Repositioning drugs for traumatic brain injury - N-acetyl cysteine and Phenserine
Hoffer et al. Journal of Biomedical Science
Repositioning drugs for traumatic brain injury - N-acetyl cysteine and Phenserine
Barry J. Hoffer 0
Chaim G. Pick 2
Michael E. Hoffer 1
Robert E. Becker 5
Yung-Hsiao Chiang 4
Nigel H. Greig 3
0 Department of Neurosurgery, Case Western Reserve University School of Medicine , Cleveland, OH , USA
1 Department of Otolaryngology, University of Miami Miller School of Medicine , Miami, FL , USA
2 Department of Anatomy and Anthropology, Sackler School of Medicine, Tel-Aviv University , Tel-Aviv , Israel
3 Intramural Research Program, National Institute on Aging, National Institutes of Health , Baltimore, MD , USA
4 Department of Neurosurgery, Taipei Medical University , Taipei , Taiwan
5 Aristea Translational Medicine , Park City, UT , USA
Traumatic brain injury (TBI) is one of the most common causes of morbidity and mortality of both young adults of less than 45 years of age and the elderly, and contributes to about 30% of all injury deaths in the United States of America. Whereas there has been a significant improvement in our understanding of the mechanism that underpin the primary and secondary stages of damage associated with a TBI incident, to date however, this knowledge has not translated into the development of effective new pharmacological TBI treatment strategies. Prior experimental and clinical studies of drugs working via a single mechanism only may have failed to address the full range of pathologies that lead to the neuronal loss and cognitive impairment evident in TBI and other disorders. The present review focuses on two drugs with the potential to benefit multiple pathways considered important in TBI. Notably, both agents have already been developed into human studies for other conditions, and thus have the potential to be rapidly repositioned as TBI therapies. The first is N-acetyl cysteine (NAC) that is currently used in over the counter medications for its anti-inflammatory properties. The second is (−)-phenserine ((−)-Phen) that was originally developed as an experimental Alzheimer's disease (AD) drug. We briefly review background information about TBI and subsequently review literature suggesting that NAC and (−)-Phen may be useful therapeutic approaches for TBI, for which there are no currently approved drugs.
Traumatic brain injury; N-acetyl cysteine; Phenserine
Traumatic brain injury
Traumatic brain injury (TBI) is the leading cause of
death and long-term disability in the developed world.
Annually, an estimated 10 million people suffer a TBI
event worldwide [
]. Projections indicate that TBI will
comprise the third largest portion of the total global
disease burden by 2020 . Within the US, an estimated
1.7 million people per year sustain a TBI, and
approximately 5.3 million people live with a TBI-induced
]. By far the majority of TBIs are mild to
moderate in nature and account for 80–95% of cases,
with severe TBI comprising the remainder . With
increases in survival rate following initial injury, TBI can
result in substantial and lifelong cognitive, physical, and
behavioral impairments that require long-term access to
health care and disability services [
vulnerable are the elderly, in which the same insult
results in greater disability and can lead to a dramatic
increase in the risk of neurodegenerative [
neuropsychiatric disorders. TBI symptoms can
occasionally resolve within the first year following injury, but
some 70% to 90% of patients continue to exhibit
prolonged and often permanent neurocognitive
dysfunctions. It is now recognized that TBI is a time-dependent
process, rather than a single static event. Emerging
evidence indicates that this process can lead to early onset
of dementia [
]. From a clinical perspective, TBI is
one of the most powerful environmental risk factors for
development of Alzheimer’s disease (AD). Recent gene
expression studies have defined the up-regulation of
pathways leading to AD and Parkinson’s disease induced
by mild, let alone moderate or severe forms of TBI [
]. In light of the lack of any available therapeutic
options, it is important to understand the mechanisms that
underlie head injury and the neuronal dysfunction and
loss that ensue as well as possible therapeutics.
TBI-associated brain damage can be classified into two
major phases. First, an initial primary damage phase
occurs at the moment of insult. This includes contusion
and laceration, diffuse axonal injury and intracranial
hemorrhage, and results in instantaneous (necrotic) cell
]. This period is followed by an extended
second phase that encompasses cascades of biological
processes initiated at the time of injury that may persist
over much longer times consequent to ischemia,
neuroinflammation, glutamate toxicity, astrocyte reactivity,
axonal shearing and apoptosis [
evidence suggests that secondary brain injury may be
reversible; depending on the biological cascades that drive
the delayed secondary phase that occurs following TBI
and how quickly and effectively these can be interrupted
or mitigated [
]. These cascades involve
neuroinflammation, oxidative stress, generation of reactive
oxygen species, inhibition of neurogenesis, apoptosis, loss of
cholinergic circuits, and glutamate excitotoxicity.
