Journal of the Neurological Sciences 419 (2020) 117217

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Journal of the Neurological Sciences

journal homepage: www.elsevier.com/locate/jns

Review Article

Strategies to prevent hemorrhagic transformation after reperfusion

therapies for acute ischemic stroke: A literature review
Yutaka Otsu a, 1, Masaki Namekawa a, 1, Masafumi Toriyabe a, b, 1, Itaru Ninomiya a,
Masahiro Hatakeyama a, Masahiro Uemura a, Osamu Onodera a, Takayoshi Shimohata c,
Masato Kanazawa a, *
a
Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan
b
Department of Medical Technology, Graduate School of Health Sciences, Niigata University, Niigata, Japan
c
Department of Neurology, Gifu University Graduate School of Medicine, Gifu, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Reperfusion therapies by tissue plasminogen activator (tPA) and mechanical thrombectomy (MT)
tPA have ushered in a new era in the treatment of acute ischemic stroke (AIS). However, reperfusion therapy-related
Thrombectomy HT remains an enigma.
Reperfusion
Aim: To provide a comprehensive review focused on emerging concepts of stroke and therapeutic strategies,
Therapeutic time window
including the use of protective agents to prevent HT after reperfusion therapies for AIS.
Hemorrhagic transformation
Methods: A literature review was performed using PubMed and the ClinicalTrials.gov database.
Results: Risk of HT increases with delayed initiation of tPA treatment, higher baseline glucose level, age, stroke
severity, episode of transient ischemic attack within 7 days of stroke onset, and hypertension. At a molecular
level, HT that develops after thrombolysis is thought to be caused by reactive oxygen species, inflammation,
remodeling factor-mediated effects, and tPA toxicity. Modulation of these pathophysiological mechanisms could
be a therapeutic strategy to prevent HT after tPA treatment. Clinical mechanisms underlying HT after MT are
thought to involve smoking, a low Alberta Stroke Program Early CT Score, use of general anesthesia, unfavorable
collaterals, and thromboembolic migration. However, the molecular mechanisms are yet to be fully investigated.
Clinical trials with MT and protective agents have also been planned and good outcomes are expected.
Conclusion: To fully utilize the easily accessible drug—tPA—and the high recanalization rate of MT, it is
important to reduce bleeding complications after recanalization. A future study direction could be to investigate
the recovery of neurological function by combining reperfusion therapies with cell therapies and/or use of
pleiotropic protective agents.

1. Introduction with tPA administration is a class I recommendation included in the

Acute ischemic stroke (AIS) is a leading cause of death and disability
worldwide. The 25th anniversary of the first tPA clinical trial and the 5th
anniversary of the first MT trial ushered in a new era in the treatment of
AIS. Currently, intravenous (IV) administration of tissue plasminogen
activator (tPA) is the only thrombolytic therapy approved to treat AIS
within 4.5 h of stroke onset [1–3]. In addition, mechanical thrombec­
tomy (MT) or endovascular therapy has shown great benefit to AIS pa­
tients when performed within 6 or 8 h of stroke onset in patients with
large vessel occlusion [4–8]. MT with a stent retriever in combination
American Heart Association/American Stroke Association guidelines
(causative occlusion of the internal carotid artery or middle cerebral
artery segment 1 [M1]; to be administered within 6 h of symptom onset)
[3]. The frequency of tPA treatment increased from 9.9% in 2006 to
21.8% in 2018 according to the Austrian Stroke Unit registry [9].
However, in the United States, between 3.4% and 5.2% of all patients
with AIS are eligible for tPA treatment because of the huge geographical
area and differences in healthcare systems [10]. MT was performed in
only 3.0% of patients with AIS in the Catalonian registry (REVASCAT
trial) [11]. In other words, patients eligible for reperfusion treatment

* Corresponding author at: Department of Neurology, Brain Research Institute, Niigata University, 1-757 Asahimachi-dori, Chuoku, Niigata 951-8585, Japan.
E-mail address: masa2@bri.niigata-u.ac.jp (M. Kanazawa).
1
These authors contributed equally to the work.

