3. Neuroprotection for Ischaemic Stroke Translation From the Bench to the Bedside

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Neuroprotection for ischaemic stroke: Translation fromthe bench to the bedside

Brad A. Sutherland1†, Jens Minnerup2†, Joyce S. Balami3, Francesco Arba1,Alastair M. Buchan1*‡, and Christoph Kleinschnitz4‡

Neuroprotection seeks to restrict injury to the brain paren-

chyma following an ischaemic insult by preventing salvage-

able neurons from dying. The concept of neuroprotection

has shown promise in experimental studies, but has failed to

translate into clinical success. Many reasons exist for this

including the heterogeneity of human stroke and the lack ofmethodological agreement between preclinical and clinical

studies. Even with the proposed Stroke Therapy Academic

Industry Roundtable criteria for preclinical development of

neuroprotective agents for stroke, we have still seen limited

success in the clinic, an example being NXY-059, which ful-

filled nearly all the Stroke Therapy Academic Industry

Roundtable criteria. There are currently a number of ongoing

trials for neuroprotective strategies including hypothermia

and albumin, but the outcome of these approaches remains

to be seen. Combination therapies with thrombolysis also

need to be fully investigated, as restoration of oxygen and

glucose will always be the best therapy to protect against

cell death from stroke. There are also a number of promising

neuroprotectants in preclinical development including hae-

matopoietic growth factors, and inhibitors of the nicotina-

mide adenine dinucleotide phosphate oxidases, a source of

free radical production which is a key step in the pathophysi-

ology of acute ischaemic stroke. For these neuroprotectants

to succeed, essential quality standards need to be adhered

to; however, these must remain realistic as the evidence that

standardization of procedures improves translational success

remains absent for stroke.

Key words: acute stroke therapy, ischaemic cascade, ischaemicstroke, neuroprotection, STAIR, translation

Introduction to neuroprotection

Currently, the only approved measures for the treatment of

acute ischaemic stroke are thrombolysis and antiplatelet

therapy. However, the concept of neuroprotection has received

significant attention over the past 30 years, with many experi-

mental neuroprotectants being trialled preclinically and clini-

cally. Where thrombolysis aims to break down the occluding

clot to restore blood flow to the ischaemic brain, neuroprotec-

tion seeks to limit ischaemic injury by preventing the salvage-able neurons in the penumbra that surrounds the core from

dying. Rapid restoration of oxygen and glucose by thromboly-

sis will always provide the most effective neuroprotection, but

directly targeting the brain parenchyma to confer neuropro-

tection may be a viable method, particularly in conjunction

with thrombolysis. Many well-defined molecular targets

(Fig. 1) now exist within the ischaemic cascade that can, in

theory, be pharmacologically altered to produce neuroprotec-

tion (1). Neuroprotective agents aim to salvage ischaemic

tissue, limit infarct size, prolong the time window for throm-

bolytic therapy or minimize post-ischaemic reperfusion

injury or inflammation. Over 1000 neuroprotective agents

have been tested in basic stroke studies (2) with many showing

promise. Despite this, neuroprotection in the clinic has failed

to eventuate, disappointing clinicians, researchers, and stroke

patients alike. Nearly 200 neuroprotection clinical trials are

ongoing or have been completed, with none achieving success-

ful translation to clinical practice so far (3).

This review attempts to define neuroprotection and outline

the current status of the neuroprotection field both preclini-

cally and clinically, while identifying the problems associated

with translating neuroprotection from the bench to bedside. It

also describes some promising neuroprotectants, the use of

Correspondence: Alastair M Buchan*, Acute Stroke Programme, Level 7

John Radcliffe Hospital, Oxford Biomedical Research Centre, Nuffield

Department of Clinical Medicine, University of Oxford, Headley Way,

Oxford, OX3 9DU, UK.

E-mail: [email protected] 1Acute Stroke Programme, Nuffield Department of Clinical Medicine,

University of Oxford, Oxford, UK2Department of Neurology, University of Münster, Münster, Germany 3Acute Stroke Programme, Department of Medicine and Clinical

Geratology, Oxford University NHS Trust, Oxford, UK4Department of Neurology, University of Würzburg, Würzburg,

Germany

Conflict of interest: The authors declare no conflict of interest with this

manuscript.

†Equal contribution;

‡ joint senior authors.

DOI: 10.1111/j.1747-4949.2012.00770.x

Review

© 2012 The Authors.

International Journal of Stroke © 2012 World Stroke OrganizationVol 7, July 2012, 407–418 407


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neuroprotection alongside thrombolysis, and finally some

concerns with the current criteria for preclinical neuroprotec-

tion studies.

