SE M I N A R S I N P E R I N A T O L O G Y ] (2015) ]]]–]]]

Available online at www.sciencedirect.com

Seminars in Perinatology

www.seminperinat.com

Drugs for neuroprotection after birth asphyxia:

Pharmacologic adjuncts to hypothermia
Frank van Bel, MD, PhDn, and Floris Groenendaal, MD, PhD
Department of Neonatology, University Medical Center Utrecht, Wilhelmina Children’s Hospital, Utrecht,
The Netherlands

article info abstra ct

Keywords: An adverse outcome is still encountered in 45% of full-term neonates with perinatal
Perinatal asphyxia asphyxia who are treated with moderate hypothermia. At present pharmacologic therapies
Pharmacologic neuroprotection are developed to be added to hypothermia. In the present article, these potential neuro-
Hypothermia protective interventions are described based on the molecular pathways set in motion
during fetal hypoxia and following reoxygenation and reperfusion after birth. These
pathways include excessive production of excitotoxins with subsequent over-stimulation
of NMDA receptors and calcium influx in neuronal cells, excessive production of reactive
oxygen and nitrogen species, activation of inflammation leading to inappropriate apopto-
sis, and loss of neurotrophic factors. Possibilities for pharmacologic combination therapy,
where each drug will be administered based on the optimal point of time in the cascade of
destructive molecular reactions, may further reduce brain damage due to perinatal
asphyxia.
& 2015 Elsevier Inc. All rights reserved.

Introduction and necrotic neuronal cell death due to secondary energy

Perinatal asphyxia in the near-term and full-term infant is
still one of the most important causes of neonatal death and
adverse motor and cognitive outcome and has an incidence
of 2–20 in every 1000 live born infants, depending in which
part of the world they are born.1,2
There is increasing evidence that not only the actual period
of antenatal and/or perinatal hypoxia-ischemia (HI) affects
the neuronal cells, but also reoxygenation and reperfusion
upon and after birth add substantially to delayed apoptotic
failure. This delayed brain damage may proceed up to days or
even weeks after birth.3,4
At the same time this biphasic or even triphasic pattern of
brain damage creates a “therapeutic” window, which is
believed to range from birth up to 6 h of life, during which
neuroprotective strategies can prevent or reduce the full
consequences of delayed brain damage.4–6 Up to now, the
only established postnatal therapy to achieve this goal to a
certain extent is moderate hypothermia.7,8 Although the
composite adverse outcome was reduced, an adverse

Herewith the authors of this article, Frank van Bel, MD and Floris Groenendaal, MD, declare that Frank van Bel and Floris Groenendaal
are, together with Cacha Peeters-Scholte, inventors of 2-iminobiotin as neuroprotective agent for neonates with cerebral hypoxia-
ischemia. They have any financial or personal relationships with other people or organizations that could potentially and
inappropriately influence their work and conclusions. They further declare to have no proprietary or commercial interest in any
product mentioned or concept discussed in this article.
n
Correspondence to: Department of Neonatology, University Medical Center Utrecht, Wilhelmina Children’s Hospital, Room KE 04.123.1,
P.O. Box 85090, 3508 AB Utrecht, The Netherlands.
E-mail address: f.vanbel-2@umcutrecht.nl (F.v. Bel).