https://doi.org/10.1016/j.jns.2020.117217
Received 1 September 2020; Received in revised form 9 October 2020; Accepted 29 October 2020
Available online 4 November 2020
0022-510X/© 2020 Elsevier B.V. All rights reserved.
Y. Otsu et al.
still account for only 5%–10% of all patients with AIS. In the pooled
analysis, tPA (alteplase) increased hemorrhagic transformation (HT),
especially when administered from 3 h to 4.5 h after onset of symptoms
[12,13]. Even within 4.5 h of stroke onset, early treatment is very
important for patients with severe stroke because of the increasing risk
of symptomatic HT or intracranial hemorrhage [13]. Therefore, strate­
gies to prevent HT that develop after reperfusion therapies might in­
crease the number of patients who are eligible for reperfusion.
Reperfusion within the therapeutic time window can reduce brain
damage by salvaging a reversibly damaged penumbra of tissue. Ische­
mia–reperfusion–induced injury has been demonstrated in a variety of
organ systems, caused by many potential mechanisms that can disrupt
the neurovascular unit and increase the blood-brain barrier (BBB)
permeability, thus leading to HT. Very recently, among patients with AIS
who underwent diffusion-weighted imaging (DWI) with fluid-
attenuated inversion recovery (FLAIR) and perfusion mismatch se­
quences between 4.5 and 9 h of stroke onset, tPA treatment improved
functional outcomes [14,15]. However, the incidence of symptomatic
HT has increased [14,15]. Another meta-analysis suggested that eligible
patients presenting with unknown symptom onset time and DWI-FLAIR
mismatch or patients with symptom onset outside the conventional time
window of 4.5 h with evidence of viable tissue on penumbral imaging
could benefit from prompt intravenous (IV) tPA administration; how­
ever, the HT increased [16]. Moreover, although the rate of symptom­
atic HT did not differ significantly between the MT and control groups in
the DAWN and DEFUSE 3 trials, the symptomatic HT ratio in the MT
group was double than that in the control group (asymptomatic HT was
unknown) [17,18]. From the findings of the Safe Implementation of
Treatments in Stroke-International Stroke Thrombolysis Registry (SITS-
ISTR) (N = 50,726), early neurological deterioration (at 24 h from
baseline scale) was most associated with HT and death (odds ratio, 3.72
and 7.32, respectively) [19]. Therefore, HT attenuation after tPA
treatment and MT is an important and safe therapeutic strategy to treat
AIS. It might enable extension of the therapeutic time window and the
probability of achieving more excellent outcomes.
This review focuses on emerging concepts of stroke and the thera­
peutic strategies to improve the outcomes of patients with AIS, including
administration of protective agents to prevent HT after reperfusion
therapies for AIS.
2. Methods
A literature review was performed using PubMed and the National
Institutes of Health clinical trial database (ClinicalTrials.gov). We
searched articles published between April 1995 and August 2020 using
the following search terms: “stroke,” “cerebral ischemia,” “thrombol­
ysis,” “tissue plasminogen activator, tPA,” “mechanical thrombectomy,
MT,” and “hemorrhagic transformation, HT,” and “pleiotropic mecha­
nism.” Based on the data of the clinical studies, we selected positive
Fig. 1. Flowchart for study inclusions.
2
Journal of the Neurological Sciences 419 (2020) 117217
outcome or ongoing studies (Fig. 1).
3. Results
3.1. HT pathophysiology after thrombolysis
To prevent the onset of thrombolysis-associated HT, it is necessary to
understand its pathophysiology. The risk of symptomatic HT also in­
creases with several factors (Table 1). However, the risk of HT
completely offset by a long-term survival benefit if treatment is
administered within 4.5 h [20–22]. These risk factors may have caused
BBB disruption and HT.
The molecular mechanisms underlying HT are thought to be caused
by the following: (1) cerebral ischemia/reperfusion injury leading to the
activation of several reactive oxygen species (ROS), (2) inflammation,
(3) remodeling factor-mediated effects, and (4) direct toxicity of tPA
(Fig. 2) [23–25]. HT-induced mediators change chronologically and
dynamically, whereas in rat models, (1) ROS production was higher in
the reperfusion group than in the middle cerebral artery occlusion
(MCAO) models [26]. In permanent MCAO models, ROS gradually
increased over the course of up to 3 h. By contrast, reperfusion after 1 h
of MCAO resulted in a significant and sustained increase in ROS for 3 h.
