Pediatric Drugs (2020) 22:95–104

https://doi.org/10.1007/s40272-019-00373-3

ORIGINAL RESEARCH ARTICLE

A Phase IIa Clinical Trial of 2‑Iminobiotin for the Treatment of Birth

Asphyxia in DR Congo, a Low‑Income Country
Thérèse Biselele1 · Jephté Bambi1 · Dieu M. Betukumesu1 · Yoly Ndiyo1 · Gabriel Tabu2 · Josée Kapinga2 ·
Valérie Bola3 · Pascal Makaya3 · Huibert Tjabbes4 · Peter Vis5 · Cacha Peeters‑Scholte4

Published online: 21 January 2020

© Springer Nature Switzerland AG 2020

Abstract
Aim The main burden of hypoxic-ischemic encephalopathy falls in low-income countries. 2-Iminobiotin, a selective inhibi-
tor of neuronal and inducible nitric oxide synthase, has been shown to be safe and effective in preclinical studies of birth
asphyxia. Recently, safety and pharmacokinetics of 2-iminobiotin treatment on top of hypothermia has been described. Since
logistics and the standard of medical care are very different in low-resource settings, the aim of this study was to investigate
safety and pharmacokinetics of Two-IminoBiotin in the Democratic Republic of Congo (TIBC).
Methods Near-term neonates, born in Kinshasa, Democratic Republic of Congo, with a Thompson score ≥ 7 were eligible
for inclusion. Excluded were patients with (1) inability to insert an umbilical venous catheter for administration of the study
drug; (2) major congenital or chromosomal abnormalities; (3) birth weight < 1800 g; (4) clear signs of infection; and (5) mori-
bund patients. Neonates received six infusions of 2-iminobiotin 0.16 mg/kg started within 6 h after birth, with 4-h intervals,
targeting an AUC​0–4h of 365 ng*h/mL. Safety, defined as vital signs, the need for clinical intervention after administration
of study drug, occurrence of (serious) adverse events, and pharmacokinetics were assessed.
Results After parental consent, seven patients were included with a median Thompson score of 10 (range 8–16). No relevant
changes in vital signs were observed over time. There was no need for clinical intervention due to administration of study
drug. Three patients died, two after completing the study protocol, one was moribund at inclusion and should not have been
included. Pharmacokinetic data of 2-iminobiotin were best described using a two-compartment model. Median AUC​0–4h was
664 ng*h/mL (range 414–917). No safety issues attributed to the administration of 2-iminobiotin were found.
Conclusion The present dosing regimen resulted in higher AUCs than targeted, necessitating a change in the dose regimen
in future efficacy trials. No adverse effects that could be attributed to the use of 2-iminobiotin were observed.
EudraCT number 2015-003063-12.

Key Points
Electronic supplementary material The online version of this
article (https​://doi.org/10.1007/s4027​2-019-00373​-3) contains 2-Iminobiotin, a selective nitric oxide synthase inhibitor,
supplementary material, which is available to authorized users. is a potential neuroprotective drug after birth asphyxia.

* Cacha Peeters‑Scholte No safety issues were found after administration of

cacha.peeters@neurophyxia.com 2-iminobiotin for birth asphyxia in DR Congo, a low-
1
income country.
Neonatal Unit, Department of Pediatrics, University Hospital
of Kinshasa, Kinshasa, Democratic Republic of Congo The dose regimen of 2-iminobiotin should be adjusted to
2
Neonatal Unit, Department of Pediatrics, Clinique Ngaliema, meet the predefined target AUC.
Kinshasa, Democratic Republic of Congo
3
Neonatal Unit, Department of Pediatrics, Hôpital Saint
Joseph, Kinshasa, Democratic Republic of Congo
4
Neurophyxia BV, Onderwijsboulevard 225,
5223 DE ’s‑Hertogenbosch, The Netherlands
5
LAP&P Consultants, Leiden, The Netherlands