Importantly, these cascades occur in combination, rather
than alone. Indeed, such combinations are likely
complexed by time dependence, the nature of the TBI, the
nature of the recipient and environmental factors. In the
light of this, it perhaps not surprising that so many
experimental therapeutics directed towards a single
mechanism whose inhibition demonstrates promise in an
animal model of TBI in a homogeneous rodent strain
have failed to demonstrate efficacy in the human
condition. In the section below, we summarize how NAC and
(−)-Phen might alter these TBI-induced cascades and
There is considerable literature on NAC as a
neuroprotective agent in preclinical models of central and peripheral
nervous system injury. NAC has been shown to have
antioxidant and neurovascular-protective effects after
preclinical TBI [
]. NAC treatment following
controlled cortical impact (CCI) increased levels of
antiinflammatory M2 microglia in white matter tracts .
Specifically, there is neuroprotective efficacy of a single
dose of NAC in ameliorating biochemical and histological
endpoints in a rat weight drop model [
] and of multiple
doses in ameliorating inflammatory sequelae in an open
skull dural impact rat model [
]. The antioxidant and
anti-inflammatory effects of NAC [
] may be
downstream consequences of inhibition of NAC-induced
nuclear factor-κB-activated pathways that include cytokine
cascades and phospholipid metabolism [
], which may
also underlie the broader efficacy of NAC in rodent
ischemia-reperfusion cerebral stroke models [
24, 27, 29
rodent sensory nerve axotomy model, and prevention of
mitochondrial damage with loss of dendritic spines in
hippocampal neurons [
]. Both NAC treatment alone and
NAC treatment with topiramate ameliorate behavioral
signs of mild weight drop TBI in rodent models [
The up-regulation of brain levels of glutathione (GSH)
by systemic administration of NAC represents another
potential neuroprotective mechanism. NAC is a
precursor for GSH, which is a tripeptide derived by linking the
amine group of cysteine to a glycine and to the carboxyl
group of the glutamate side-chain. GSH is a critical
intracellular antioxidant that prevents damage caused by
reactive oxygen and nitrogen species (ROS and RNS).
GSH is generated within its target cells from the amino
acids, L-cysteine, L-glutamic acid and glycine.
Importantly, the sulfhydryl (thiol) group (SH) of cysteine acts
as a proton donor, and in this role is responsible for the
antioxidant activity of GSH (Fig. 1). This cysteine
represents the rate-limiting factor in cellular GSH production,
since cysteine is relatively scarce, except in specific
foods. Supporting the potential role of GSH in the
effects of NAC, it has been reported that, in spite of its
poor penetration into the central nervous system, NAC
can significantly elevate GSH levels in the brain
following oxidative stress [
] and GSH deficiency .
Moreover, it has recently been demonstrated in a unique
animal model of mTBI, involving thinning of the skull
and compression, that GSH from the periphery can
enter the brain and exert neuroprotective activity [
The cellular basis for memory and regulation of
motivation associated with the nucleus accumbens may also
be improved via NAC-induced neuronal activation of
cysteine-glutamate exchange, augmented by the indirect
effects of NAC on the metabolic glutamate receptors,
mGluR2/3 and mGluR5, as reported for amelioration of
cocaine-induced disruption of memory and regulation of
motivation in rodents [
We have also been evaluating NAC as a
countermeasure for the neurosensory sequelae of mTBI in military
]. The rationale underpinning this
approach is based on the fact that NAC’s mechanism of
action can ameliorate or prevent the cascade of
pathological events seen after mTBI, as noted above. In
addition, NAC is the active ingredient in the brand name
medication Mucomyst, a compound with a thirty-year
safety history in U.S. hospitals, used for cystic fibrosis,
acetaminophen poisoning, and high dye load x-rays as
both an oral and an intravenous treatment. As such, our
work represents repositioning of a “proven” medication
whose tolerability/safety is well characterized, as
opposed to introducing previously non-utilized or
nonFDA approved pharmaceuticals. Historically, this has