ROS, presumably, play a role in the early stages of reperfusion injury. (2)
Inflammation is also an important factor mediating reperfusion injury.
tPA-induced reperfusion resulted in increased numbers of neutrophils,
microglia and macrophages [27–30], and proinflammatory M1 macro­
phages after 3 h [31]. Further, inflammation mediates BBB disruption
and induces HT. (3) Angiogenesis after ischemia could be a pathological
process contributing to neuropil injury after ischemia, or might be a
mechanism to limit the ischemic injury, or both, or perhaps a mecha­
nism to recover tissue function [32,33]. During angiogenesis, remodel­
ing factors, such as vascular endothelial growth factor (VEGF),
angiopoietin-1, and matrix metalloproteinase (MMP)-9, play impor­
tant roles in the chronic phase of ischemia. However, alternation in the
levels of these factors in the acute phase induces HT [34–36]. In hy­
perglycemic mice, tPA treatment increased peroxisome proliferator-
activated receptor-γ upregulation, activation of inflammasomes, and
VEGF and MMP-9 signaling [37]. HT after thrombolytic therapies might
be coupled with VEGF and MMP-9 signaling [31,34,37,38]. Remodeling
factors mediate BBB disruption and delay HT (> 18–24 h). Inhibition of
the VEGF signaling pathway and the administration of angiopoietin-1
attenuates HT after tPA treatment of ischemic stroke [34,35].
Although this treatment can enable vascular protection, it cannot reduce
the cerebral infarct volume [34,35]. Pleiotropic target, which includes
vascular protection, neuroprotection, and anti-inflammation, is an ideal
therapeutic strategy for ischemic stroke. It has been reported that the
growth factor, progranulin, could protect against acute focal cerebral
ischemia by neuroprotection, suppression of neuroinflammation, and
attenuation of BBB disruption via VEGF inhibition [27]. Intravenously
administered recombinant progranulin significantly reduced the vol­
umes of cerebral infarction and edema, suppressed HT, and improved
motor outcomes in thromboembolic rats with delayed administration of
Table 1
Risk factors of hemorrhagic transformation after tPA treatment.
Risk factors
Delayed initiation of tPA (alteplase) treatment
Higher baseline glucose level
Diabetes mellitus
Hypertension
Older age
Greater stroke severity
Large infarct volume
Excessive alcohol consumption
Episode of TIA within 7 days
tPA, tissue plasminogen activator; TIA, transient ischemic attack.
Y. Otsu et al.
Fig. 2. Mechanisms underlying intracerebral hemorrhagic transformation after
tPA treatment and the therapeutic targets (revised figure from Kanazawa et al.
[25]). BBB, blood-brain barrier; ROS, reactive oxygen species; tPA, tissue
plasminogen activator.
tPA treatment. It has also been reported that progesterone, a sex steroid
hormone, attenuates HT after tPA treatment in an ischemic stroke model
of rats [31,38]. Intraperitoneal administration of progesterone reduces
volumes of cerebral infarction and edema, suppresses HT via inhibition
of VEGF, MMP-9 and MMP-3 activity, and inflammation. Progesterone
may be also effective for suppression of HT. Although a phase IV clinical
trial is currently ongoing in hemorrhagic stroke with progesterone
(ClinicalTrials.gov; NCT04143880), clinical trials for ischemic stroke
are not planned. Pleiotropic growth factors could inhibit HT and effi­
ciently promote functional recovery. (4) Recently, two clinical trials
investigating the direct effects of MT without tPA demonstrated a lower
incidence of HT compared to MT with tPA [39,40]. Thus, tPA (alteplase)
could directly accelerate HT. Modulating these variable pathophysio­
logical mechanisms is a direct and promising therapeutic strategy to
prevent HT. Several mechanisms underlie an ischemic stroke. Therefore,
therapeutic agents with pleiotropic protective mechanisms, which target
the underlying molecular pathways, are ideal to prevent HT.