Vol.:(0123456789)
96
1 Introduction
The world has made substantial progress in reducing child
mortality in the past several decades. However, globally
2.5 million newborns still died in 2017, and neonatal
deaths accounted for 47% of all deaths of under-5-year-
olds, increasing from 41% in 2000 [1]. Twenty-three per-
cent of these deaths are being caused by birth asphyxia
[2]. Additionally, birth asphyxia leads to a substantial
burden of care due to long-term neurological disability
and impairment.
Currently, there are no established neuroprotective
treatment strategies for birth asphyxia in low-income
countries (LIC). Standard care mainly consists of medi-
cation to relieve symptoms of pain, epilepsy, and infection.
In the past few years, therapeutic hypothermia (TH; whole
body hypothermia or selective head cooling) has been
introduced to reduce morbidity and mortality following
birth asphyxia in term-born neonates in the Western world.
However, in LIC, treatment with TH has not proven its
efficacy yet [3], and a rescue treatment is urgently needed.
2-Iminobiotin (2-IB), a selective inhibitor of induc-
ible and neuronal nitric oxide synthase and a vitamin B7
analog with anti-apoptotic properties, has recently been
evaluated as a potential drug to reduce the devastating
effects of hypoxic-ischemic encephalopathy (HIE) [4].
After being tested in two piglet HIE models [5, 6], several
models in rats [7, 8], and in a human phase I study in
adult male volunteers [9], it was shown that 2-IB had an
excellent safety profile and was well tolerated up to high
dosages.
Based on the dose-escalation study in newborn piglets
subjected to HIE, and treated with a 2-IB dose of 0.1, 0.2,
or 1.0 mg/kg every 4 h or placebo, it was shown that all 2-IB
doses significantly improved short-term outcome. Short-
term outcome was defined as increased survival with a nor-
mal amplitude integrated electroencephalography (aEEG),
and decreased nitrotyrosine staining of parietal cortex, tem-
poral cortex, and thalamus at 48 h after HIE [6]. A dose
comparable to a 0.2 mg/kg dose in the piglet with a target
area under the concentration–time curve from 0 to 4 h (AUC​
0–4h) of 365 ng*h/mL was suggested to be the most appropri-
ate dose for use in future human clinical HIE trials.
When combining pharmacokinetics (PK) data obtained
from both animal models and adult humans, clearance
appeared to increase linearly with body weight and a con-
stant mg/kg dose achieved similar exposure across dif-
ferent species [10]. Therefore, a dose of 0.2 mg/kg every
4 h was predicted to be the optimal dose for a study in
normothermic asphyxiated neonates.
For that reason, a pilot study was conducted to investi-
gate the safety and pharmacokinetics of 2-IB in six (near)
T. Biselele et al.
term neonates with moderate to severe HIE with a dose of
0.2 mg/kg/dose every 4 h [11]. Five out of the six neonates
experienced severe HIE. 2-IB was well tolerated, espe-
cially no signs of hypotension were observed and no rel-
evant changes in hematology or biochemistry were noted,
other than those that were related to the condition of HIE.
Two patients died because of hypoxic-ischemic-related
brain injury. In all patients, no (severe) adverse events
that could be attributed to 2-IB were reported. Although
limited PK data were available, 2-IB exposure in terms of
AUC​0–4h was higher than expected (566–712 ng*h/mL).
In retrospect, the estimated allometric exponent of 1.0,
based on interspecies scaling, was too high [10]. Using
adult human data with an accepted allometric coefficient
of 0.75 and correcting for immaturity would have been
more precise in determining the neonatal clearance and
thereby the dose [12]. The Data Safety Monitoring Board
(DSMB) advised to conservatively decrease the 2-IB dose
from 0.2 to 0.16 mg/kg/dose every 4 h in the current TIBC
study based on the sparse PK data obtained in neonates
and simulation experiments (see also supplemental Table 1
in the electronic supplementary material [ESM] [13]; H.
Tjabbes, of Neurophyxia B.V., personal communication).
Since logistics and the standard of medical care are very
different in LIC compared with the Western world, the aim
of the current TIBC study was to study the safety and PK of
2-IB in a phase IIa clinical study in normothermic neonates
after moderate to severe birth asphyxia in the Democratic
Republic Congo (DRC), a LIC.
2 Methods
2.1 Setting
This trial was performed in three large hospitals in Kinshasa,
the capital of the DRC, between June and October 2016.
The University Hospital of Kinshasa is in the south-western
part of Kinshasa. The Clinique Ngaliema is a well-equipped
hospital in the north-west of Kinshasa, and the Saint Joseph
Hospital is a conventional Catholic hospital in the center of
Kinshasa. There is no ventilation equipment available in the
three centers.
A medical mobile team was alerted when an asphyxiated
baby was admitted in one of the three hospitals. Asphyxia
was defined as a Thompson score of 7 or higher at 1–3 h
of age. The Thompson score is a clinical measure of the
severity of the HIE and ranges between 0 (normal) and 22
(severely affected) [14]. The medical mobile team helped
doctors on duty to obtain informed consent, and in the con-