How to define and measure neuroprotection

As described earlier, neuroprotection is designed to restrict

injury to the brain following an injurious ischaemic insult by

preventing neuronal cell death, especially in the salvageable

penumbral region. This leads to the working definition of

neuroprotection as ‘any strategy, or combination of strate-

gies, that antagonizes, interrupts, or slows the sequence of

injurious biochemical and molecular events that, if left

unchecked, would eventuate in irreversible ischemic injury’

(4). According to this definition, protection from injury

originates at the neuron itself (endogenous or direct neuro-

protection). Consequently, it does not include treatment

approaches that primarily target the cerebral vasculature,

such as thrombolytics, antithrombotics, and antiplatelet

drugs (extrinsic or indirect neuroprotection) (4). Even

though these agents do protect the brain by restoring blood

flow and preventing clot formation, their mechanisms of

action are vascular-based and do not target the brain paren-

chyma itself. Nevertheless, given the wide array of biochemi-

Fig. 1 The cascade of biochemical events leading to apoptosis or necrosis following cerebral ischaemia. Vascular occlusion in a blood vessel initiates a

complex signalling cascade that leads to neuronal cell death. The reduction in blood flow produces ionic pump failure and anoxic depolarization leading

to enhanced glutamate release and a sudden increase in intracellular calcium. This rise in calcium triggers mitochondrial collapse, free radical production,

cytotoxic oedema, and increased NO generation. Reperfusion also produces injury by augmenting BBB breakdown, inflammation, and free radical

production leading to apoptosis. Red borders signify important events in the cascade. The blue border indicates reperfusion. This figure has been adapted

from Durukan & Tatlisumak (1). AA, arachidonic acid; AMPA, a -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; BBB, blood-brain barrier; iNOS,

inducible nitric oxide synthase; NMDA, N-methyl-D-aspartate; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; PLA 2, phospholipase A2.

Review B. A. Sutherland et al .


International Journal of Stroke © 2012 World Stroke Organization408 Vol 7, July 2012, 407–418


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cal pathways that have been elucidated to play a role inischaemic cell death (Fig. 1), this working definition still

covers an extensive variety of potential neuroprotective

agents.

In the scope of published neuroprotective preclinical

studies, there is significant variability in the quantity of neu-

roprotection achieved. Some agents produced substantial pro-

tection of the brain following ischaemia (e.g. NXY-059), while

others showed minimal neuroprotection (e.g. edaravone) (2).

Also, based on the Stroke Therapy Academic Industry Round-

table (STAIR) criteria (5,6), many neuroprotective studies

exhibit low methodological quality (2) with a wide heteroge-

neity in the methodology used. This has led to increasingly variable results meaning many neuroprotective agents pro-

ceeded to clinical investigation with only weak preclinical evi-

dence and so were doomed to fail. There are many other

reasons why clinical neuroprotection has not eventuated given

the preclinical success, which are described in Table 1 and in

many other reports (7–9).

It is hard to gauge from the experimental evidence and the

low methodological quality whether there are really any

drugs that can induce ‘true’ neuroprotection. It is possible

that many of the neuroprotective effects observed could be

due to a manifestation of physiological/pathophysiological

changes following ischaemia. These changes could include

modulating temperature (hypothermia), cerebral blood flow

(CBF; hyperperfusion), inflammation (anti-inflammatory

effects), and blood-brain barrier (BBB) damage (reducing

BBB disruption and vascular permeability). One example is

the N-methyl-D-aspartate (NMDA) receptor antagonist

MK-801, which induced hypothermia to produce neuropro-

tection instead of directly targeting the neuron (10). Inter-

estingly, the same compound also raised CBF in the

ischaemic region which may have contributed to its neuro-

protective effects (11). The concept of neuroprotection pro-

duced by enhancing CBF rather than inhibiting the

ischaemic cascade has recently been discussed (12). Sophis-ticated imaging tools might help to delineate genuine neu-

roprotection from within the neuron from secondary or

non-specific effects in the future. This was demonstrated in a

mouse model of multiple sclerosis where a new form of early

but reversible axonal damage caused by oxidative stress and

successive mitochondrial dysfunction was visualized in vivo

by serial two-photon microscopy (13).

Another problem in animal studies is how neuroprotection

is assessed. Infarct volume is most commonly used as the

primary end-point and is quantified by using a histological

stain. The majority of experiments claim that neuroprotection

has been achieved when there is a reduction in infarct volume.However, this is difficult to translate to human studies where

neuroprotection would be reached if stroke patients received

sustainable functional benefit. So what does neuroprotection

really mean if infarct volume is reduced but functional

improvement is lacking? Clearly, functional assessment

including mortality rates in preclinical studies should be a

mandatory outcome parameter for the investigation of any

neuroprotective drug, as outlined by STAIR (5,6). Unfortu-

nately, meaningful functional testing in small laboratory

animals, especially mice, is frequently hampered by stroke

severity and the limited correlation with higher brain func-

tioning in humans, and so representative tests must be care-

fully selected.

The current status of neuroprotection

Even after 30 years of neuroprotection research, no neuro-

protective therapy has been brought into clinical practice.

However, there are some exciting new developments in the

neuroprotection field, and this section will describe the

current status of neuroprotection in both preclinical animal

research and clinical human research.

Table 1 Reasons for translational failure of neuroprotective agents from pre-clinical to clinical studies

Animal models Human studies

Highly control led, homogeneous population Variable, heterogeneous population

Younger animals Older patients

Limited comorbidities Numerous comorbidities

Induced onset of stroke Spontaneous onset of strokeUniform aetiology Variable aetiologies

Ischaemic territory usually from middle cerebral artery Ischaemic territory not restricted to middle cerebral artery

Control over therapeutic time window (usually early treatment) Less control over therapeutic time window (usually delayed treatment)

Controlled occlusion duration Variable occlusion duration

Adequate sample size Inadequate sample size

Wide scope for dose optimization Reduced scope for dose optimization

Multiple routes of administration Limited routes of administration

Rapid availability of the drugs to the target area Slow availability of the drugs to the target area

Infarct volume as outcome Function as outcome

ReviewB. A. Sutherland et al .