3.2. Agents with potential benefits in clinical studies of patients treated
with tPA
Studies have investigated the concomitant use of various agents,
which reduce hemorrhagic events and exert neuroprotective effects,
alongside tPA. Because monotherapies might be insufficient to attenuate
HT after tPA treatment and improve the therapeutic outcome of AIS
patients, the efficacy of several—but not single—potential target drugs
have been evaluated (Fig. 3). The drugs that have shown potential
benefits in clinical trials are described below.
Edaravone, a free radical scavenger, exerts a neuroprotective effect
in ischemic brain models by inhibiting vascular endothelial cell injury
and ameliorating neuronal damage [41]. Combined treatment with tPA
and edaravone might suppress MMP-9 expression in and around the
cerebral microvessels and inhibit BBB damage in a rat focal cerebral
ischemic model. Moreover, edaravone might protect cerebral micro­
vascular integrity because it safeguards the basement membrane from
excess free radicals and MMP-9, leading to a subsequent decrease in HT
and functional improvement. Edaravone was approved for use in pa­
tients with AIS in Japan [42] in 2001 and the compound is widely used
in China [43], although combined use of tPA with a free-radical­
–trapping agent—NXY-059—did not show any evidence of efficacy
3
Journal of the Neurological Sciences 419 (2020) 117217
Fig. 3. Drug candidates to prevent hemorrhagic transformation after tPA
treatment and their underlying therapeutic mechanisms. MMP, matrix metal­
loproteinase; tPA, tissue plasminogen activator.
against HT [44]. Another clinical trial reported that the administration
of edaravone before tPA did not affect the rate of early recanalization,
symptomatic HT, or favorable outcomes after tPA treatment [45].
Because edaravone is widely used in Japan, it was difficult to create a
control group (tPA without edaravone treatment). Although edaravone
may be a good candidate for combined therapy with tPA and MT, a
worldwide clinical trial is needed to assess the efficacy of edaravone and
tPA with and without MT.
Fingolimod is a sphingosine analog that binds to the sphingosine-1-
phosphate receptors. It inhibits the release of lymphocytes from lymph
nodes and limits their recirculation [46]. Inflammation caused by brain
ischemia contributes to HT and reperfusion injury and worsens the
clinical outcomes of stroke associated with tPA treatment. In animal
models of thromboembolic MCAO, fingolimod and tPA attenuated the
neurological deficit and reduced infarct volume and HT [47]. In addi­
tion, although a small number of patients received tPA and fingolimod
combined therapy 4.5 to 6 h after ischemic onset, improved functional
outcomes were observed [48]. Fingolimod is approved for use and is
being used as a disease-modifying drug for multiple sclerosis; therefore,
drug repurposing is easy. However, the neutrophil-to-lymphocyte ratio
is associated with a greater risk of HT in tPA-treated patients with AIS
[29,30]. Combinating fingolimod with alteplase bridging with MT in
acute ischemic stroke (FAMTAIS) trial was withdrawn on March 2020
(ClinicalTrials.gov; NCT02956200). Therefore, based on these results,
the effects of combined treatment with tPA and fingolimod remain
unknown.
Minocycline, apart from its antibacterial properties, has neuro­
protective effects in various neurological disease processes [49]. Mino­
cycline decreases plasma MMP-9 levels and reduce infarction and HT
[36]. Two pilot clinical trials have shown that minocycline, in combi­
nation with tPA, is potentially effective to treat AIS [50,51]. In 2018,
two systematic reviews showed that use of minocycline with tPA had
favorable functional outcomes [52,53]. However, phase III large clinical
trials are needed to confirm the potential treatment effect of minocycline
in AIS therapy.
Natalizumab is a monoclonal antibody targeting α4 integrin within
the adhesion molecule very late antigen-4 (VLA-4), leading to reduced
transmigration of leucocytes, excluding neutrophils, across the vascular
endothelium [54]. Natalizumab is approved for the treatment of mul­
tiple sclerosis and Crohn’s disease and is highly effective in reducing
inflammatory lesions [55–57]. Safety and efficacy of intravenous nata­
lizumab in AIS (ACTION and ACTION II) trials were performed
(ClinicalTrials.gov; NCT01955707 [58] and NCT02730455 [59],
respectively). The administration of natalizumab was combined with
tPA and MT. However, natalizumab did not reduce infarct volume
growth from baseline to day 5 compared with that of the placebo.