duct of trial-related procedures. Written informed consent
was obtained from one of the parents in the local language
(Lingala) or French before start of treatment. When the
2-Iminobiotin for Birth Asphyxia in Low-Income Countries
consenting parent was illiterate, informed consent was given
verbally in the presence of an impartial witness.
2.2 Study Design
Dual ethical review was performed in both the sponsor and
the host country. The protocol was approved by the ethics
committee of the University of Kinshasa, DRC and received
a positive advice from the ethics committee of Utrecht, the
Netherlands. The study was registered at http://www.clini​
caltr ​ialsr​egist​er.eu (EudraCT number 2015-003063-12).
This study is a phase IIa clinical study investigating the
safety and pharmacokinetics of 2-IB administered to neo-
nates with moderate to severe birth asphyxia treated with
standard care without hypothermia in a low-resource setting.
An independent DSMB, consisting of three independ-
ent members with sufficient expertise in the clinical care
of neonates with perinatal asphyxia, biostatistics in early-
phase clinical studies, medical ethics, and pharmacology,
advised on the safety of study subjects and the overall sci-
entific merit of trial continuation, and assessed the results
of this clinical phase IIa study. The strategy for safety sur-
veillance by the DSMB was defined in the DSMB charter
as follows: (a) The DSMB reviewed the safety data during
the two planned meetings; also, the sponsor and the Coor-
dinating Investigator asked the DSMB to review individual
serious adverse events (SAEs). (b) The DSMB informed on
any suspected unexpected serious adverse reaction (SUSAR)
that had occurred involving 2-IB within 7 days for fatal and
life-threatening SUSARs and 15 days for other SUSARs. (c)
However, at any time during the study, if there was convinc-
ing evidence of harm based on the (unblinded) data includ-
ing, but not limited to, SAEs and SUSARs, the DSMB could
recommend that the sponsor consider such options as (but
not limited to) halting the study temporarily or definitely or
adapting the dosage regimen for all or selected populations.
(d) The DSMB made a recommendation to the sponsor after
review of the data. The stopping rule for safety was defined
as discontinuation of the study because major safety issues
were found that were related to the use of the study drug.
Additionally, the DSMB of the current PK and safety study
also advised on the ultimate dose regimen to be used in a
future randomized controlled phase IIb study, investigating
the efficacy of 2-IB for moderate to severe birth asphyxia
in LIC.
2.3 Patients
Near-term neonates (gestational age ≥ 36 weeks) with a
Thompson score ≥ 7 between 1 and 3 h after birth and the
ability to start treatment within 6 h after birth were included
in this study. Exclusion criteria were as follows: (1) inability
to insert an umbilical venous catheter for administration of
97
the study drug; (2) major congenital or chromosomal abnor-
malities such as diaphragmatic hernia, omphalocele, clini-
cally identified trisomy 13 or trisomy 18; (3) birth weight
< 1800 g; (4) clear signs of infection (defined as a rectal
temperature > 37.5 °C or skin rash such as pustules); (5)
moribund patients.
2.4 Study Drug
2-IB is a stable small molecule organic compound (243 Da)
with an adequate aqueous solubility at lower pH values.
There are no metabolites formed either in humans or piglets,
and the compound is completely cleared via the kidney. The
compound is a high clearance compound with a volume of
distribution slightly above total body water volume and the
kinetics appeared linear.
2-IB drug product formulations were temperature-logged
transported to DRC and stored in a chocolate cooler (not
exceeding temperatures of 25 °C) in the university hospi-
tal in Kinshasa. Earlier stability studies using storing at
25 ± 2 °C/60% relative humidity (RH), 30 ± 2 °C/65% RH
and 40 ± 2 °C/75% RH showed a very stable 2-IB drug prod-
uct. Samples were tested for appearance, pH, osmolality,
content, impurities, (sub)visible particles, closure integrity,
and sterility. All parameters remained unchanged during the
course of 6 months at 40 ± 2 °C/75% RH and of 5 years at
25 ± 2 °C/60% RH and 30 ± 2 °C/65% RH. No increase of
impurities/degradation products were observed over the time
course of the studies (data not shown).
2.5 Dose Regimen
After parental consent, neonates received six IV infusions of
0.16 mg/kg 2-IB, with 4-hourly intervals. 2-IB was admin-
istered as a slow intravenous bolus in a solution of 0.75 mg/
mL via an umbilical catheter. The first dose had to be admin-
istered within 6 h after birth.
2.6 Outcome
For safety analysis, the following parameters were
monitored:
1. Vital signs (heart frequency, respiratory rate, and non-
invasive blood pressure) using a vital signs monitor
(Mindray Bio-Medical Electronics, iMEC series, Shen-
zhen, China) from before start until 4 h after the last
study drug administration, at 36 h and 48 h after the first
study drug administration.
2. Need for clinical intervention due to respiratory and cir-
culatory insufficiency from the start until 15 min after
administration of the study drug.
98 T. Biselele et al.