represented a more rapid and successful translational
strategy than developing new previously untested drug
candidates. Notably, we have demonstrated NAC to
be efficacious in reducing the sequelae of mTBI in a
randomized, double-blind, placebo-controlled study
examining mTBI after blast injury [
]. Mild traumatic
brain injury (mTBI) secondary to blast exposure is the
most common battlefield injury in the Middle East. There
has been little prospective work in the combat setting to
test the efficacy of new countermeasures. The goal of our
study was to compare the efficacy of NAC versus placebo
on the symptoms associated with blast exposure mTBI in
a combat setting. This study was a randomized double
blind, placebo-controlled study that was conducted on
active duty service members at a deployed field hospital in
Iraq. All symptomatic U.S. service members who were
exposed to significant blast and who met the criteria for
mTBI were offered to participate in the study, and 81
individuals agreed. Individuals underwent a baseline
evaluation and then were randomly assigned to receive either
NAC or placebo for seven days. Each subject was
reevaluated at 3 and 7 days. Outcome measures were the
presence of the following symptoms of mTBI: dizziness,
hearing loss, headache, memory loss, sleep disturbances,
and neurocognitive dysfunction. The resolution of these
symptoms 7 days after the blast exposure was the main
outcome measure in this study. Logistic regression on the
outcome of ‘no day 7 symptoms’ indicated that NAC
treatment was significantly better than placebo (OR = 3.6,
p = 0.006). Secondary analysis revealed subjects receiving
NAC within 24 h of blast had an 86% chance of symptom
resolution with no reported side effects versus 42% for
those seen early who received placebo. This study
demonstrates that NAC, a safe pharmaceutical countermeasure,
has beneficial effects on the severity and resolution of
symptoms of blast induced mTBI. This was the first
demonstration of an effective short-term countermeasure for
mTBI (Fig. 2). Further work on long term outcomes and
the potential use of NAC in civilian mTBI is warranted
focusing on sports head injuries and traffic accidents. To
highlight the value of work on NAC, the U.S. Army
recently published its new strategic research plan for
developing improved drug therapy for TBI [
]. In this
Number of clinical symptoms at seven days
document, the authors clearly indicate that NAC is one of
the only safe medicines that has reasonable pilot data for
the treatment of mTBI in a human, clinical setting and
strongly recommend expanded clinical trials.
Studies of experimental TBI models as well as post
mortem human TBI samples have demonstrated losses in key
features of the cholinergic system [
inhibitors have, for example, been appraised in preclinical
and clinical TBI studies, but have generated largely mixed
43–46, 14, 47
]. Paradoxically, rapid elevations in
acetylcholine (ACh) levels within CSF of animal models
and humans have been reported following TBI [
with higher levels associated with greater injury . This
trend supported the early experimental and clinical use of
anticholinergic agents, particularly muscarinic antagonists,
for the mitigation of ACh-related toxicity to ameliorate
TBI-induced deficits [
We evaluated the actions of an experimental and
reversible anti-acetylcholinesterase (AChE) agent,
(−)-phenserine tartrate ((−)-Phen) [
] in a well-characterized mild
concussive model of TBI in mouse [
]. Notably, in
addition to its anti-AChE activity, (−)-Phen is able to
inhibit the synthesis of amyloid precursor protein (APP) and
alpha-synuclein (α-syn), proteins of consequence in the
pathology of AD and PD, respectively, and of currently
increasing relevance to TBI in light of the up regulation of
pathways leading to AD and PD in animal models of TBI
] and in light of increased risk for early onset
dementia and PD in humans suffering TBI [
7, 8, 65–67
In addition, (−)-Phen possesses anti-inflammatory
], also a phenomenon of significance in TBI [
although the majority of anti-inflammatory approaches
have failed [
]. Furthermore, (−)-Phen possesses an array
of trophic and anti-apoptotic actions via mechanisms that
are now being characterized, as detailed below.