Y. Otsu et al. Journal of the Neurological Sciences 419 (2020) 117217

Although no difference was noted between the natalizumab and placebo use with tPA to treat AIS. Phase III clinical trials are inevitably needed to
groups in the National Institutes of Health Stroke Scale (NIHSS) and confirm the possibilities of preventing HT and to identify the functional
incidence of HT, more patients in the natalizumab group than in the efficacy of the candidate drugs.
placebo group had modified Rankin scores of 0 or 1 at day 30 (p = 0.024)
and day 90 (p = 0.18) in ACTION trial [58]. However, the effect of two
doses of natalizumab on functional outcomes was not observed in the 3.3. HT in MT
phase IIb ACTION II trial [59]. Therefore, larger trials are currently not
planned. It is important to review the significance of reperfusion injuries
Otaplimastat is a small molecule with a quinazoline-2,4-dione scaf­ presenting after MT. Five trials (MR CLEAN [4], REVASCAT [5],
fold that improves neurological outcomes through anti-excitotoxic ef­ ESCAPE [6], SWIFT PRIME [7], and EXTEND-IA [8]) showed superior
fects via N-methyl-(D)-aspartate (NMDA) receptor-mediated efficacy and safety of MT compared to medical (tPA) treatment in pa­
excitotoxicity [60]. In a phase IIa trial examining the safety and efficacy tients with AIS [77]. Another recent study reported a high reperfusion
of combined otaplimastat and tPA treatment in AIS patients, otaplima­ ratio (80%) after MT [40]. HT after MT occurred at a rate of 11.6% in
stat was found to be safe for use and tended to show better functional another multicenter study [78]. However, there was no increase in
outcomes than the placebo group [61]. Currently, a phase IIb trial is mortality or symptomatic HT between the IV tPA and MT with tPA
ongoing to obtain more evidence of the clinical efficacy of otaplimastat. groups [79]. Although HT is common because of high reperfusion after
However, the functional efficacy of otaplimastat needs to be investi­ MT, symptomatic HT after MT is uncommon.
gated in larger trials. Several risk factors are thought to cause HT after MT (Table 2).
IV glyburide (glibenclamide) is a specific blocker of the adenosine Significant factors that cause HT after MT include current smoking, a
triphosphate (ATP) sensitive potassium (K+) channel (KATP channel) low Alberta Stroke Program Early CT Score (ASPECTS), use of general
and sulfonylurea receptor 1 (SUR1)-transient receptor potential mela­ anesthesia, unfavorable collaterals on angiography, and thromboem­
statin 4 (TRPM4) channel. Results of preclinical studies suggest that bolic migration [78,80]. The TICI-ASPECTS-glucose (TAG) score, which
blockade of the SUR1-TRPM4 channel in neurons, astrocytes, and is the combined score of thrombolysis in cerebral ischemia (TICI), AS­
endothelium substantially reduces cerebral edema in rodent models of PECTS, and glucose level, was associated with symptomatic HT after MT
stroke [62,63]. In addition, it was shown that, in activated brain endo­ [81]. Other factors causing HT include vitamin K antagonists (VKAs) and
thelium, tPA induced phasic secretion of MMP-9 and causes activation of high UA levels [82]. An advanced age and a high NIHSS score are
SUR1-TRPM4 channels [64]. Therefore, glyburide acts as a partial in­ relatively significant causative factors for HT after MT [78]. However,
hibitor of MMP-9, which is expected to be beneficial in the treatment of prior use of direct oral anticoagulation (DOAC) [83] and the presence of
stroke, especially in patients treated with tPA. A phase II trial of a severe leukoaraiosis [84,85] were not significantly associated with
combination of tPA and glyburide was performed. Midline shift, an symptomatic HT after MT.