3. The occurrence of AEs/SAEs, including mortality.
2.7 Pharmacokinetic Analysis
For PK analysis, five PK samples of 0.4 mL were taken via
heel prick; directly after completion of the first infusion,
just before the second and last infusion, and 1 h and 4 h
after the last infusion. Blood samples were centrifuged as
soon as possible after sampling, and the supernatant was
frozen at − 20 °C until temperature-logged transfer to the
Netherlands for determination of the concentrations. Bio-
analysis of plasma samples for 2-IB levels was performed
by validated liquid chromatography-mass spectrometry/mass
spectrometry method (Charles River Labs, ‘s-Hertogen-
bosch, the Netherlands). The lower limit of quantification
(LLOQ) of 2-IB in human EDTA plasma was 5.00 ng/mL,
with a dynamic range of 5.00–5000 ng/mL. Between-run
and within-run coefficients of variation were < 20%. Samples
were stored at − 80 °C until analysis.
PK analysis was performed using nonlinear mixed effects
modeling (NONMEM; version 7.4; ICON Development
Solutions, Ellicott City, MD, USA), with the main aim to
obtain individual exposure metrics for 2-IB [15]. As the
dataset was very small (27 observations from 7 subjects),
the dataset was enriched with interim data after completion
of part A of the 2-STEP study (31 observations from 6 sub-
jects), which became available during the execution of the
current trial [13]. An overview of the main characteristics of
the 2-STEP and current study is provided in Table 1.
Model development included testing of one- and two-
compartment models and the optimization of the interin-
dividual variability for the PK model parameter, as well as
the residual variability. The model was subjected to a visual
predictive check.
Table 1  Main characteristics of the model developmental dataset
Parameter Study
2-STEP part A [13] TIBC
Dose (mg/kg) 0.16 0.16
Infusion duration (min) 1 1
Treatment duration (h) 24 24
Number of doses administered 4 6
Dose interval (h) 6 4
Number of samples 31 27
Number of subjects 6 7
Race Caucasian Black
Income country High Low
Body temperature Hypothermic Normothermic
TIBC Two-IminoBiotin in the Democratic Republic of Congo
Given the small dataset, a formal statistical covariate
analysis was not considered to be reliable, hence a graphi-
cal exploratory analysis of the influence of selected covari-
ates (birth weight, gestational age, and Thompson score)
of interest on clearance was performed for the subjects
in the current TIBC study. Additionally, individual clear-
ance estimates from the subjects in the model develop-
ment dataset were compared between the 2-STEP study
and the current TIBC study. The main aim of the modeling
exercise was to obtain individual model-based exposure
metrics. Interindividual variability was modeled on clear-
ance, and a proportional residual error model was applied.
Individual estimates for the PK profiles were obtained
using the population parameter estimates and random
effects. Through simulation of the predicted concentra-
tions the following exposure metrics were computed for
each patient:
1. Cmax (observed maximum plasma concentration over the
full treatment duration).
2. AUC​ 0–4h (area under the plasma concentration–time
curve from time 0 to 4 h after infusion).
3. AUC​ 0–∞ (area under the plasma concentration–time
curve from time 0 to infinity).
4. Tmax (time at maximum plasma concentration).
5. t½ (terminal elimination half-life).
6. CL (clearance, model estimate).
7. Vss (volume of distribution at steady state, model esti-
mate).
2.8 Data Collection and Statistical Analysis
For this phase IIa study, no formal statistical analysis was
performed. Data were collected regarding the pregnancy,
delivery, and neonatal admission including postmen-
strual age, biometry, time of birth, Apgar at 5 min, time
of admission at the neonatal ward, and Thompson score
at age 1–3 h. The aEEG (CFM Olympic Brainz Moni-
tor; Natus Medical Incorporated, Pleasanton, CA, USA)
was recorded during the first 48 h and analyzed using
visual interpretation of the background pattern [16] and
presence of seizures on the raw EEG signal [17]. For the
assessment of safety and tolerability, vital signs, including
breathing and heart frequency, blood pressure, transcuta-
neous oxygen saturation, and temperature were monitored
and recorded 15 min before, just before, 15 and 30 min
after the administration of the study drug, every hour until
24 h after start of the study drug, and at 36 h and 48 h
for a maximum of 7 days using assessments done as part
of the standard care. The clinical course of each patient
was followed closely, and local tolerance and AEs/SAEs
including mortality were monitored. Data are reported
2-Iminobiotin for Birth Asphyxia in Low-Income Countries 99