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Preclinical animal research

The development of new neuroprotective therapies for stroke

involves the evaluation of candidate drugs from in vitro

models to animal experiments and, finally, testing in clinical

trials. Animal studies not only allow the determination of a

drug’s efficacy but also can elucidate its underlying mecha-

nisms in stroke pathophysiology. Up to now, numerous drugstargeting different aspects of the ischaemic cascade (Fig. 1)

were tested in animal models of focal cerebral ischaemia. Drug

mechanisms that were found to be successful in experimental

studies regarding both infarct size reduction and improved

functional outcome include impeding excitotoxicity, local

inflammation, neuronal apoptosis, free radical damage, and

calcium influx into cells. As reviewed by O’Collins et al . (2),

some of these mechanisms of brain injury were targeted by

more than 10 different agents in hundreds of experiments. For

example, the authors identified 277 studies of 21 drugs aiming

to attenuate excitotoxicity in experimental stroke and 114

studies relating to nine drugs with anti-inflammatory activi-ties (2). Overall, 1026 candidate stroke drugs have been iden-

tified in the period covering 1957 to 2003 (2). A more recent

survey of Pubmed-referenced publications showed that the

number of experimental studies of candidate neuroprotective

drugs for stroke therapy has particularly increased over the last

15 years (4). Nearly two-thirds of the published studies report

an improved outcome with a neuroprotective compound

compared to control treatment in animal models of focal cer-

ebral ischaemia (2). However, there appears to be significant

publication bias in preclinical stroke studies which may

account for approximately one-third of the efficacy reported

in meta-analyses, leading to an overstatement of efficacy (14).

In contrast, in clinical trials, drugs targeting only one key

mechanism of cerebral ischaemia have failed to improve

outcome as discussed later in this review. One plausible reason

for this failure might be the multiplicity of mechanisms

involved in causing neuronal damage following stroke (Fig. 1).

Therefore, a novel approach for the development of neuropro-

tective drugs includes the evaluation of compounds with a

multimodal mode of action. This concept also considers the

use of agents with recovery-enhancing properties in addition

to neuroprotective actions (15,16). A recent trend for judging

the potency of neuroprotectants is to pool results from differ-

ent animal studies for meta-analysis. This method, originally

applied for clinical trials in humans, was recently used toobtain further information on the efficacy, the dose–response

relationship, and the therapeutic time window of promising

stroke drugs for potential guidance of clinical trials (17–19).

Clinical human research

A considerable number of neuroprotection clinical trials for

ischaemic stroke are ongoing or have been completed. Viewing

the Internet Stroke Centre Stroke Trials Registry (3), a number

of agents possessing a wide variety of mechanisms of action

have been tried. Unfortunately, none of these agents have

achieved clinical success for neuroprotection. However, there

are some promising ongoing studies that, among others,

include the use of hypothermia, albumin, magnesium, mino-

cycline, and statins as potential approaches to neuroprotection

in the clinical setting. The ongoing clinical trials investigating

neuroprotection for acute ischaemic stroke have been outlinedin Table 2. A list of neuroprotective strategies that have under-

gone completed studies for acute ischaemic stroke and that

have all failed to show neuroprotection in the clinic is shown

in Supporting Information Table S1.

Hypothermia

Hypothermia is one of the most promising neuroprotective

approaches, which has consistently shown benefit in animal

models of cerebral ischaemia, reducing infarct volume by

more than 40% (21). Hypothermia is thought to be neuropro-

tective through several mechanisms including decreasing exci-

tatory amino acid release, reducing free radical formation,enhancing small ubiquitin-related modifier (SUMO)-related

pathways, attenuating protein kinase C activity, and slowing

cellular metabolism (22–24).

The Cooling for Acute Ischaemic Brain Damage (COOL-

AID) studies, COOL-AID I (using surface cooling) (25), and

COOL-AID II (using endovascular cooling) (26) showed that

mild therapeutic hypothermia for acute ischaemic stroke was

feasible, but with no change in clinical outcome. The recent

Intravascular Cooling in the Treatment of Stroke – Longer

recombinant tissue plasminogen activator (rtPA) window

study showed that catheter-based cooling within 6 h of

symptom onset of acute stroke was well tolerated in patients

given rtPA, but there were no differences in 90-day outcomes

(27). Other safety and efficacy clinical studies such as Control-

led Hypothermia in Large Infarction and Cooling in Acute

Stroke are ongoing (3).

Despite the encouraging results from hypothermia studies

in humans, there are a number of limitations in applying

hypothermia to stroke patients. Stroke patients are generally

awake and do not tolerate cooling in contrast to cardiac arrest

and brain injury patients. Attaining target temperature and

prolonging or maintaining that temperature stroke patients

while awake is challenging. There are frequent complications

such as pneumonia, hypotension, cardiac arrhythmias, elec-

trolyte derangements, and infections (27–29). A number of patients also experience shivering during cooling which can

be controlled with anti-shivering agents such as buspirone

and meperidine (28). Another problem is the rebound

increase in intracranial pressure experienced during re-

warming; a phenomenon that is not well studied in laboratory

models (30).