The potential mechanisms for (−)-Phen to be repositioned
for TBI are summarized as follows
(−)- (−)-Phen, developed as a drug candidate for AD at
the NIA, is a low molecular weight (mw 487.5), (−)-
chirally pure, lipophilic (Log D 2.2) orally bio-available
agent. The compound was originally developed as an
acetyl- cholinesterase selective inhibitor with a high
brain delivery [
]; importantly it is administered in
the form of its tartrate salt to support its required
aqueous solubility for pharmacological action [
]. In this
regard, (−)-Phen and three active first-pass hepatic
metabolites readily enter brain (approx. 7:1 to 1.25:1
brain/plasma ratios (Fig. 3) and, in dose-dependent
relationships (EC50 = 26 to 100 nM), produce a broad range
of pharmacological benefits of relevance to the effective
treatment of disorders such as TBI and AD. The actions
include anti-inflammatory; neutralizing oxidative stress;
neuroprotection from anecrotic cell death and neuronal
stem cell augmentation, as well as AChE, APP and α-syn
(−)-Phenserine’s active metabolites
Preclinical and clinical studies have recently demonstrated
that the broad range of beneficial pharmacological actions
provided by (−)-Phen administration derive from the
combined actions of (−)-Phen together with its stepwise
metabolism to it primary metabolites (−)-N1- and/or
(−)-N8-norphenserine to (−)-N1, N8-bisnorphenserine
(also called (−)-N1,N8-bisnorphenylcarbamoyl-eseroline)
]. The differing plasma concentrations,
brain:plasma distributions, t1/2 elim rates, and ranges of EC50s of
(−)-Phen and these key metabolites have been evaluated.
There is strong evidence of several relevant activities:
1. Antiinflammatory activities
Phytohemagglutinin (PHA) is a lectin present in
particular legumes, particularly red kidney bean
(Phaseolus vulgaris), and has potent, cell
agglutinating and mitogenic activities that cause
the immune activation of peripheral blood
mononuclear cells (PBMCs), and their subsequent
generation of cytokines. PHA is often used as a
tool to challenge PBMCs in culture, and as shown
in Fig. 4, results in the production and secretion of
pro- and anti-inflammatory cytokines, represented
by IL-1β and IL-10, respectively. As illustrated in
Fig. 4, (−)-Phen (0.1 to 10 uM) substantially
mitigated the PHA-induced elevation in
proinflammatory IL-1β levels without impacting
PHA-induced anti-inflammatory IL-10 levels;
thereby mitigating inflammation. Recent in vivo
studies in experimental TBI demonstrate that the
anti-inflammatory actions seen in ex vivo studies
of PBMCs translate into animals by mitigating
neuroinflammatory markers associated with
microglial cell activation. In the light of extensive studies
indicating that chronic neuroinflammation is a
common characteristic across neurodegenerative
disorders (including AD, PD, TBI and stroke) that drives
disease progression, its mitigation by well tolerated
agents can be considered beneficial .
2. Suppression of glutamate induced excitotoxicity:
Glutamate is a key excitatory neurotransmitter in
mammalian brain, and when intensely activated
can be toxic to neurons over a range of acute CNS
injury conditions that encompass TBI, stroke,
hypoglycemia and status epilepticus. Excess
glutamate is likewise implicated in chronic
neurodegenerative disorders, particularly AD.
Excessive glutamate activates its postsynaptic
receptors, N-methyl-D-aspartate (NMDA),
(AMPA) and kainate (KA). Such activation of
AMPA receptors depolarizes the cell and
concurrently unblocks the NMDA channels (releasing the
Mg2+ block), and thereby allowing Ca2+ entry.
Such depolarization opens voltage-activated
calcium channels, causing Ca2+ ion and water influx
into the cell down the osmotic gradient and
leading the cells to cytotoxicity. Illustrated in Fig. 5,
(−)-Phen treatment provides protection against
glutamate-induced excitotoxicity in rat primary
hippocamcal cultures. Specifically, glutamate
significantly reduced cell viability of cultured primary
hippocampal cultures by 53.5%, which was
mitigated by (−)-Phen as a return to 73.5% of control
levels and protection against anecrotic cell death
]. Similar neuroprotection was found in human
immortal neuronal (SH-SY5Y) cells with phenserine
analogs following glutamate challenge.
In relation to the in vivo relevance of cellular
studies indicating protection against glutamate
excitotoxicity, (−)-Phen has been evaluated in rats
challenged with a lethal dose of the
organophophate soman, where (−)-Phen both
increased the survival rate of animals and provided
neuroprotection of neuronal cells in the
hippocampus, basolateral amygdala and cingulate
]. In soman-induced toxicity, the sudden
substantial loss of AChE leads to abnormal
accumulation of ACh within cholinergic synapses and
results in the excessive stimulation of muscarinic
and nicotinic receptors within the central and
peripheral nervous systems. In the brain, such
excessive stimulation of cholinergic neurons induces the
release of glutamate, leading to the overactivation
of the NMDA receptor, and excessive influx of Ca2+
resulting in excitotoxic neuronal cell death [
These studies, together, support the notion that
neuroprotection provided by (−)-Phen in cellular
studies is of in vivo relevance, as additionally
supported in anoxia (stroke) in vivo studies in the rat.