http://dx.doi.org/10.1053/j.semperi.2015.12.003
0146-0005/& 2015 Elsevier Inc. All rights reserved.
2 SE M I N A R S I N
Table – Information of dosages of drugs and gasses used or being used in (ongoing) clinical studies investigating
pharmacologic neuroprotection after perinatal hypoxia-ischemia.
Drug or gas
Maternal allopurinol
Torrance et al.30
Kaandorp et al.26
Postnatal allopurinol
Van Bel et al.62
Benders et al.99
Gunes et al.63
Postnatal 2-iminobiotin (CT: NCT01626924)
Postnatal rhEPO
Elmahdy et al.82
Zhu et al.83
Wu et al.84 (þHT) Postnatal
Darbepoetin
Baserga et al.88 (þHT)
Postnatal xenon ventilation
TOBYXe study (þHT) (CT: NCT00934700)
CoolXeno 3 study(þHT) (CT: NCT02071394)
Postnatal Topiramate
NeoNATI study (þHT) (CT: NCT1241019)
Postnatal MgSO4
Hemen study (þHT) (CT: NCT02499393)
CT, www.ClinicalTrials.gov; HT, hypothermia; IT, infusion time.
outcome of 45% is still high8 and it is conceivable that the
outcome can be improved further when moderate hypother-
mia will be combined with other neuroprotective strategies.
In order to achieve this goal it is important to delineate the
potentially harmful cascade of molecular pathways induced
by fetal hypoxia and upon and after reoxygenation and
reperfusion. With this knowledge additional pharmacother-
apeutical interventions can be considered which may be
capable to inhibit these destructive mechanisms. In this
article we will particularly focus on those pharmacological
interventions which can be expected to be effective in the
human infant within the near future. Information of proposed
dosages of the drugs and gasses discussed in this article will be
provided, when available, in a separate table (Table).
Destructive molecular pathways initiated during
and after HI
1. Fetal hypoxia, an important determinant of perinatal
asphyxia, sets in motion the excessive production of
excitatory neurotransmitters which gives rise to the
activation of N-methyl D-aspartate (NMDA)- and
voltage-regulated ion channels on the cell membranes
resulting in a surge of extracellular calcium into neuro-
nal and microglial cells and astrocytes.9,10 This will not
only lead to direct cell damage but also to a drop in the
intra-and intercellular pH leading to production of pro-
radicals liberated from their binding proteins and accu-
mulation of xanthine.
P E R I N A T O L O G Y ] (2015) ]]]–]]]
Dosage
500 mg iv; IT: 10 min
500 mg iv; IT: 10 min
20 mg/kg/iv o 4 h (IT: 10 min); repeat 20 mg/kg/iv after 12 h
20 mg/kg/iv o 4 h (IT: 10 min); repeat 20 mg/kg/iv after 12 h
20 mg/kg/iv o 6 h (IT: 10 min); repeat every 12 h (total 120 mg/kg)
0.2 mg/kg/dose iv, o6h, repeat every 4 h up to 6 doses in 20 h
2500 U/kg/sc o 6 h;repeated daily for 5 days
300 U/kg or 500 U/kg/sc o 48 h; every other day for 2 week
250, 500, 1000, or 2500 U/kg/iv o 24 h; up to 6 doses every 48 h
2 or 10 μg/kg/iv o 12 h; second dose at day 7
30% Xenon gas for 24 h (start o 6 h)
50% Xenon gas for 18 h (start o 5 h)
10 mg/kg/orally-at admission; repeat at days 2 and 3
250 mg/kg/iv o 6 h (IT: 60 min); repeat on days 2 and 3.
2. Upon reoxygenation and reperfusion xanthine will be
metabolized to uric acid at the expense of excessive
formation of the superoxide free radical (O2
d ), which
plays a central role in the further formation of free
radicals and toxic compounds11–13: it reacts with pro-
radicals such as non-protein bound iron to form the very
toxic hydroxyl free radical (OH∙).14 It furthermore reacts
with nitric oxide (NO∙), because of the HI-increased
formation of neuronal and later also inducible nitric
oxide synthase (nNOS; iNOS),15,16 to form the toxic
peroxynitrite (ONOO
).
The initially high levels of excitatory neurotransmit-
ters and the surge of free radicals, especially upon
and early after reperfusion and reoxygenation con-
tribute to activation of transcription factors such as
nuclear factor kappa B (NFkB) and c-jun N-terminal
kinase (JNK) with subsequent activation of an
inflammatory response with abundant formation of
pro- and anti-inflammatory cytokines, and leading to
production of iNOS setting in motion a pre-apoptotic
pathway with further (delayed) brain damage.17–19
3. Finally, a substantial part of the delayed brain damage is
related to downregulation of the formation of neuro-
tropic, maturational, and growth factors inhibiting neu-
rogenesis and repair.20,21
In the Figure A, the four patterns of above mentioned
destructive molecular pathways are schematically depicted
as a function of postnatal age. The proposed pharmacological
 SEM I N A R S I N P E R I N A T O L O G Y ] (2015) ]]]–]]] 3