established marker of brain swelling, was significantly reduced in gly­ Presence of unfavorable collaterals on angiography is one of the
buride group vs. controls at 72–96 h (P = 0.0006) [65]. Thus, the plasma main reasons for HT after MT. Collateral flow by angiogenesis and
levels of MMP-9 in glyburide group was lower than those in controls at arteriogenesis after ischemic stroke is stimulated by various chemokines
24–72 h (P = 0.006) [66]. However, primary outcome, modified Rankin and growth factors and supports ischemic border and functional re­
Scale 0–4, and the frequency of HT was no different between the groups covery after ischemic stroke [32,86]. However, the presence of and
(ClinicalTrials.gov; NCT01794182) [65,66]. Although the results of mechanisms underlying collateral development prior to ischemic stroke
primary efficacy outcome and suppression of HT were disappointing, are still unknown. Angiographic early venous filling is also a predictive
glyburide will serve to guide a phase III trial. factor of HT after MT [87,88]. Severe HT is also more likely to occur
Statins, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) when recanalization has failed [89]. This indicates that acute reperfu­
reductase inhibitors, have multifaceted effects, such as anti- sion stress may demand collateral flow and is more likely to induce HT
inflammatory and antioxidant effects [67]. Statins reduce the risk of under poor collateral flow. A recent study that used direct arterial
intracranial hemorrhage [68]. In addition, there is increasing evidence damage model with rough suture insertion showed that VEGF and MMP-
that early use of simvastatin and atorvastatin in animal models reduces
HT through anti-inflammatory effects, such as suppression of MMP Table 2
expression and protective effects on the BBB [69,70]. Low dose of statins Risk factors of hemorrhagic transformation after mechanical thrombectomy.
(for example, simvastatin; 20 mg, once daily) may be useful in reducing Risk factors References
HT [71]. In contrast, the STARS trial showed no HT suppression or Significant TAG score Montalvo et al. [81]
therapeutic effect of combined simvastatin (40 mg, once daily) and tPA Angiographic early venous Ohta et al. [87], Cartmell et al.
therapy [72]. Therefore, the effect of thrombolytic therapy on HT re­ filling [88]
Smoking Ohta et al. [87], Cartmell et al.
mains clinically unknown. Furthermore, the effects of statins with tPA in
[88]
the acute phase varied depending on each condition. ASPECTS Ohta et al. [87], Cartmell et al.
Uric acid (UA) is an endogenous antioxidant derived from purine [88]
metabolism. Administration of UA in the MCAO mouse model reduced General anesthesia Boisseau et al. [78]
infarction volume and ROS production [73]. UA also prevented the Angiographic unfavorable Boisseau et al. [78] Bang et al.,
collaterals [80]
production of superoxide in the ischemic arterial wall, suggesting that it
Thromboembolic migration Boisseau et al. [78]
might protect the cerebral vasculature after AIS [74]. Chamorro et al. VKAs Meinel et al. [83]
first described that higher endogenous levels of UA in patients with AIS Higher uric acid level Yuan et al. [82]
at clinical onset were associated with better stroke outcomes [75]. A Relatively Failed recanalization Parrilla et al. [89]
significant High age Boisseau et al. [78]
phase IIb/III trial (URICO-ICTUS) that recruited patients with AIS on tPA
Higher NIHSS Boisseau et al. [78]
treatment with UA or placebo did not demonstrate a significant differ­ Not significant DOAC Meinel et al. [83]
ence in clinical outcomes between the two patient cohorts. However, Severe leukoaraiosis Atchaneeyasakul et al. [84],
secondary analysis showed that UA administration decreased the inci­ Boulouis et al. [85]
dence of early neurological worsening [76]. Thus, a larger study is TAG, the combination score of thrombolysis in cerebral ischemia (TICI), AS­
planned to analyze the effects of combined therapy with UA and MT PECTS, and glucose level; ASPECTS, Alberta Stroke Program Early CT Score;
[74]. NIHSS, National Institutes of Health Stroke Scale; VKAs, vitamin K antagonists;
In summary, several candidates have been clinically evaluated for DOAC, direct oral anticoagulants.

4
Y. Otsu et al.
9 upregulation may cause MT-induced BBB disruption in a rat stroke
model [90]. The association of collateral angiogenesis by VEGF and
MMP-9 may be the cause of HT after MT. However, further studies
mimicking human MT models are essential to investigate the precise
mechanisms underlying HT and unfavorable collaterals.