3. Protection against oxidative stress:
Fig. 6 shows that (−)-Phen provides protection
against H2O2-induced oxidative toxicity in human
immortal SH-SY5Y cells. Human SH-SY5Y cells
were plated and after 24 h, cells were exposed to
(−)-Phen (10 or 30 uM) followed by oxidative
stress (100 uM H2O2). Cell viability was quantified
at 24 h (MTS assay). (−)-Phen treatment
significantly ameliorated the H2O2-mediated neuronal
toxicity and provided protection against apoptotic
cell death [
4. Inhibition of APP synthesis:
Multiple studies have demonstrated across
different laboratories that (−)-Phen lowers levels of
APP in neuronal cell cultures [
appears to be a non-cholinergically facilitated
action, as it is shared by its (+)-enantiomeric form,
Posiphen ((+)-Phenserine tartrate) that lacks
anticholinesterase activity, and is mediated
posttranscriptionally via an iron response element
within the 5′-untranslated region (5’UTR) of APP
]. The EC50 of this APP lowering
action appears to be in the order of 0.64 uM and
1.14 uM to lower secreted versus intracellular
levels of APP, respectively, in human immortal
neuronal (SH-SY5Y) cells [
]. Notably, primary
neurons appear to be more sensitive, with
(−)-Phen mediated APP lowering actions occurring
at far lower drug doses (100 nM) [
]. As Fig. 7a
and b document, (−)-Phen inhibits APP synthesis
in vivo, and importantly lowers brain tissue levels
of Aβ42. Fig. 7b shows the action of (−)-Phen on
Aβ levels in the cortex of transgenic (APPSWE + PS1)
AD mice over-expressing human Aβ, in which a
daily dose of 2.5 mg/kg substantially (p < 0.05)
lowered APP as well as Aβ. Such (-)-Phen induced APP
lowering action in brain translated to rats (Fig. 7a).
By contrast, neither donepezil nor (−)-physotigmine
(a structural analog of (−)-Phen) shared this action.
In line with the described APP lowering actions of
(−)-Phen, a similar dose in rats lowered nucleus
basalis lesion-induced elevations in APP, as
evaluated in CSF samples [
]. Figure 8 shows the
suppression of Aβ42 after administration of (−)-Phen to
humans in a study of healthy volunteers
administered the agent twice daily over 35 days in which
the dose was elevated to 15 mg BID ([
], Fig. 8).
This same (−)-Phen dose provided an efficacy signal
in mild to moderate AD patients [
]. A proof of
mechanism clinical study of Posiphen likewise has
demonstrated APP and Aβ lowering actions, as
evaluated in time-dependent CSF samples obtained after
10 day dosing. Notably, (−)-Phen’s APP lowing action
appears to be shared by not only its [+]-enantiomer
but also by its 3 primary metabolites at
concentrations as low as 100 nM [
]. Furthermore, these
actions on APP by (−)-Phen and analogs additionally
result in significant reductions in α-syn, which
similarly appears to have a regulatory element controlling
its translational efficiency within its 5’UTR [
In the light of several epidemiological studies
reporting that a history of brain trauma places a
patient at greater risk of developing AD and/or PD
7, 8, 65, 66, 67
], (−)-Phen mediated reductions in
APP, Aβ and α-syn may translate into potential
therapeutic value. In this regard, diffuse axonal
injury (DAI) is one of the most frequent and key
pathologies that occurs in TBI in both humans
and animal models (87). APP, in particular, is
routinely present in high concentrations in axons and is
conveyed through neurons via fast axonal transport.