A) Time profile destructive pathways induced by perinatal asphyxia

Superoxide FR (O2-•)
(Pro-)radical formation (i.e.OH•)

pH Inflammation/cytokines / iNOS/Apoptosis
nNOS >NO° > ONOO-
FREE-Iron

Ca 2+

glutamate

Hypo-
Xanthine Modulation trophic factors

30 min 3h 6h 12h 24h days

Fetal
hypoxia Birth
B) Optimal neuroprotective (combination) therapy
Maternal/neonatal allopurinol (xenon/argon)
Hypothermia (72h)
Melatonin/rhEPO Melatonin/rhEPO
Sel-nNOS/iNOS inhib (2-IB)
rhEPO/IGF-1/repair with (M)SCs

Fig – (A) The (post-ictal) time profiles of the potentially destructive molecular pathways are depicted as a function of postnatal
age (hours). During fetal hypoxia there is an increase in excitatory neurotransmitters inducing calcium influx into brain cells;
formation of pro-radicals (free iron) due to lowering of the interstitial pH and accumulation of hypoxanthine. Upon
reoxygenation/reperfusion an early surge of O2
d (white line) and consequently of other free radicals (OH∙) (orange line) and
nitric oxide synthase-induced ONOO
(blue line) occurs. This chain of events as well activates the inflammatory pathway with
increased formation of pro- and anti-inflammatory cytokines (purple line) from about 6 h onward with switches on
inappropriate apoptotic activity. Finally the neurotrophic factors are downregulated (yellow line) leading to a diminished
possibility of recovery of affected neurons (O2
d , superoxide free radical; OH∙, hydroxyl free radical; ONOO
, peroxynitrite). (B)
Possible combination therapy paradigms with optimal points of time for start and cessation of pharmacological compounds
directed to the different destructive molecular pathways. If recognized, maternal allopurinol can be started during substantial
fetal hypoxia and continued during early life. Free radical and ONOO
formation can be reduced by free radical scavenging by
melatonin, rhEPO and 2-IB respectively (start within 6 h of age), whereas anti-inflammatory activity can be challenged by
melatonin and rhEPO up to 48 h of age; reduction of apoptosis and stimulation of production of (neuro)trophic factors can be
achieved with rhEPO, IGF-1 and/or (M)SCs, administered for longer periods (days or weeks) (rhEPO, recombinant human
erythropoietin; 2-IB, 2-iminobiotin; IGF-1, insulin growth factor-1; (M)SCs, (mesenchymal) stem cells). (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)