Albumin

Albumin, a protein involved in the transport of small mol-

ecules in the blood, plays a key role in restricting fluid leaking





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from the vasculature into the tissue (31). In animal studies,

albumin was shown to diminish infarct volume significantly

with a therapeutic time window of four-hours poststroke (32).

Albumin produces its neuroprotective effect through several

mechanisms including ameliorating brain swelling, enhancing

blood flow to sub-occlusive microvascular lesions, maintain-

ing vascular patency, and preventing re-occlusion after suc-

cessful thrombolysis (4).

The pilot study Albumin in Acute Stroke (ALIAS) demon-

strated that high-dose human albumin therapy is safe and may

confer a neuroprotective effect within five-hours after acute

ischaemic stroke (33,34). These encouraging results have led

to a large placebo-controlled randomized multicentre phase lll

trial of albumin therapy in acute ischaemic stroke – ALIAS-

Part 2 – which is ongoing (35).

The preclinical evidence for the validity of albumin as a

neuroprotective agent is limited in that albumin efficacy in

focal cerebral ischaemia was mainly described by only one

group and independent confirmation by others is pending.

Magnesium

Magnesium may have significant neuroprotective properties

in stroke, with preclinical evidence revealing a 25% level of

protection (36). Magnesium produces this protection through

a number of mechanisms including antagonism of calcium

channels, noncompetitive antagonism of NMDA receptors,

inhibition of excitatory neurotransmitter release, and vascular

smooth muscle relaxation (37). However, magnesium can

also produce post-ischaemic hypothermia which could con-

tribute to its neuroprotective effects in studies that were not

temperature-controlled (38). Looking at studies that were

temperature-controlled, magnesium was largely ineffective

(38) suggesting that magnesium may only produce neuropro-

tection in concert with hypothermia.

Much of the failure of previous neuroprotective trials may

be due to the delayed delivery of agents to stroke patients. The

Field Administration of Stroke Therapy–Magnesium (FAST-

MAG) Pilot Trial attempted to overcome this by having para-

medics initiate magnesium sulphate therapy in acute stroke

patients in the field before arrival to the hospital (39). The

field-based magnesium intervention was feasible and safe with

no serious adverse effects, and was associated with a beneficial

functional outcome at three-months. Based on these positive

results, a large phase lll clinical trial is already in progress

(FAST-MAG) (3).

Although magnesium might act pleiotropically on ischae-

mic neurons, powerful effects of this naturally occurring elec-

trolyte on stroke outcome in clinical practice may be

surprising, especially when given as a monotherapy. A combi-

natory approach with other neuroprotective or clot-breakingagents may be more promising.

Minocycline

Minocycline is a tetracycline antibiotic, which has been shown

to produce a 30% reduction in infarct size in models of cer-

ebral ischaemia (36). The proposed mechanisms of action of

minocycline include anti-inflammatory effects, reduction of

microglial activation, matrix metalloproteinase activity, and

nitric oxide (NO) production, and inhibition of apoptosis

(40). Moreover, via its antibacterial properties, minocycline

Table 2 Neuroprotective compounds currently undergoing clinical trials*

Category Name(s) Mechanism

Clinical

phase Manufacturer

Antioxidant Ebselen Free radical scavenger III Daiichi Pharmaceutical Co., LTD

Edaravone (MCI-186) Free radical scavenger III Mitsubishi Pharma Corporation

Anti-apoptotic/ regeneration AX200 (filgrastim, G-CSF analogue) Growth factor II Sygnis Bioscience GmbH & Co KGHuman Chorionic Gonadotropin

(hCG)/Erythropoietin (NTx-265)

Growth factors, oxygen delivery I I Stem Cel l Therapeutics Corp.

Excitotoxicity Magnesium sulphate NMDA ion channel blocker III Many manufacturers (Abbott

Laboratories for FAST-MAG)

Fluid regulators Albumin Haemodiluting agent III Baxter Bioscience

Others Citicoline (CDP choline) Membrane stabilizer III Ferrer Grupo

Deferoxamine mesylate Iron chelator II Novartis Pharma

DP-b99 Metal ion chelator III D-Pharm Ltd

Hemicraniectomy Reduce cerebral oedema and intracranial

pressure

III None

Hypothermia Reduce cerebral oxygen metabolism,

synaptic inhibitor

III None

Insulin Reduce glucose and brain damage III Eli Lilly

Lovastatin HMG CoA reductase inhibitor, antioxidant II Many manufacturers

Minocycline Antibiotic, pleiotropic protective effects III WyethSimvastatin HMG CoA reductase inhibitor, antioxidant III Many manufacturers

*Information gathered from Stroke Trials Registry (3), O’Collins et al . (2), and Cochrane Clinical Trials Database (20).





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could reduce infections such as pneumonia or urinary tract

infections resulting from stroke-induced immunosuppression

(41).