MT alone (without tPA) may counter one of the mechanisms of
reperfusion injury. Results of the SKIP study, a randomized study of MT
with and without IV tPA in patients with AIS, demonstrated that the
favorable outcome rates at 90 days were not significantly different be­
tween the two groups (p = 0.18 for non-inferiority). On the other hand,
HT was significantly lower in the MT group without IV tPA (direct MT)
than in the MT group with IV tPA (33.7% and 50.5%, respectively, p =
0.02) [39]. In addition, another study demonstrated that the functional
outcome at 90 days in the direct MT group within 206 min of stroke
onset was non-inferior to that in the MT group with IV tPA within 215
min of stroke onset [40]. This study demonstrated that asymptomatic
HT in the direct MT (33.3%) and MT with IV tPA (36.2%) groups, and
symptomatic HT in the direct MT (4.3%) and MT with IV tPA (6.1%)
groups, were not statistically different. Although the ratio of asymp­
tomatic HT in both groups was higher than that reported in other studies
in the HERMES reanalysis (4%) [91], tPA had no effect on HT within the
therapeutic time window. These results show that MT with and without
tPA within 4.5 h after symptom onset did not increase the risk of HT. A
time-dependent mechanism might be very important for HT after MT.
4. Discussion
4.1. Next-generation therapeutic strategies
Patients with AIS, which developed 4.5–9 h from stroke onset, or
those with wake-up stroke with salvageable brain tissue identified by
DWI-FLAIR mismatch or perfusion image who were treated with alte­
plase achieved better functional outcomes than those who received
placebo [14,15]. However, the rate of symptomatic HT was high with
alteplase treatment, although this increase did not negate the overall net
benefit of thrombolysis. The guidelines for MT were revised to extend
the therapeutic time window (within 16 or 24 h after the onset of
symptoms) if patients met the DAWN or DEFUSE 3 eligibility criteria
[17,18]. The next-generation therapeutic strategies are as follows: (1)
development of new thrombolytic agents, and (2) combination treat­
ments with protective and thrombolytic agents or with thrombolytic
agents and MT.
4.2. New thrombolytic agents
Some new thrombolytic agents that have superior effects than alte­
plase have been described recently.
Desmoteplase (Desmodus rotundus salivary plasminogen activator)
was thought to be an alternative to alteplase. It has demonstrated
minimal neurotoxicity, fibrin specificity, and a long half-life. However, a
randomized, placebo-controlled, phase III clinical trial (the prematurely
terminated DIAS-4 trial), together with a post hoc pooled analysis of the
concomitant DIAS-3, DIAS-4, and DIAS-J (Japan) trials that enrolled
patients with AIS presenting within 3–9 h after onset, did not show
beneficial outcomes when administered to patients with ischemic stroke
[92].
Stachybotrys microspora triphenyl phenol-7 (SMTP-7) may be a
candidate thrombolytic agent. The small SMTP-7 molecule promotes
thrombolysis and suppresses inflammation after reperfusion by inhib­
iting the effects of proinflammatory cytokines [93,94]. Although SMTP-
7 decreased infarction volume, HT, mortality, and neurological deficits
in rodents and primate focal cerebral ischemia models [95], another
report showed that a small number of embolic strokes in monkeys ten­
ded to cause HT-associated premature death [96]. However, a phase II
clinical trial is currently under investigation (JapicCTI-183,842; Japan
Pharmaceutical Information Center Clinical Trials Information). This
5
Journal of the Neurological Sciences 419 (2020) 117217
study might reveal the therapeutic potential of SMTP.
Tenecteplase is a genetically engineered variant of tPA with a longer
half-life and more fibrin specificity than tPA. These properties confer
tenecteplase with more complete clot lysis abilities and fewer bleeding
complications. Among patients with AIS who received tenecteplase or
alteplase treatment within 4.5 h after the onset of the ischemic stroke
(EXTEND-IA TNK trial, a phase III clinical trial), the tenecteplase group
had greater reperfusion rates and better clinical improvement at 24 h
than the tPA group [97]. The other phase III clinical trial, the Norwegian
Tenecteplase Stroke Trial (NOR-TEST), showed a similar efficacy and
safety of tenecteplase, compared with alteplase, in larger groups of pa­
tients [98]. However, intravenous tenecteplase administration prior to
MT in patients with large vessel occlusion ischemic stroke (The
EXTEND-IA TNK Part 2 Randomized Clinical Trial) showed that a high
dose of tenecteplase did not significantly improve cerebral reperfusion
prior to MT as compared to a low dose of tenecteplase [99]. Neverthe­
less, a low dose of tenecteplase was safe, and high reperfusion was noted
before MT in patients with large vessel occlusion ischemic stroke. Ten­
ecteplase, which has fewer bleeding complications than other agents, is
clinically replacing alteplase, and a next-generation thrombolytic agent
is also being investigated.