as mean ± SEM or as percentages, when appropriate. 3.2 Outcome

Not normally distributed data were reported as median
(range). The vital signs from admission until 48 h after HIE are
shown in Fig. 1. No relevant change in heart rate, non-
invasive blood pressure, transcutaneous oxygen saturation,
3 Results or spontaneously respiratory rate was observed over time.
At admission the mean rectal temperature of the patients
3.1 Patients was 34.9 ± 0.6 °C, increasing to a stable temperature of
35.9 ± 0.4 °C at 3 h after start of 2-IB treatment. No hypo-
Seven infants were included in the trial during the 5-month thermia was instituted.
study period. All mothers had prenatal consultations. No relevant effects on heart rate and blood pressure were
Three infants were admitted at the University Hospital of seen during and after administration of 2-IB. There was
Kinshasa, two at the Saint Joseph Hospital and another two no need for intervention due to respiratory and circulatory
at the Clinique Ngaliema, of which two patients were out- insufficiency from the start until 15 min after administration
born. Maternal and neonatal characteristics of the included of the study drug.
patients are shown in Table 2. All the infants needed resus- Three patients died: two after completing the study proto-
citation until 10 min after birth with facial mask and room col (patient 1 at 3 days after birth due to a neonatal infection
air. Blood gas analysis for pH and base excess was not and patient 4 at 2 days after birth due to resistant hypoglyce-
available in all hospitals. None of the patients had intra- mia with severe metabolic acidosis); patient 3 was moribund
uterine growth restriction, neither were there clinical signs at inclusion and died 1 h after inclusion, and should have
of chorioamnionitis. been excluded from this study (for vital signs, see Online
All the infants experienced HIE with a median Thomp- Resource 1 in the ESM). None of these (severe) adverse
son score of 10 (range 8–16) between 1 and 3 h after birth. events were judged by the investigators as being (potentially)
Clinical seizures were observed in three infants at the time related to the study drug.
of admission. All seven neonates had aEEG recordings. A continuous
normal voltage pattern was seen in four out of seven patients
at inclusion, which remained normal until 48 h after birth.
Table 2  Maternal and neonatal characteristics
One of these patients (patient 2) had repetitive seizures at
Maternal factors 36 h after inclusion and was treated with phenobarbitone
Maternal age, years 31 ± 2.8 with good effect. All these four patients had sleep–wake
Primiparity, n (%) 2 (29) cycling at 48 h after birth. Patient 1 had a burst suppression
Preeclampsia, n (%) 1 (14) pattern with seizures at admission that remained identical at
Urogenital infection, n (%) 0 (0) 48 h after birth, despite phenobarbitone treatment. Patient 3
Premature rupture of membranes, n (%) 2 (29) had a continuous low voltage pattern with seizures at admis-
Emergency caesarean section, n (%) 5 (71) sion, 1 h before death. Patient 4 had a discontinuous normal
Inborn, n (%) 5 (71) voltage with severe seizures at admission and evolved to
Neonatal factors continuous low voltage at 48 h after birth.
Male gender, n (%) 2 (29)
Postmenstrual age (weeks) 38.8 ± 0.7
Biometry 3.3 Pharmacokinetics
Birth weight (g) 3150 ± 189
Length (cm) 48.2 ± 1.1 Full PK data of 2-IB in six subjects were described using a
Head circumference (cm) 34.1 ± 0.8 two-compartment model based on a parameter estimation
Apgar score of the dataset combined with part A from the 2-STEP study.
1 min 3 (1–7) The final model was a two-compartment model similar to
5 min 4 (2–6) that of part A from the 2-STEP study with interindividual
10 min 6 (4–7) variability detected on clearance alone. In Fig. 2, the clear-
Thompson score between 1–3 h after birth 10 (8–16) ance estimates from the subjects in the model develop-
Duration of resuscitation (min) 14.8 ± 3.1 (unknown ment dataset are shown of the patients in the current TIBC
in 1 patient) study and group A of the 2-STEP study. Median clear-
Admission at neonatal ward (min) 49 ± 11 ance was 0.539 L/h in normothermic patients, treated with
Data are presented as mean ± standard error of mean or when appro- 0.16 mg/kg every 4 h for 24 h compared with a clearance
priate median (range) and number (percentage) of 0.468 L/h in hypothermic patients, treated with the same
100 T. Biselele et al.