In an open-label evaluator study, minocycline administra-

tion led to a significantly better outcome in acute stroke

patients compared to placebo (40). Similarly, in Minocycline

to Improve Neurological Outcome in Stroke, minocycline wassafe and well tolerated alone and in combination with rtPA

(42). Encouraging results from these trials have led to the

ongoing Phase III Neuroprotection with Minocycline Therapy

for Acute Stroke Recovery Trial (43).

While the efficacy and neuroprotective potential of mino-

cycline in acute ischaemic stroke still need to be established,

this antibiotic has been used in clinical practice for many

years without serious safety concerns. Nevertheless, wide-

spread and uncritical application of anti-infective agents

could promote the occurrence of multiresistant and invasive

pathogens especially in the setting of intensive care units or

stroke wards.

Statins

Hydroxymethylglutaryl–coenzyme A (HMG-CoA) reductase

inhibitors (statins) are the most widely used cholesterol-

lowering drugs. In addition to their well-established role for

stroke prevention, statins may also be protective in acute

ischaemic stroke (2,44). The main proposed mechanism of

action is due to an increase in NO bioavailability that regulates

cerebral perfusion and improves endothelial function (45).

Other possible mechanisms include antioxidant properties,

atherosclerotic plaque stabilization, and anti-inflammatory

effects (45).

Neuroprotection with Statin Therapy for Acute Recovery

Trial (NeuSTART) was a phase 1B dose-escalation study that

showed that lovastatin administration was safe and feasible up

to three-days after an acute ischaemic stroke (46). Now, a

phase ll trial (NeuSTART II) is in progress to confirm lovas-

tatin safety and efficacy in improving functional outcome after

stroke (3).

Although HMG-CoA reductase inhibitors clearly act

beyond their sole lipid-lowering properties, the concept of

statins as powerful neuroprotectants or anti-inflammatory

drugs was recently called into question in another frequent

neurological disease. Surprisingly, patients suffering from

multiple sclerosis and treated with statins in combination withinterferon-b showed a trend towards increased disease activity

and lesion size compared with patients receiving placebo plus

interferons (47).

DP-b99

DP-b99 is a novel therapeutic that chelates membrane-

activated divalent metal ions such as calcium and zinc (48). As

cell death following cerebral ischaemia is in part mediated by

these toxic metals, DP-b99 administration was shown to

provide significant neuroprotection in animal models of

stroke (48). This promising compound underwent a phase II

trial showing that patients receiving DP-b99 had improve-

ments in a number of secondary end-points following acute

ischaemic stroke (49). Now, the phase III Membrane Activator

Chelator Stroke Intervention trial is underway investigating

the capacity of DP-b99 to improve functional outcome follow-

ing acute ischaemic stroke (3).

Difficulties in translation intoclinical practice

Examples of neuroprotective therapies frompreclinical to clinical

As outlined earlier, a wide variety of neuroprotective drugs

have been tested in preclinical animal studies with about 100

of these being trialled in human studies (2). Even in cases

where the drug showed neuroprotection in animal experi-

ments, all have failed to achieve the primary end-point of neuroprotection in humans. Described later are two case

examples, tirilazad and NXY-059, which are drugs that have

shown a good level of protection preclinically, that have pro-

ceeded into clinical trials with limited success.

Tirilazad

Tirilazad (U74006F) is a 21-aminosteroid that can inhibit

lipid peroxidation by acting as a free radical scavenger (50). In

transient focal ischaemia, Xue et al . (51) showed that tirilazad

reduced cortical infarct size in rats, but this was not observed

in permanent ischaemia. An overall analysis of all tirilazad

preclinical studies showed that tirilazad reduced injury by 29% and had more significant effects following transient

occlusion compared to permanent ischaemia (52). This sug-

gests that tirilazad required revascularization so that it could

reach the ischaemic penumbra to achieve neuroprotection.

The efficacy of tirilazad was greater when given pre-ischaemia,

but some efficacy was observed in delayed treatment out to

six-hours post-ischaemia (52).

The preclinical evidence earlier convincingly showed pro-

tection, while the clinical evidence failed to reproduce these

results. The Randomized Trial of Tirilazad mesylate in patients

with Acute Stroke (RANTTAS) trial (53) was a multicentre,

randomized, double-blinded, vehicle-controlled trial investi-

gating tirilazad in acute stroke patients. Patients were not

thrombolysed, and so were not stratified between transient

and permanent ischaemia, although preclinical evidence sug-

gested that this was important. Tirilazad was administered

within-six hours (median time of 4·3 h) with subsequent

administrations every six-hours for 11 additional doses. The

study was prematurely terminated after inclusion of 556

patients due to lack of any functional benefit at three-months.

Further systematic analysis of all clinical trials investigating

tirilazad showed that tirilazad actually increased disability and

death in acute stroke patients (54). This is at odds with the





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preclinical data and may be due to not using patients that had

recanalization, and administering the treatments much later

compared to the preclinical setting (median over all studies:

five-hours clinical vs. 10 mins preclinical) (52). In order for

neuroprotection to translate, methodologies between animal

and human studies need to be more consistent and tightly

controlled.