interventions as described below will be discussed on basis of
the course of the postnatal time pattern of these pathways.
Pharmacologic neuroprotective strategies (on top
of hypothermia)
Antenatal neuroprotective therapy
The formation of excitatory neurotransmitters, free (pro-)
radicals starts already during the actual hypoxia and accel-
erates upon birth. In this setting antenatal therapy via the
mother can be considered to be the “first line of defense,” at
least when fetal hypoxia has been recognized.
The only drug investigated in a randomized blinded clinical
trial in pregnant women just before delivery and diagnosed
with fetal hypoxia is the xanthine-oxidase inhibitor allopur-
inol and its active metabolite oxypurinol.22 Allopurinol and
oxypurinol readily pass the placenta and are detectable in
umbilical blood in therapeutic concentrations as early as
20 min after maternal oral or intravenous administration.23
Especially in high dosages allopurinol is supposed to be a
free-iron chelator and free-radical (hydroxyl) scavenger.24,25
Based on short-term outcome markers, allopurinol has been
shown to be potentially effective, but effects were gender
specific: only females showed benefit from antenatal therapy
with allopurinol.26 Sex differences with respect to neuro-
protective therapy after HI were already suggested in earlier
reports27–29 A drawback of the study was that in the vast
majority of fetuses hypoxia was mild. The long-term results
of this study are currently under investigation. An earlier
pilot study preceding the randomized trial by the same
investigators in fetuses with more severe hypoxia showed
the potency of allopurinol as a neuroprotective agent.30
A second potentially promising neuroprotectant is the
noble gas xenon. Xenon is an antagonist of the NMDA
4 SE M I N A R S I N P E R I N A T O L O G Y
receptor but has also strong anti-apoptotic properties.31–33
And, most important, xenon is considered to be a safe
anesthetic which rapidly crosses the placenta.34 This makes
xenon very attractive for its use in mothers undergoing an
emergency cesarean section because of severe fetal hypo-
xia.35 However, unlike for postnatal use (see also below),
there are to date no studies performed to investigate and
confirm the neuroprotective actions of xenon as an antenatal
neuroprotective strategy.
Ascorbic acid (vitamin C),36,37 tetrahydrobiopterin (BH4),38
phenobarbital,39 N-acetylcysteine,40 and melatonin41 were or
are proposed to be of value for antenatal neuroprotection, but
an unfavorable safety profile (N-acetylcysteine), inconclusive
or disappointing clinical results (vitamin C and phenobarbi-
tal) or no clinical studies (melatonin) prevent clinical use of
these compounds up to now.42
Magnesium has been shown to reduce excitotoxicity fol-
lowing hypoxia-ischemia by inhibition of the NMDA-recep-
tor/ion channel complex.43 The mode of action of magnesium
in fetal neuroprotection remains largely unknown, since
in vivo concentrations are much lower than those needed
for inhibition of the NMDA receptor.44 Although magnesium
administration before 33 weeks of gestation to women with
high-risk pregnancies have resulted in a reduction of cerebral
palsy and adverse motor development 45 is in term pregnan-
cies no definite beneficial effects of maternal magnesium
demonstrated.46
Early postnatal neuroprotective therapy
As summarized above, the immediate postnatal reoxygena-
tion–reperfusion phase leads to further to the activation of
NMDA- and ion channels with subsequent calcium influx in
neurons, excessive formation of free radicals, and the nNOS-
related toxic ONOO
.
Therefore, NMDA/ion channel antagonists, inhibition of
xanthine-oxidase, prevention of nNOS (and subsequently
also of iNOS) formation, and chelation of pro-radicals are
critical to reduce early reperfusion–reoxygenation damage to
the brain.
In recent studies noble gases, which are NMDA/ion channel
antagonists, have been used for neuroprotection. Xenon, a
known anesthetic has been tested in several experiments.47–49
Xenon shows a rapid passage of the blood–brain barrier, and
has no reported negative cardiovascular side effects. Xenon as
such did not appear to be effective, but in combination with
hypothermia neuroprotection by xenon has been demon-
strated in several species.42
In a feasibility study 14 neonates with perinatal asphyxia
and therapeutic hypothermia received up to 50% xenon for
18 h and no adverse side effects were demonstrated.50
Several dosing strategies of xenon are explored, including
50% xenon for 18 h (ClinicalTrials: NCT02071394), or 30%
xenon for 24 h (ClinicalTrials: NCT00934700). At present
follow-up of the patients included in these studies is ongoing.
Prolonged use of xenon in neonates faces several chal-
lenges. Most important of all xenon is very expensive, and
therefore adjustments in neonatal ventilators need to be
made to recruit xenon.51 To reduce loss of xenon cuffed tubes
are used which might enhance the risk of tracheal lesions.
] (2015) ]]]–]]]
To avoid these latter potential problems other noble gases
have been tested for neuroprotective properties.52 In partic-
ular argon was neuroprotective after neonatal brain hypoxia-
ischemia.52,53 A study in piglets showed cardiovascular stabil-