4.3. Combination treatments from experimental studies to clinical
applications
To conduct successful clinical trials of protective agents, animal
models and experimental methods that mimic real patient populations
are essential. Although preclinical studies of protective agents in animal
models have shown many positive outcomes, only a few successful drugs
can be assessed clinically [100,101]. One of the reasons for this is the
dissociation between the animal model and the actual patient popula­
tion. Existing models of animal experiments mostly involved young
males with no complications [100]. In many cases, the endpoint of
treatment response was the infarction volume, which is dissociated from
clinical trials that often evaluate treatment efficacy using the modified
Rankin score at 90 days [101]. Models and methods of animal experi­
ments should be considered to find effective therapeutic agents in
clinical practice. Long-term functional assessment is needed in both
male and female animal models with older age and complications. For
clinical application of combination treatments, evaluation by suitable
models should be essential in pre-clinical studies.
Theoretically, the increased risk of HT after MT does not differ
significantly from that after tPA treatment. However, some recent
studies have shown that the risk of HT in MT without tPA may be lower
than that with tPA treatment alone [39,40]. Thus, the neuroprotectant,
nerinetide, which interferes with post-synaptic density protein 95, did
not show efficiency in combination with IV tPA and MT in the primary
endpoint [102]. However, the MT without IV tPA group showed sig­
nificant functional improvement following the administration of ner­
inetide (59.3%) compared to placebo (49.8%). The absence of benefit of
nerinetide in the alteplase group was likely because of enzymatic
cleavage of nerinetide by plasmin, leading to subtherapeutic concen­
trations of nerinetide. From these results, the effects of MT with candi­
date protectants without tPA should be further investigated.
5. Conclusion and future research direction
Clinical trials for MT showed that 71 to 80% of patients presented
recanalization [40,91]. However, only half or fewer patients presented
favorable neurological outcomes. These results suggest that MT provides
insufficient therapeutic efficacy. Patients eligible for tPA should receive
IV tPA even if MT is being considered. To take advantage of the easily
accessible drug tPA and the high recanalization rate of MT, it is
important to reduce bleeding complications after recanalization. In
addition, cell therapies are gaining attention in the treatment of stroke
because they seem to have a protective function [103]. The
Y. Otsu et al.
endovascular suture model in rodents with occlusion of the MCA may
closely mimic the situation of MT [90,100]. Cell therapies involving
protective phenotypes in a pre-clinical suture model facilitate functional
recovery after post-reperfusion treatment [104,105]. It is unknown
whether administrations of both bone marrow-derived progenitor cells
(MASTERS) [106] and stem cells [107] in phase II clinical trials show
functional improvement and suppression of HT. However, a half of the
patients underwent IV tPA and MT in these trials. The safety of the
combination of reperfusion therapies and cell therapies has previously
been shown. Following the results, clinical trials are ongoing to evaluate
the safety and efficacy of administration of bone marrow-derived pro­
genitor cells and umbilical cord mesenchymal stem cells after IV tPA or
MT (ClinicalTrials.gov; NCT02961504 (MASTERS-2) and
NCT04434768, respectively). Future research should facilitate the re­
covery of neurological function by combining reperfusion therapies with
cell therapies and/or investigate pleiotropic protective agents for
treatment of HT.
Declaration of competing interest
TS is an academic adviser for ShimoJani LLC, a biotechnology
company.
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research
(Research Project Number: 18K07493), a grant from SENSHIN Medical
Research Foundation, Takeda Science Foundation and Astellas Foun­
dation for Research on Metabolic Disorders, and Medical Research
Encouragement Prize of the Japan Medical Association and Niigata
Medical Association (Dr. Kanazawa).
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