Fig. 1  Vital parameters a heart

rate, respiratory rate and trans-
a 180 100
cutaneous oxygen saturation, 160 90
b systolic and diastolic blood 80
pressure, and c temperature, 140
from admission until 48 h after 70

Saturaon (%)
120

Rate (min-1)
start of 2-iminobiotin treatment. 60
100
Data is mean ± SD. X-axis is 50
not linear 80
40
60
30
40 20
20 10
0 0

heart rate respiratory rate oxygen saturaon

b 90
80
Blood pressure (mm Hg)

70
60
50
40
30
20
10
0

systolic BP diastolic BP
c
38.0
Temperature (°C)

37.0
36.0
35.0
34.0
33.0
T= 12H 30MIN
T= -15 MIN
T= 0H 15MIN
T= 1H
T= 3H
T= 3H 59MIN
T= 4H 30MIN
T= 6H
T= 7H 45MIN

T= 9H

T= 11H 59MIN

T= 14H
T= 15H 45MIN
T= 16H 15MIN
T= 17H
T= 19H
T= 19H 59MIN
T= 20H 30MIN
T= 22H
T= 24H
T= 36H
T= 48H
T= 8H 15 MIN

T= 11H

temperature

.
Data is mean ± SD X-axis is not linear.

dose every 6 h for 24 h. The residual variability was modeled
proportionally.
The goodness-of-fit plots are given in Online Resource
2 (see ESM). A visual predictive check is given for the two
studies that were included in the model development dataset
separately (Online Resource 3, see ESM). The model was able
to accurately predict both the medians as well as the variability
of the observations for both the 2-STEP and the current study
as given by the precision in both the predicted and observed
5th and 95th percentiles. Model parameter estimates are given
in Table 3. Individual plots show high peak concentrations and
little to no accumulation of 2-IB, except for patient 1 who was
hypothermic from 13 to 48 h after inclusion and was shown to
have a neonatal infection afterwards (Fig. 3).
Individual exposure metrics based on the individual model
parameter estimates are described in Table 4. Median AUC​
0–4h was 664 ng*h/mL (range 414–917), nearly twice as high
as targeted. The median clearance of 2-IB treated patients
was 0.539 L/h. There was no apparent relationship observed
between clearance and body weight, gestational age, and dis-
ease severity, defined as Thompson score at admission, in this
group of patients (Online Resource 4, see ESM).
2-Iminobiotin for Birth Asphyxia in Low-Income Countries 101