ity of argon administration after hypoxia and during ther-
apeutic hypothermia.54 Blood levels of argon could be
measured and were dose dependent. The noble gas argon is
much cheaper than xenon, and may be a useful alternative
for xenon.
Another clinically potential pharmacologic intervention for
the near future may be inhibition of excessive NOS formation.
Whereas non-selective inhibition of NOS is detrimental,
probably due to vasoconstriction, selective inhibition of
neuronal and inducible NOS appears to be neuroprotec-
tive.55,56 A selective nNOS inhibitor that has been used in
many experiments is 7-nitroindazole. However, some addi-
tional inhibition of eNOS may occur, which is not desirable in
cerebral hypoxia-ischemia.57 Therefore, compounds have
been developed that have much higher in vitro specificity
and potency than available nNOS inhibitors.58 Combination
of a selective nNOS inhibitor with a selective iNOS may be
more effective. 2-Iminobiotin is an in vitro inhibitor of both
nNOS and iNOS and provides neuroprotection after hypoxia-
ischemia in neonatal rat and pig models, although the neuro-
protection may be independent of NOS inhibition in vivo.59–61
A phase 2 study is ongoing (ClinicalTrials.gov Identifier:
NCT01626924).
Allopurinol may have its most optimal indication for
antenatal use in case of proven fetal hypoxia, especially to
inhibit the excessive production of xanthine-oxidase during
birth. However, several clinical studies reporting the use of
intravenous allopurinol in the (early) postnatal period
showed a beneficial effect on short-term cerebral biomarkers
and even on long-term outcome, especially in moderately
asphyxiated infants and seems to have no serious adverse
effects.62–64
However, in a Cochrane review in 2008 no conclusions
could be drawn about the efficacy of postnatal neuroprotec-
tion with allopurinol after perinatal asphyxia since the
clinical trials reported up to now were underpowered.65 An
international trial aiming to treat asphyxiated newborns on
the resuscitation table with allopurinol is under considera-
tion at present.
Other neuroprotective drugs with potential for future clin-
ical use, particularly in the early postnatal period, are mela-
tonin and N-acetylcysteine (NAC). Melatonin has been
thought to have anti-oxidative properties on top of its anti-
inflammatory and anti-apoptotic effects.66–68 Experimental
studies showed neuroprotective properties in the mid- and
late-gestation sheep fetus.68–69 A small clinical study in
asphyxiated newborns showed a reduction of free radical
formation.70 Although melatonin seems to be safe and with-
out serious adverse effects, there is no consensus on the
optimal dosing.71 NAC is a precursor of glutathione, has the
properties of an anti-oxidant and is supposed to exert also a
direct free radical scavenging activity.72 The neuroprotective
properties are controversial and (hemodynamic) adverse
reactions are reported.42 Neuroprotective actions of NAC have
been reported in neonatal rodents as well as in piglets with
and without additional hypothermia.73,74 Although clinically
 SEM I N A R S I N P E R I N A T O L O G Y
used in preterm newborns, mostly in relation with preven-
tion of bronchopulmonary dysplasia, no clinical studies are
done yet in asphyxiated (term) newborns.
Studies with tetrahydrobiopterin being a neuroprotective
agent after perinatal asphyxia are actually non-existent,
although its safety profile is very attractive for further study
in relation with perinatal hypoxia-ischemia.42 Topiramate, a
well-known anticonvulsant, has sparsely investigated for its
neuroprotective actions after birth asphyxia.75 A safety and
efficacy study (NeoNATI) was recently completed in combi-
nation with moderate hypothermia76 (ClinicalTrials:
NCT01241019). Postnatal magnesium administration has not
been uniform in demonstrating neuroprotective effects of
magnesium in full-term affected neonates.44 Earlier trials for
neuroprotection after perinatal hypoxia-ischemia were dis-
continued because Magnesium administration carried the
risk of severe hypotension.77 However, currently studies are
performed of magnesium combined with moderate hypo-
thermia (ClinicalTrials: NCT02499393 and NCT01646619).
Anti-inflammatory and anti-apoptotic therapy
The transcription factor nuclear factor kappa B (NFkB) regu-
lates the expression of genes involved in inflammation and
apoptotic activity. Its activation gives rise to abundant for-
mation of pro- and anti-inflammatory cytokines, production
of inducible NOS (iNOS), and inappropriate apoptosis.18
Of the neuroprotective agents with an anti-inflammatory
action and a potential for clinical application in the near
future erythropoietin (EPO) is the most and up to now only
attractive candidate.78 EPO exerts its anti-inflammatory
action after binding to its receptor (EPOR) which is expressed
on the membranes of neurons, astrocytes, and microglial
cells.79 Recombinant Human EPO (rhEPO) is already quite
extensively used in the very and extremely preterm infant in
increasing dosages and in a pilot study in term neonates with
neonatal stroke and has a safe pharmacological profile.80,81
Numerous experimental studies in experimental studies in