As a consequence, a rapid and substantial
accumulation of APP is routinely evident in damaged axons
following experimental and human TBI. In fact,
immunohistochemical evaluation of APP
accumulation associated with axons, particularly in white
matter regions, is routinely used to detect DAI in human
brain tissue [
]. The accumulation of APP in axons
following TBI is considered an early event, and is
associated with upregulated APP gene expression [
]. The extensive co-distribution of APP with
Aβ accumulations and plaques has been
described in swollen axons associated with DAI
within days of an experimental TBI [
likewise, has been reported in human TBI [
with Aβ plaques evident within the gray matter
and notably also in the white matter in close
proximity to swollen axons. Together, these observations
indicate that damaged axons provide a prime source
of Aβ following a TBI. Such TBI-induced actions,
whether occurring early or later in life, can readily
increase the vulnerability of the brain to other
neurodegenerative events as detailed in the ‘two hit’
hypothesis of the ‘Latent Early-life Associated
Regulation’ (LEARn) model of Lahiri et al., [
whereby genetic and environmental risk factors
combine in an epigenetic pathway to trigger the
etiology of a later neurobiological disorder (such as
TBI leading to AD). Interestingly, the direct infusion
of an anti-APP antibody into the affected brain
region following TBI in rats resulted in a reduction in
neuronal loss, less astrocyte activation, a smaller
region of brain damage and less behavioral
impairment than was evident in vehicle treated TBI
animals , supporting a reduction in APP as a
therapeutic strategy worth investigating.
Similarly, TBI has been reported to alter the
distribution of α-Syn, and post-translationally
modified it. An abnormal accumulation of α-Syn
has been noted in axonal swellings and dystrophic
neurites in TBI brains, with the creation of
nitrated as well as conformationally modified
forms. In rodent TBI models, striatal axons exhibit
the most extensive accumulations of α-Syn forms
]. Albeit the role of this synaptic protein
requires greater elucidation, such TBI-induced
changes are likely to impair its physiological role,
and potentially induce a pathological one. Thus
mechanisms to lower α-Syn may be of importance.
5. (−)-Phenserine augments neurogenesis:
Extensive studies have demonstrated that
neurogenesis continues to occur throughout life
within key areas of the brain that include the
subventricular zone (SVZ) of the lateral ventricles
and the subgranular zone (SGZ) of the
hippocampal dentate gyrus (DG) in rodents, in
non-human primates as well as in humans
]. Newly generated neural stem cells (NSCs)
can differentiate into functional mature neurons
and integrate into neuronal networks, including
those involved in cognitive function [
Ischemic brain injury as well as TBI stimulates the
proliferation of NSCs localized to the SVZ and
SGZ of adult brain, and the resultant newborn
cells can migrate to damaged brain areas to
potentially differentiate into mature neuronal cells [
]. However, the process of neurogenesis is not
particularly efficient, and is impaired by numerous
factors initiated and amplified by ischemia and
TBI, such as the presence of neuroinflammation
]. Strategies and in particular, drugs that
enhance neurogenesis therefore hold the potential to
mitigate TBI and other neurodegenerative disorders.
First, Fig. 9 demonstrates, (−)-Phen enhances
neural precursor cell viability in cell culture –
increasing neurosphere size and augmenting their
survival. Second, in cellular and animal studies
high levels of APP (which are elevated by TBI as
well as in AD) induce the differentiation of NSCs
towards a glial phenotype, and away from a
neuronal one. This action is reversed by (−)-Phen
]. Third, (−)-Phen elevates neurotrophic factor
levels in brain – as assessed by measuring BDNF, a
key regulator of neurogenesis [
]. In both wild
type and AD transgenic mice; administration of
(−)-Phen analogs has been demonstrated to
augment neurogenesis [
] and, notably,
enhance the survival of neurospheres as well as
neuronal cells in culture .
6. Protection of neurons from anoxia:
The most consistent postmortem discovery in fatal
head injury is the presence of cerebral ischemia
], which appears to be a key outcome
predictor. Whereas there are numerous studies
documenting reductions in cerebral blood flow in
models of severe TBI where there is significant
tissue and microvascular failure consequent to
endothelial swelling, perivascular edema, and
microthrombosis, particularly contiguous to focal
lesions, its impact on mild and moderate TBI has
remained more difficult to conclusively define.
However, it is becoming increasingly appreciated
that tissue hypoxia following a TBI occurs in a
widespread manner in the brain, including within
regions that appear to be structurally normal.
Additionally, cerebral tissue hypoxia seems to arise
independent of ischemia, sometimes in areas of no
overlap, which suggests a microvascular etiology.