due to respiratory and circulatory insufficiency during/after

2-IB administration. In this study, three patients died, of
which one patient was moribund at inclusion with a Thomp-
son score of 16 and should not have been included. This is
comparable with the mortality rate (44%) in the study that
was conducted in the same hospitals to investigate the fea-
sibility of neuroprotective treatment in the DRC [18]. None
of these SAEs were judged by the investigators and DSMB
as being (potentially) related to the study drug, but were
caused by neonatal infection (patient 1) at 3 days after birth,
and resistant hypoglycemia and severe metabolic acidosis
(patient 4) at 2 days after birth. Although only seven patients
were included in this trial, no 2-IB-related side effects were
reported by the principal investigator experienced in treat-
ing patients with moderate to severe HIE in LIC. In total, 25
neonates with HIE have now been treated in the pilot study
(n = 6), the 2-STEP study (n = 12), and the current TIBC
study (n = 7). Although no formal adverse drug reaction
Fig. 2  Boxplot for individual clearance determined using the cur-
rent model for individuals participating in the 2-STEP study cohort probability scale was performed in this or the other studies,
A and in the current study. The horizontal line indicates the median, no time-related reactions to the study drug infusion were
the grey area the interquartile range and the whiskers the range of the found in any of the studies.
data. Clearance of patients of the 2-STEP study [13] cohort A, treated
The present dosing regimen resulted in an AUC nearly
with a dose of 0.16 mg/kg/dose every 6 h for 24 h during hypother-
mia, and clearance of patients in current study, treated with a similar twice as high as that targeted based on the piglet model [6].
dose every 4 h for 24 h during normothermia. Data from patient 3 The discrepancy between expected versus observed AUC
of the current study have been excluded since this patient should not in this population is in all likelihood caused by the fact that
have been included
the pilot data was based on only two subjects. The vari-
ability in clearance could not be explained by bodyweight,
Table 3  Pharmacokinetic model parameters of 2-IB from 2-STEP gestational age, or Thompson score at admission (Online
and TIBC study Resource 4, see ESM). As serum creatinine concentrations
Parameter Estimate (%SE) 95% CI during the first few days of life are confounded by maternal
transfer, determination of individual renal function based on
Structural model parameters serum creatinine is subject to a high degree of uncertainty
CL (L/h) 0.435 0.303–0.567 in neonates, and is not part of the standard care in an LIC.
Vc (L) 0.740 0.467–1.01 Moreover, the present study was a pilot study and not pow-
Q (L/h) 2.12 1.69–2.56 ered for statistical inference.
Vp (L) 1.07 0.638–1.50 The current study leads to a better understanding of the
Interindividual variability PK of 2-IB in normothermic neonates. In future, larger scale
CL (%) 48 (31) 31–61 trials will allow the development of a model that is more pre-
Residual variability cise and less sensitive to potential bias so that the presence
Proportional 36 (25) 26–44 of clinically relevant covariates can be determined, thereby
CI confidence interval, CL clearance, Q intercompartmental clear- optimizing the dosing schedule of 2-IB.
ance, SE standard error of estimate, Vc volume of the central com- For a randomized study aimed at looking at effectivity
partment, Vp volume of the peripheral compartment of 2-IB after moderate to severe HIE in LIC, the dose regi-
men has to be adjusted to meet the AUC​0–4h target criteria
of 365 ng*h/mL. To achieve this, either the dose can be
4 Discussion reduced to 0.09 mg/kg per dose with 4-hourly intervals or
the dose interval can be prolonged to 6-hourly intervals with
This is the first PK and safety study of 2-IB in newborns a dose reduction to 0.14 mg/kg.
with moderate to severe hypoxia-ischemia in a low-resource In order to administer the neuroprotective treatment
setting, such as in the DRC. within the therapeutic window, the baby has to be trans-
Regarding safety, during and until 30 min after 2-IB ported to a neonatal ward facility within 6 h of birth. In LIC
administration, no relevant differences in vital parameters such as sub-Saharan Africa, this is a challenge, since there is
were detected. There was no need for clinical intervention a lack of good public transport and communications systems,
102 T. Biselele et al.