different neonatal species showed its neuroprotective proper-
ties with respect to post-hypoxic-ischemic brain damage.79
Clinically a pilot study of 45 patients showed that treatment
of asphyxiated newborns with rhEPO seemed safe and fea-
sible.82 A blinded randomized phase III trial in asphyxiated
term newborns in China showed that repeated (low) doses of
intravenous administered rhEPO decreased the incidence of
disabilities at 18 months of age, although there was a
suggestion that females but not necessarily males benefit
from this therapy.83 A pharmacokinetic study in 24 severely
asphyxiated newborns who underwent hypothermia were
treated with additional rhEPO and showed that a quite high
dose of 1000 U/kg was well tolerated. Plasma concentrations
were similar as reported in earlier neuroprotective studies in
newborn animals.84 A randomized trial with hypothermia
with high-dose rhEPO as add-on therapy is currently running
(ClinicalTrials: NCT01913340) These randomized trials in
newborns undergoing hypothermia are necessary to ulti-
mately confirm the neuroprotective actions of additional
rhEPO and to determine the optimal timing, dose, and
duration of treatment. Up to now no serious adverse effects
are reported during the clinical use of rhEPO in (preterm)
] (2015) ]]]–]]] 5
newborns. However, especially when higher dosages are used
and/or administered for longer periods of time there may be
concerns with respect to safety and more research is war-
ranted here.85,86 EPO derivatives which have less unwanted
effects such as asialo-EPO 87 or longer half-life such as
darbepoietin88 are less well investigated but may be clinically
interesting because of their specific properties.
In addition to its anti-oxidative and free radical scavenging
actions melatonin is also supposed to have anti-
inflammatory properties by preventing activation and trans-
location of NFkB to the nucleus89 (also see above).
The development of inhibitors of pro-inflammatory cyto-
kines90 or anti-apoptotic strategies (NFkB inhibition)91 are
extremely interesting and potentially very forceful but
beyond the focus of this review.
Suppletion of (neuro)trophic and growth factors
Neurotrophic, maturational, and growth factors are impor-
tant for a normal development of the immature brain by
stimulation of the endogenous neurogenesis.92 Downregula-
tion of these factors occurs after severe perinatal HI and may
be deleterious for recovery and proper development of the
post-asphyxial newborn brain.
Besides the anti-oxidative and anti-inflammatory actions of
EPO, as described above, it has been supposed that EPO has
also important neurotrophic properties.93–95
It is suggested that for a sustained effect of EPO on neuro-
and angiogenesis and thus improved long-term outcome
higher doses (up to 5000 U/kg) and prolonged periods of
treatment are necessary, which may raise safety
concerns.85,86
An important related issue is the role of hypoxia-induced
factor 1α (HIF-1α), activated during and upon hypoxia-ische-
mia, which gives rise to the formation of transcriptional
targets, leading to an enhanced production of EPO and
endothelial growth factor and consequent brain repair.96
Other neurotrophic factors such as IGF-1, basic fibroblast
growth factor (bFGF), and BDNF in experimental studies in
neonatal animals are reported to be also effective to reduce
post-asphyxial brain damage.97,98 However, use of autologous
or allogeneic mesenchymal stem cell therapy after perinatal
hypoxia-ischemia seems more effective in this respect but is
discussed separately in this issue.99
Combination therapy, the way to go
It is unrealistic to assume that single or add-on therapy
without taking into account the time frame of the sequential
potentially destructive molecular pathways, will be the ulti-
mate answer toward a substantial reduction of brain damage
after (severe) perinatal asphyxia. Moreover, appropriate dos-
ing of the drugs, pharmacologic compounds, and gasses
discussed above, the duration and site of their administra-
tion, and a possible gender specificity of the compounds used
needs ongoing research. However, when we can solve the
above mentioned questions and are able to determine the
optimal post-ictal time of intervention in each specific
molecular pathway, a major achievement can be added to
6 SE M I N A R S I N P E R I N A T O L O G Y
the treatment of hypoxia-ischemia-induced neonatal
encephalopathy.78
The Figure B shows a tentative (pharmacological) treatment
paradigm, which may have clinical relevance in the (near)
future.
Concluding remarks
Ongoing research in neuroprotective pharmacology after
perinatal asphyxia has offered us potentially forceful com-
pounds to further reduce and to repair damage to the
immature brain during and after severe birth asphyxia as
add-ons to the only established neuroprotective therapy—
moderate hypothermia. There is still a long way to go to
determine the optimal dose of these compounds and to find
the optimal point of time of start and duration of pharmaco-
logical therapy. A definite decision on the clinical use
depends, of course, first of all on the safety profile of the
potentially neuroprotective drug.
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