NXY-059

NXY-059 is a nitrone that exhibits free radical scavenging

properties and inhibits many stages of the ischaemic cascade

(55). NXY-059 has shown significant neuroprotective effects

in animal models of both transient and permanent occlusion

of the middle cerebral artery (MCA) (56), with an overall

reduction in infarct volume of 43% (18). The protective effects

of NXY-059 in rodents were confirmed in nonhuman pri-

mates (marmosets), which follow the STAIR criteria (57). The

time course of effects of NXY-059 is similar to rtPA with

efficacy within four-hours of occlusion (58). Therefore, due to

their distinct mechanisms, NXY-059 could potentially be usedin concert with thrombolytic treatment for acute ischaemic

stroke in humans.

Two trials were performed to assess the neuroprotective

activity of NXY-059 on human stroke: SAINT I and SAINT II.

The SAINT I trial (59) was a phase III double-blinded, rand-

omized, placebo-controlled trial that revealed a small but sig-

nificant improvement in disability (modified Rankin scale)

with NXY-059 three-months following stroke, but it did not

improve neurological outcome (National Institute of Health

Stroke Scale). Post hoc analysis showed that patients who

underwent thrombolysis and treatment with NXY-059 had

reduced incidence of haemorrhagic transformation. SAINT II(60) was a statistically more powerful study but disappoint-

ingly failed to confirm the data reported in SAINT I. In SAINT

II, there was no difference in disability at three-months, and

the reduction in haemorrhagic transformation with rtPA by

NXY-059 could not be reproduced. There was a higher fre-

quency of rtPA use in SAINT II (44% vs. 28%) (61) which may

have contributed to the difference in results. Only a small

proportion of NXY-059 may cross the BBB, and the neuropro-

tective action of NXY-059 may be mediated in the endothe-

lium and neurovascular unit (58,62). This would also explain

the reduced risk of haemorrhage in thrombolysed patients

(62). However, there was no preclinical evidence that NXY-059

exerted its effects by altering CBF in experimental models

(63).

There were a number of methodological weaknesses in the

preclinical NXY-059 studies that potentially affected the

human trials. Methodological quality was low (18) and only

9% of studies with NXY-059 measured CBF (64). The lack of

infarction observed in many NXY-059 studies may be due to

not confirming MCA occlusion with CBF measurements,

rather than a neuroprotective effect of NXY-059 (65). Many

studies were not blinded or randomized (18), and further

analysis suggests that there may have been significant publica-

tion bias (56). Interestingly, out of all the current drugs in

phase II/III trials,only NXY-059 fulfilled the STAIR criteria for

adequate translation into clinical trials (64), even with these

methodological weaknesses.

A potential reason for clinical failure of the SAINT trials is

the difference in methodology compared to animal studies.

The SAINT trials enrolled patients up to six-hours post-ischaemia onset, while a maximum of four-hours time

window was chosen in animal studies (66). In animal studies,

only occlusion of the MCA was performed, while the SAINT

trials enrolled patients with different types of stroke, such as

posterior or lacunar strokes. The SAINT trials perhaps should

have selected stroke patients that more closely resembled what

had previously worked in animal experiments, e.g. patients

with MCA occlusion (66).

Both tirilazad and NXY-059 are antioxidants that were sup-

posedly neuroprotective by scavenging free radicals and pre-

venting oxidative stress. Although oxidative stress has been

suggested for many years to cause tissue damage and neuronal

death, there is still no successful therapeutic application. Todate, all clinical attempts to scavenge reactive oxygen or nitro-

gen species (ROS/RNS) by applying antioxidants have not

resulted in clinical benefit and have even caused harm. Given

that ROS/RNS are extremely short-lived molecules that form

at multiple sites of the brain upon ischaemia, this is in fact not

surprising. However, the characterization of the relevant enzy-

matic sources of oxidative stress such as nicotinamide adenine

dinucleotide phosphate (NADPH) oxidases (see later) may

allow therapeutic targeting of oxidative stress by preventing

the formation of ROS initially instead of scavenging ROS after

they have been formed (67).

Clinical neuroprotection – why it has failed

The two case examples of preclinical to clinical neuroprotec-

tion assessment outlined the problems associated with trans-

lation. The reasons for translational failure are numerous

(Table 1), ranging from flaws in clinical trial design, delayed

treatment time window, small sample sizes, different

outcome measures, insufficient dosing, and failure to achieve

adequate plasma levels of study medications (7,68,69). Also,

the heterogeneous nature of human stroke is at odds with

the homogeneous animal models currently used (70).

Animal models of stroke mimic at best less than 25% of all

strokes, with the rat model of MCA occlusion probably

reflecting the Total Anterior Circulation Stroke Syndrome in

humans (71). Therefore, there is a need to create new animal

models that better reflect the heterogeneity of ischaemic

stroke in humans. In addition, preclinical studies are usually

performed on young healthy animals, whereas patients are

mostly elderly with possible comorbidities. Ageing is associ-

ated with significant structural and functional changes in the

brain, which affects outcome and the ability to recover after

an ischaemic event (72).





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Experimental studies require a more rigorous design with

higher quality standard levels to avoid bias, and a careful

control of physiological variables to distinguish genuine drug

mode of action from other non-specific effects (4,56). In clini-

cal studies, the treatment time window should be restricted to

a period similar to that shown to be effective experimentally.

