
ECG QTcF interval (ms)
50 mg
100 mg
200 mg
Placebo
H
0 4 8 12 16 24 96 168
0
100
200
300
400
Time ( h)
BIA 5-961 (ng/ml)
50 mg
100 mg
200 mg

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Table 1. Mean arterial blood pressure (MABP), RR interval and QTcQ interval of the electrocardiogram following oral administration of vehicle, then etamicastat at
15, 45 and 90 mg/kg in the conscious Cynomolgus monkey monitored by telemetry.
Measurement times before and after administration (h)
Etamicastat (mg/kg)
-0.5
1
2
4
6
8
10
14
18
22
MABP (mmHg)
0
15 mg/kg
45 mg/kg
90 mg/kg
80 ± 3
79 ± 4
81 ± 7
83 ± 9
82 ± 6
78 ± 4
83 ± 6
80 ± 7
78 ± 4
88 ± 8
89 ± 7
85 ± 10
79 ± 5
74 ± 2
74 ± 8
77 ± 6
75 ± 4
74 ± 6
73 ± 6
72 ± 6
78 ± 5
77 ± 4
76 ± 7
76 ± 7
60 ± 6
59 ± 4
58 ± 6
61 ± 7
64 ± 4
64 ± 3
59 ± 5
64 ± 6
65 ± 5
65 ± 3
65 ± 6
64 ± 5
79 ± 6
76 ± 6
74 ± 6
68 ± 7 *
RR interval (ms)
0
15 mg/kg
45 mg/kg
90 mg/kg
405 ± 40
421 ± 22
395 ± 30
385 ± 18
430 ± 6
455 ± 9
422 ± 34
428 ± 11
461 ± 32
417 ± 17
431 ± 27
398 ± 28
420 ± 16
465 ± 19
454 ± 28
423 ± 18
412 ± 21
429 ± 26
445 ± 22
470 ± 28
436 ± 14
446 ± 11
438 ± 16
456 ± 4
572 ± 10
592 ± 31
596 ± 20
552 ± 20
603 ± 38
621 ± 51
666 ± 26
588 ± 43
613 ± 24
602 ± 30
588 ± 4
561 ± 37
412 ± 39
426 ± 18
407 ± 20
464 ± 16
QTcQ interval (ms)
0
15 mg/kg
45 mg/kg
90 mg/kg
250 ± 2
242 ± 5
253 ± 9
249 ± 6
242 ± 7
248 ± 4
252 ± 11
247 ± 5
248 ± 8
255 ± 3
253 ± 8
252 ± 5
247 ± 7
246 ± 6
251 ± 11
253 ± 3
251 ± 8
246 ± 5
247 ± 9
241 ± 6
245 ± 5
241 ± 4
243 ± 11
239 ± 2
251 ± 5
245 ± 9
248 ± 13
257 ± 10
254 ± 7
236 ± 5
252 ± 10
251 ± 9
253 ± 5
245 ± 12
251 ± 13
254 ± 10
247 ± 6
266 ± 15
259 ± 5
242 ± 6
The placebo and etamicastat were administered orally, by capsule, at time 0. Values are mean  SEM of results obtained from 4 animals. * denotes P < 0.05 etamicastat vs vehicle.