The measurement of brain tissue PO2, particularly
in humans, by using oxygen 15-labeled positron
emission tomography (15O PET) has recently
provided definitive evidence of cerebral ischemia
occurrence after early TBI [
], which may
persist for up to a week after injury. Hence
diffusion hypoxia in seemingly normal tissue,
distinct from macrovascular ischemia in injured
tissue, provides potential targets in TBI for
Evaluation of (−)-Phen in a classical rodent model
of ischemic stroke has been undertaken by two
separate research groups to appraise its protective
actions in conditions of anoxia. In anesthetized
male Sprague-Dawley rats with their right middle
cerebral artery ligated and the common carotids
clamped to induce focal infarction in the cerebral
cortex after 60 min of ischemia, treatment with
(−)-Phen (1 mg/kg/day) for four days, compared
to placebo, reduced the area of infarction as
assessed by digital scanner evaluation of brain
slices (p = 0.001). This neuroprotective activity, in
which anoxia leads to a focal lesion is supportive
of beneficial actions in mild to moderate TBI, in
which anoxia is considered less severe than in
middle cerebral artery occlusion-induced
7. Counters Cholinergic losses from Nucleus Basalis of
Meynert (NBM) Injuries:
One important pathological loss following head
injury results from trauma to the basal midbrain
and loss of cholinergic cells located in the NBM
and/ or loss of axons providing cholinergic input
to the cerebral hemispheres, hippocampus, and
other critical brain structures. (−)-Phen has
demonstrated efficacious benefits in the presence
of NBM cell losses, an early feature of AD
neuropathology that leads to elevated levels of
APP and Aβ [
Illustrated in Fig. 10 is the AChE inhibition
induced by (−)- Phen and its metabolites achieved
after a single acute administration of (−)-Phen to
]. As discussed above, (−)-Phen is a
highly potent inhibitor of AChE (IC50 = 22-36 nM)
in plasma and brain, as are its N1-nor and
N1,N8bisnor metabolites [
]. In rats, at a dose of 1 mg/
kg, (−)-Phen achieved a maximal inhibition of
73.5% at 5 min, and this only gradually decreased
to 43% at termination of the study at 8 h, with an
apparent t1/2 = 8.25 h (Fig. 10).
Notably, these levels of AChE inhibition reflect the
drug plus metabolite concentrations and result in
elevated brain levels of ACh [
]. As noted in
Fig. 3, (−)-Phen after oral dosing is first-pass
metabolized by hepatocytes into three active (−)
compounds: the N-1 Nor-, N-8 Nor-, and
N-1,N-8Bisnor-phenserine derivatives, which can all
readily enter brain. Thus any given initial
concentration of (−)-Phen will provide longer AChE
inhibition effects than other non-cholinergic
mediated drug and metabolite effects. In Fig. 10b,
(−)-Phen inhibits AChE in a dose-response
relationship in humans [
] that has been shown
efficacious in improving cognition lost in AD where
NBM lesions cause ACh deficiencies and at least
partially reversible cognitive losses [
8. Preservation of visual memory in mTBI mice [
In Fig. 11, as evaluated by the novel object
recognition (NOR) test at 7 days following a
mild concussive TBI (a 30 g free falling weight
from 80 cm striking a 30 g mouse on the left
side of the head in the area of the parietal
cerebral cortex above the hippocampus),
(−)-Phen at two clinically relevant doses (2.5
and 5.0 mg/kg BID for 5 days initiated after
mTBI) mitigated mTBI-induced cognitive
]. These TBI conditions (a 30 g
weight and 30 g mouse) were created to mirror
a human falling on their head from a three foot
fall, and are considered mild concussive injuries
], which certainly instigate
]. Notably, the rapid metabolic
degradation of (−)-Phen and metabolites ensures that
no pharmacological concentrations are evident
in brain during cognitive evaluations performed
2 days and later after cessation of (−)-Phen
dosing, thereby ensuring that mitigation of TBI
cognitive deficits are not symptomatically
induced by (−)-Phen’s known cholinergic actions.
9. Preservation of spatial memory in mTBI mice [
In Fig. 12, Y-maze testing was used to evaluate
spatial memory and performed 7 days and after
head injury. The mitigation of mTBI-induced
deficits by (−)-Phen in the Y-maze cross validates the
beneficial actions of the agent in the NOR
paradigm, described above.