Fig. 3  Pharmacokinetic plots of 2-iminobiotin (2-IB) for each indi- individual model fit, and black dashed lines the population model fit.
vidual patient. Grey lines indicate the nominal dosing times, red dots Time is the time after first administration of 2-IB. Patient 3 is omitted
are the observed 2-IB plasma concentrations. Blue lines represent the since he died 1 h after birth

Table 4  Exposure metrics for individual patients of current TIBC study

Patient BW (kg) Cmax (ng/mL) AUC​0–4h (ng*h/mL) AUC​inf (ng*h/mL) Tmax (h) t½ (h) CL (L/h) Vss (L)

1 2.95 387 863 2284 21.0 6.28 0.207 0.74

2 3.10 167 534 676 0.1 1.93 0.734 0.74
4 3.40 407 917 1958 21.0 4.72 0.278 0.74
5 2.30 NA 414 538 19.9 2.05 0.685 0.74
6 2.90 359 691 1183 21.0 3.41 0.392 0.74
7 3.75 56 637 800 21.0 1.89 0.750 0.74
Median 3.03 359 664 992 21.0 2.73 0.539 0.74
Range 2.3–3.75 56–407 414–917 538–2284 0.1–21.0 1.89–6.28 0.207–0.750 0.74–0.74

AUC​ area under the curve, BW birth weight, CL clearance, Cmax maximal serum concentration, inf infinity, NA not available, t½ half-life, Tmax
time to maximal serum concentration, Vss volume of distribution at steady state model estimate
2-Iminobiotin for Birth Asphyxia in Low-Income Countries
and many patients cannot afford private transport. However,
in the setting of Kinshasa, we showed earlier that about 45%
of the patients are able to reach the hospital in time [18]. In
this study, five out of seven patients were inborn, and the
mean time to admission was 49 min.
Several neuroprotective treatment strategies are currently
being investigated to reduce cerebral damage after HIE. TH
has become the standard of care for neonatal encephalopa-
thy in high-income countries [19]. However, a meta-analysis
on TH in LIC using low-cost cooling techniques did not
show a significant reduction in neonatal mortality [3]. It was
concluded that adequately randomized controlled trials are
required before cooling can be offered in LIC [20].
Pharmacological strategies to reduce cerebral brain injury
following perinatal asphyxia in LIC should be affordable,
cost effective, and easy to be administered. Also, storage
conditions have to be compatible with a high-temperature
environment, since power shutdowns often occur in LIC.
2-IB proved to be a very stable drug that can be stored for
at least 6 months at temperatures up to 40 °C. It can be
easily administered either through an umbilical catheter or
a peripheral infusion, has a predictable PK profile, and is
affordable.
Based on the concepts of value-based healthcare, it is
important to perform a cost-benefit analysis for the addi-
tive treatment with 2-IB after birth asphyxia. In 2017, about
2.3% of the global burden of disease was being caused by
encephalopathy due to birth asphyxia and trauma [21]. For
TH in the Western world, a number needed to treat of seven
was found [19]. Although the efficacy of 2-IB still needs to
be tested in a future randomized clinical trial, the production
and administration costs of 2-IB are low, making a potential
treatment with 2-IB even in LIC feasible.
5 Conclusion
This is the first study investigating safety and PK of 2-IB
after moderate to severe HIE in LIC. The present dosing
regimen resulted in higher AUC levels than targeted. The
dose regimen will be adjusted to target the AUC in future
efficacy trials, which will inform improved PK models. No
adverse effects that could be attributed to the use of 2-IB
were observed.
Acknowledgements We greatly thank Laurent Favié and Floris
Groenendaal, the investigators of the 2-STEP study, who included
patients in the 2-STEP study and provided PK data of patients from
Group A to enrich the PK population model.
Compliance with Ethical Standards
Funding This study was funded by Neurophyxia BV.
103
Conflict of interest Authors T. Biselele, J. Bambi, D. Betukumesu, Y.
Ndiyo, G. Tabu, J. Kapinga, V. Bola, P. Makaya, and P. Vis have no dis-
closures. Authors H. Tjabbes and C. Peeters-Scholte are co-founders,
consultants for and shareholder of Neurophyxia BV. Author H. Tjabbes
is Vice-President Regulatory and Medical Affairs of Neurophyxia, and
author C. Peeters-Scholte is Chief Scientific Officer.
Informed consent All procedures performed in studies involving
human participants were in accordance with the ethical standards of
the ethical committee of the Public Health School of the University
of Kinshasa (DR Congo) and the ethical committee of the Univer-
sity of Utrecht (The Netherlands), and in line with the 1964 Helsinki
declaration and its later amendments or comparable ethical standards.
Written informed consent was obtained from one of the parents in
local language (Lingala) or French before start of the study. When the
consenting parent was illiterate, informed consent was given verbally
in the presence of an impartial witness.
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