Unfortunately, most neuroprotective agents target early eventsin the ischaemic cascade, which require rapid administration

following stroke onset, which is challenging in acute ischaemic

stroke patients. Therefore, agents that have long therapeutic

time windows are optimal.

Combining neuroprotection with thrombolysis

A crucial protective strategy following stroke is the early reca-

nalization of the blood vessel to restore flow back into the

ischaemic region of the brain. This is usually achieved by

thrombolysis, and rtPA is currently the only United States

Food and Drug Administration-approved thrombolytic

therapy. Unfortunately, less than 15% of patients actually receive this therapy (73) because of the short time window of

treatment (4·5 h) and the risk of haemorrhagic transforma-

tion. Therefore, treatment strategies that can improve post-

ischaemic CBF, reduce cerebral injury, restrict adverse effects,

and extend the therapeutic time window may prove useful to

improve rtPA therapy. Thrombolysis may improve delivery of

the neuroprotectant to the penumbral region increasing the

chances of a beneficial effect. Many neuroprotective agents

exhibit synergistic effects with rtPA preclinically including

matrix metalloprotease inhibitors (74), free radical scaven-

gers (75,76), NMDA receptor antagonists (77), a -amino-3-

hydroxy-5-methyl-4-isoxazolepropionic acid receptor antago-nists (78), antioxidant agents (79), anti-inflammatory agents

(80), and antiplatelet agents (80–83).

The concept of thrombolysis combined with neuroprotec-

tive therapy has not been extensively explored in human

clinical trials. As outlined earlier, SAINT I showed that NXY-

059 administration in stroke patients who had received rtPA

reduced haemorrhagic transformation compared to NXY-059

alone (59). However, there was no additive benefit observed

with lubeluzole (sodium channel blocker) and rtPA, even

though combination therapy did not increase adverse effects

(84). Other combination studies with rtPA include clom-

ethiazole (g -aminobutyric acid agonist that showed no

benefit) (85) and UK-279276 (neutrophil inhibitory factor

that showed no benefit) (86). Much like neuroprotection

itself, the promise of combination therapy of thrombolysis

and neuroprotection is tempting, but this needs further

investigation in clinical trials before the viability of this strat-

egy can be confirmed.

One major drawback of testing combination therapies is

that any observed effect or side effect often cannot be

unequivocally assigned to one or the other partner. Moreover,

it is difficult to foresee whether the two (or more) compounds

will act synergistically (multiplicative effect), independently

from each other (additive effect) or even in an antagonistic

manner (neutralizing effect). Even though rtPA is a thrombo-

lytic, it has many effects independent of thrombolysis, which

may be due to the L-arginine present in the rtPA formulation

(87). Every stroke patient receiving rtPA also receives

L-arginine, which is a substrate for NO synthesis, and may

affect outcome following ischaemia (87). Therefore, it will bedifficult to elicit individual effects of compounds, even if rtPA

is part of the combination therapy.

Promising neuroprotectants

There are still many potential neuroprotective compounds

that are currently being investigated for the treatment of acute

ischaemic stroke. Discussed later are two groups of neuropro-

tective agents that have shown promising effects.

Haematopoietic growth factors

Haematopoietic growth factors are a group of regulatory molecules that are responsible for the mobilization, prolif-

eration, maturation, and survival of bone marrow-derived

cells (88). Receptors of several haematopoietic growth factors

are expressed on neurons. Moreover, functions of growth

factors paralleling those in the haematopoietic system were

identified in the brain (89,90). Among the haematopoietic

growth factors, the granulocyte-colony-stimulating factor

(G-CSF) and erythropoietin (EPO) were particularly well

investigated for their effects in cerebral ischaemia. Both

G-CSF and EPO reduced glutamate-induced neuronal cell

death in vitro and prevented apoptosis of neurons in vivo by

activating several anti-apoptotic pathways (89,91). G-CSFand EPO also demonstrated anti-inflammatory actions after

ischaemia (92,93). Besides having neuroprotective properties,

G-CSF and EPO facilitate functional recovery poststroke by

enhancing neurogenesis and angiogenesis (89,94,95). Meta-

analyses of EPO and G-CSF in animal experimental stroke

showed that both factors reduced infarcts and improved

functional outcomes (19,96–98). However, when the impact

of common sources of bias, e.g. unblinded outcome assess-

ment, was considered for analysis, the efficacy of EPO was

lowered, suggesting that the benefit of EPO might be over-

estimated (97).

A small clinical trial showed that EPO is safe and might be

beneficial in acute ischaemic stroke (99). However, these

promising results were not confirmed by a larger phase II/III

German multicentre stroke trial which investigated stroke

patients that had received either EPO or placebo within six-

hours of symptom onset (100). The primary end-point,

change in Barthel Index on day 90, and all secondary out-

comes failed to show any benefit of EPO. Moreover, an

increased mortality rate was observed after EPO treatment.