10. Antioxidant action by augmenting endogenous
antioxidant proteins [
Our prior studies highlighted in Figures illustrated
above, additionally demonstrated in brain
subjected to mild TBI and treated with (−)-Phen
that markers of oxidative stress Thiobarbituric
Acid Reactive Substances (TBARS) were reduced
vs. mTBI alone, when evaluated in hippocampus 5
and 14 days after injury. This reduction in
oxidative stress was consequent to a (−)-Phen
induced upregulation in the activity/expression of
the endogenous antioxidant proteins superoxide
dismutase [SOD] 1 and 2, and glutathione
peroxidase [GPx] [
These studies demonstrate that clinically relevant
doses of (−)-Phen can provide a unique and broad
range of beneficial pharmacological actions that
may favorably impact the programmed cell death
that results following a TBI, with such apoptosis
being a common feature across many
neurodegenerative disorders. (−)-Phen illustrates
how a drug that was opportunistically developed
to supplement cholinergic activity in AD, and that
has proven well-tolerated and provided a
consistent evidence of efficacy [
], can –
consequent to its more recently discovered important
broad spectrum of pharmacological actions – be
optimized to not only provide potential efficacy in
TBI, but also provide a pharmacological tool to
understand how TBI can lead to AD. It has
become increasingly clear that multiple processes
combine together following an insult (whether an
acute TBI or a chronic degenerative disorder such
as in AD or PD) to induce the programed cell
death of neurons. The modulation of (i)
inflammation either directly or via cholinergic mechanisms,
(ii) oxidative stress, (iii) neurosphere/NPC
apoptosis/survival, (iv) glutamate excitotoxity, and (v)
APP/Aβ/α-syn over-expression, as well as ability to
augment endogenous trophic factors like BDNF
and stimulate other such mechanisms, provides a
means to both limit cell death and optimize
endogenous regenerative actions. Clinical trials in
TBI and AD of experimental drugs that act via a
single mechanism only, such as anti-inflammatory
or Aβ lowering approaches, have failed to address
the full range of pathologies that lead to neuronal
loss and cognitive impairment. (−)-Phen’s described
activation of multiple pathways, including the
augmentation of endogenous antioxidant, neurotrophic,
neuroprotective, anti-inflammatory, pro-angiogenesis,
APP/Aβ/α-syn-lowering as well as cholinergic and
others provide neuroprotection across multiple
animal models. The revelation of these multiple activities
of (−)-Phen and analogs over many years exemplifies
how initial notions of a drug’s mechanism of action
may mislead investigators away from its full spectrum
of benefits for human health.
This overview provides a broad horizon of mechanisms
linked to animal models and human data supportive of
drug interventions having potential clinical efficacy
against TBI. Many problems hinder progress identifying
the mechanisms behind the interesting potential of these
and other drugs and their efficacy. The criteria for
identifying that a concussion has occurred does not
necessarily capture head injuries with even more minor
symptomatology, which may be associated with later
unfavorable consequences. The duration of impairments
from concussions prove highly variable and only some
affected persons go on to display a post concussive
syndrome, later neurological impairments, or the serious
complication of chronic traumatic encephalopathy. In
spite of these and other difficulties, the availability of
diverse animal models with face validity for human
concussions/TBI, the many affected patients, and the
responsiveness of animal models and humans to the drugs
we have reviewed give medical research a chance to help
resolve the conundrum of TBI decisively and hopefully
better define the pathologies most closely associated
with the neuronal dysfunction and deaths behind
postconcussive/ TBI injuries. Perhaps it is time to develop
new peripheral, blood accessible, markers of TBI
pathologies so that investigators can recruit human subjects
for studies of TBI mechanisms. In that way we may
answer why many species, used as animal models, benefit
from candidate treatments for concussions while these
drugs fail to meet regulatory requirements for
registration for use in humans.
(−)-Phen: (−)−Phenserine; MWM: Morris Water maze; NAC: N-acetyl cysteine;
TBARS: Thiobarbituric Acid Reactive Substances; TBI: Traumatic brain injury
Partial support by MOST 104-2923-B-038-004-MY2.
Larimee R. Cortnik for her editorial assistance.
The data in this review article came from studies supported by the US Public
Health Service and Department of Defense, the Sackler School of Medicine,
Tel-Aviv University, and Taipei Medical University.
Availability of data and materials
BJH and NHG organized and wrote most of this article. CGP, MEH, REB, and
YC revised the paper and supplied additional data and figures. All authors
read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
Robert E. Becker does not have a competing interest but a conflict of
interest. For disclosure, Robert E. Becker holds a patent on the use of
phenserine in concussion/TBI and Alzheimer’s disease assigned to Aristea
Translational Medicine Corporation of Utah. None of the other authors have
any competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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