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Table 2. ECG parameters in female and male Cynomolgus monkeys before and following oral administration of vehicle and etamicastat at 15 and 45 mg/kg.
Pre-test
Pre-test
Heart rate (bpm)
P amplitude (mV)
P duration (ms)
P-Q interval (ms)
QRS interval (ms)
Q-T interval (ms)
Vehicle
(0 mg/kg/day)
Females
Males
243±20
227±46
0.09±0.02
0.10±0.00
41±2
43±5
58±4
64±7
50±0
52±4
180±11
180±21
Etamicastat
(15 mg/kg/day)
Females
Males
250±8
258±17
0.09±0.01
0.10±0.01
46±5
44±5
60±0
60±8
50±0
53±5
173±10
170±8
Etamicastat
(45 mg/kg/day)
Females
Males
223±23
263±24
0.10±0.00
0.06±0.03 *
43±5
44±3
63±9
63±5
55±6 *
50±0
185±13
166±8
Week1
Week1
Vehicle
(0 mg/kg/day)
 before dosing
1 h after dosing
Females
237±26
258±21
0.09±0.03
0.08±0.02
40±0
40±0
60±0
59±2
50±0
51±2
183±15
178±12
 before dosing
1 h after dosing
Males
238±42
238±31
0.11±0.02
0.10±0.03
44±4
46±5
66±8
67±8
57±5
53±5
183±14
180±11
Etamicastat
(15 mg/kg/day)
 before dosing
1 h after dosing
Females
245±6
233±21
0.09±0.01
0.08±0.05
41±3
43±3
63±3
65±4
50±0
53±5
183±5
185±10
 before dosing
1 h after dosing
Males
240±41
225±31
0.08±0.03
0.09±0.01
43±5
40±0
56±5
60±0
53±5
53±5
176±11
180±12
Etamicastat
(45 mg/kg/day)
 before dosing
1 h after dosing
Females
225±39
208±30 *
0.10±0.01
0.09±0.03
48±10
43±5
68±10
64±8
53±5
50±0
190±18
198±17
 before dosing
1 h after dosing
Males
220±40
233±22
0.09±0.01
0.09±0.03
40±0
43±5
63±5
63±5
53±5
55±6
190±20
193±15
Week 13
Week 13
Vehicle
(0 mg/kg/day)
 before dosing
1 h after dosing
Females
252±18
230±24
0.09±0.02
0.07±0.02
40±0
40±0
58±4
58±4
58±4
58±4
177±12
187±21
 before dosing
1 h after dosing
Males
246±36
210±44
0.12±0.07
0.10±0.03
42±4
45±5
61±5
67±8
60±0
60±0
183±15
197±26
Etamicastat
(15 mg/kg/day)
 before dosing
1 h after dosing
Females
240±24
218±15
0.10±0.01
0.10±0.01
41±3
44±5
59±3
63±5
60±0
60±0
190±12
200±0
 before dosing
1 h after dosing
Males
250±22
225±44
0.08±0.03
0.08±0.03
40±0
40±0
59±3
60±0
60±0
60±0
185±13
193±22
Etamicastat
(45 mg/kg/day)
 before dosing
1 h after dosing
Females
213±49
203±25
0.11±0.02
0.10±0.01
40±0
43±5
59±3
68±10 #
63±5
63±5
195±31
203±17
 before dosing
1 h after dosing
Males
253±38
243±38
0.10±0.00
0.08±0.02
43±5
43±5
59±3
63±5
60±0
60±0
180±14
183±21
Values are means ± SEM. Significantly different from corresponding values in vehicle treated animals (* P<0.05) following Dunnett test.
Significantly different from corresponding values before dosing 1 (# P<0.05) following Dunnett test.

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Table 3. Pharmacokinetic parameters of etamicastat in female and male Cynomolgus monkeys after single and 91 once daily administrations of
etamicastat by oral gavage.
Day 1
Day 91
Dose
(mg/kg)
15
45
Ratio
15
45
Ratio
Females
Cmax
(ng/ml)
686.0±118.8
2497.5±284.3
4.3±1.4
868.8±122.3
3145.0±228.5
3.9±0.7
tmax
(h)
2.5±0.5
3.5±0.5
1.6±0.4
3.5±0.5
4.0±0.0
1.3±0.3
AUC0-24
g•h/l
5591.8±1014.5
23022.0±1898.3
4.9±1.5
6688.0±1016.4
26180.8±943.0
4.2±0.7
AUC0-∞
g•h/l
6136.0±1130.7
25678.3±2336.9
5.1±1.6
7142.0±1150.3
28024.5±989.9
4.3±0.7
t½
(h)
6.9±0.3
6.9±0.4
1.0±0.1
6.0±0.3
5.8±0.1
1.0±0.0
Males
Cmax
(ng/ml)
935.5±71.4
1875.0±179.8
2.0±0.0
834.3±101.7
2895.0±306.9
3.5±0.2
tmax
(h)
3.0 ±0.6
3.5±0.5
1.3±0.3
3.5±0.5
3.0±0.6
0.9±0.1
AUC0-24
g•h/l
7902.0±339.7
16137.8±2294.1
2.0±0.2
7125.0±1159.0
24168.8±1426.4
3.7±0.7
AUC0-∞
g•h/l
8873.5±358.0
18549.3±2787.9
2.1±0.3
7671.3±1232.2
25662.8±1420.6
3.7±0.6
t½
(h)
7.4±0.4
7.4±0.7
1.0±0.1
6.4±0.4
5.6±0.4
0.9±0.1
Values are means ± SEM of 4 animals per group.

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Table 4. Summary statistics of demographic and baseline characteristics of study participating human subjects.
Study
Etamicastat group (n)
Age (years)
Height (cm)
Weight (kg)
BMI (kg/m2)
SBP (mmHg)
DBP (mmHg)
HR (bpm)
Single ascending dose study
Placebo (20)
30.8 (19-44)
178.2 (166-195)
72 (61-92)
22.7 (20.4-27.1)
106 (86-116)
61 (46-73)
54 (38-70)
2 mg (6)
32.0 (21-38)
182.5 (173-194)
82 (68-97)
24.7 (18.1-29.9)
116 (102-127)
68 (60-80)
60 (44-70)
10 mg (6)
30.3 (20-44)
182.2 (175-191)
79 (72-90)
23.7 (20.8-25.8)
107 (96-113)
62 (57-70)
48 (40-55)
20 mg (6)
26.8 (19-32)
178.8 (170-186)
66 (56-75)
20.6 (18.5-21.7)
99 (88-107)
56 (53-63)
52 (44-62)
50 mg (6)
28.8 (20-36)
175.7 (173-179)
70 (62-81)
22.5 (20.2-25.3)
111 (100-120)
58 (47-66)
57 (45-80)
100 mg (6)
29.8 (19-40)
175.5 (171-183)
68 (60-80)
21.9 (18.8-25.8)
104 (95-111)
55 (50-58)
56 (53-61)
200 mg (6)
30.7 (20-42)
176.3 (169-191)
72 (63-83)
23.2 (21.7-25.1)
109 (96-144)
60 (56-74)
58 (46-70)
400 mg (6)
28.2 (25-32)
173.2 (167-181)
70 (60-79)
23.5 (21.0-26.9)
119 (102-146)
60 (53-66)
59 (47-80)
600 mg (6)
31.3 (22-41)
174.7 (161-183)
74 (63-84)
24.3 (22.7-26.8)
114 (97-138)
59 (51-69)
74 (63-84)
900 mg (6)
37.7 (22-43)
172.0 (164-178)
64 852-73)
21.5 (19.0-25.6)
107 (102-117)
61 (46-68)
60 (48-72)
1200 mg (6)
31.7 (19-38)
179.0 (172-184)
77 (63-88)
23.8 (21.3-26.5)
111 (99-123)
57 (49-61)
61 (57-66)
Young & Elderly study
100 mg Young group
(13)
32.6 (18-44)
175.8 (162-185)
79.0 (57.0-102)
25.8 (20.9-34.4)
117 (103-137)
61 (50-75)
58 (47-70)
100 mg Elderly group
(12)
69.3 (65-75)
166.9 (160-179)
69.2 (52-86)
25.2 (19.8-29.0)
115 (98-143)
64 (56-68)
66 (52-76)
Hypertensive patients study
Placebo (5)
59.6 (53-64)
169 (162-180)
83.2 (74-102)
29.0 (25.0-33.5)
160.6 (123-174)
92.6 (89-99)
70.7 (55-82)
50 mg (5)
55.8 (51-61)
177 (169-184)
84.3 (64-101)
26.8(22.4-32.2)
150.2 (141-161)
94.0 (84-99)
64.5 (56-68)
100 mg (6)
56.5 (49-64)
176 (168-181)
84.8 (73-101)
27.3(24.1-30.8)
164.8 (151-187)
96.8 (86-109)
63.8 (55-72)
200 mg (6)
59.8 (58-61)
171 (165-179)
83.3 (71-105)
28.3 (24.6-32.8)
147.2 (129-163)
87.2 (78-93)
60.0 (49-72)
BMI, body mass index; SBP, supine systolic blood pressure; DBP, supine diastolic blood pressure; HR; heart rate beats per min
Values are means with range values in parenthesis.

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Table 5. Summary pharmacokinetic parameters of etamicastat.
Study
Etamicastat group (n)
Cmax (ng/ml)
Cmin (ng/ml)
tmax (h)
AUC0-t (g•h/l)
AUC0-∞ (g•h/l)
t1/2 (h)
Single ascending dose study
Placebo (20)
ND
ND
ND
ND
ND
ND
2 mg (6)
ND
ND
ND
ND
ND
ND
10 mg (6)
6±1
ND
2.0 (1.0-2.0)
4±2
ND
ND
20 mg (6)
23±8
6.1±1.5
1.0 (0.5-2.0)
84±36
148±25
6.6±1.6
50 mg (6)
49±13
6.1±1.2
1.0 (0.5-2.0)
304±181
421±238
12.6±6.0
100 mg (6)
106±24
6.3±0.9
1.5 (1.0-3.0)
957±212
1134±250
19.7±5.7
200 mg (6)
202±24
7.0±1.5
1.0 (1.0-3.0)
2026±663
2230±701
19.8±3.3
400 mg (6)
475±119
9.9±2.5
2.0 (1.0-2.1)
3914±1637
4171±1733
17.3±3.7
600 mg (6)
766±161
10.5±4.9
3.0 (1.0-5.0)
7111±2480
7355±2566
15.7±1.6
900 mg (6)
1469±327
26.6±9.4
3.0 (1.0-5.0)
14197±4231
14908±4428
18.8±1.5
1200 mg (6)
1638±423
26.7±17.1
2.5 (1.0-3.0)
13997±5687
14707±6147
18.2±1.6
Young & Elderly study*
100 mg Young group (13)
105±37
15.0±11.1
1.5 (1.0-4.0)
1142±692
1497±825
17.3±5.8
100 mg Elderly group (12)
124±33
18.5±11.9
1.0 (0.5-5.0)
1666±1026
2199±1262
28.1±12.2
Hypertensive study**
Placebo (5)
ND
ND
ND
ND
ND
ND
50 mg (5)
62±24
12.7±2.0
1.0 (0.5-2.0)
769±456
1123±676
18.5±9.5
100 mg (6)
168±56
14.3±3.2
1.0 (0.5-2.0)
2387±1366
2906±1603
24.6±9.2
200 mg (6)
277±118
16.5±5.9
1.0 (1.0-2.0)
3633±1845
4307±2050
28.0±3.1
ND = not detected
Values are means±SD or means with range values in parenthesis.
* PK parameters refer to last day of once-daily administration = 7 days
** PK parameters refer to last day of once-daily administration = 10 days