Potential reasons for the differences between preclinical

studies and the clinical trial could be due to an overestimated

efficacy of EPO in animal studies through neglected quality





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characteristics and unexpected side effects of EPO in patients,

i.e. an intrinsic stroke-inducing capacity of EPO (101). A

recent phase IIa clinical trial demonstrated that G-CSF is well

tolerated even at high doses in stroke patients (102). Explora-

tory analysis revealed a dose-dependent beneficial effect of

G-CSF in patients with large infarcts (102). A phase II trial on

AX200 (filgrastim, a G-CSF analogue) for ischaemic strokepatients (AXIS-2) is currently ongoing, and results are

expected soon (103).

NADPH oxidase inhibitors

The concept that free radicals including ROS are involved in

the pathophysiology of acute ischaemic stroke and account for

secondary infarct growth is over 30 years old (104) but still

valid and based on solid experimental data (105,106). If ROS

are the trigger that causes neurotoxicity in the ischaemic brain,

the question regarding the main sources of ROS arises. Some

of the most attractive candidates are NADPH oxidases, the

only known enzyme family solely dedicated to ROS produc-

tion. Many other enzymes are able to form ROS, e.g. xanthineoxidase, uncoupled NO synthase, and cytochrome P450

enzymes. However, in all these cases, ROS formation requires

an initial oxidation step to occur and none of them forms ROS

natively (107). The structure and function of the NADPH

oxidases were initially characterized in neutrophils, where the

enzyme plays a pivotal role in immunological host defence.

Recently, it has been discovered that the catalytic sub-unit of

the phagocytic NADPH-oxidase is only one member of a

family of four homologous proteins known as NOX1-4 (for

NADPH-oxidase).

By strictly adhering to current quality standards in experi-

mental stroke research (5), we could demonstrate thatNOX4-derived oxidative stress is a crucial player in the

pathophysiology of cerebral ischaemia (67,108). NOX4 was

massively induced in neurons and brain vessels in human

stroke patients and mice subjected to transient MCAO. Mice

deficient in NOX4, but not those deficient for NOX1 or

NOX2, were largely protected from oxidative stress and neu-

ronal apoptosis, after both transient and permanent cerebral

ischaemia. This was independent of gender and age as elderly

mice were equally protected. Interestingly from a transla-

tional perspective, application of the only validated pharma-

cological NADPH oxidase inhibitor, VAS2870, several hours

after ischaemia had the same beneficial effect as deleting

NOX4 (67). The extent of neuroprotection was exceptional

(~70% reduction of stroke volumes), resulting in signifi-

cantly improved long-term neurological function and

reduced mortality.

Targeting the right enzymatic source of ROS rather than

applying non-specific antioxidants after radicals have already

been generated may represent an attractive treatment option

in acute ischaemic brain damage and other disease states

related to oxidative stress (109). Novel and sub-type-specific

NADPH oxidase inhibitors on a small molecule base with

improved pharmacological properties are currently under

development and bear a realistic chance to enter clinical trials

within the next few years.

Conclusions – the future of neuroprotectionin ischaemic stroke

The translational disappointments have created a great deal of

pessimism regarding the future of neuroprotection trials in

humans and have cast doubt on the neuroprotection hypoth-

esis. Some have even suggested that the initially favourable

results of several of the trials, e.g. SAINT I, were likely chance

findings and that the idea of neuroprotection as a form of

treatment for acute stroke should be abandoned. However,

looking into the past, despite the initial scepticism about

stroke care, the landmark National Institute of Neurological

Disorders and Stroke alteplase trial (110) not only revolution-

ized stroke treatment but also reinvigorated enthusiasm in

stroke care and research. While recent progress in stroke trials

has not directly yielded new clinical drugs, they have providedimportant mechanistic insights into the complex pathophysi-

ology of ischaemic stroke which will pave the way for upcom-

ing studies. Future neuroprotection experiments must be

methodologically sound and learn from previous failed

attempts, while clinical trials must take into account the

success achieved in preclinical studies.

With this respect, the implementation of essential quality

standards in experimental stroke research is without doubt a

meaningful measure. However, one has to keep the balance

between high quality on the one hand, and practicability in an

academic laboratory environment on the other hand. The

postulation to validate rodent findings in primates, forexample, is easily spoken but nearly impossible to realize given

that only a few primate facilities are available across Europe

(111). Ethical aspects have to be carefully considered as well

when using higher animal species in injuring and disabling

disease models. In order to provide proof-of-principle evi-

dence, it is in most cases neither necessary nor feasible to plan

and perform basic stroke studies like large controlled rand-

omized clinical trials. This is also frequently prevented by the

increasing numbers of budget cuts the scientific community

currently has to face, limited animal housing and breeding

space, lack of qualified staff, rough scientific competition, and

the strict domestic and international regulations for animal

care and use. Finally, more than 10 years after the first STAIR

recommendations were published, the ultimate proof that

plain standardization of procedures in fact increases the rate

of successful translation from bench to bedside in stroke

research is still missing. Some critics even raise the provocative

question of whether excessive methodological uniformity

counteracts innovation and only prevents promising drug

candidates from entering into clinical trials and renowned

scientific journals. Positive experiences from other neurologi-

cal diseases like multiple sclerosis should teach the stroke com-

munity that even imperfect animal models can serve as a basis





10/12

for hypothesis-driven research that ultimately facilitates the

development of new drugs.

Acknowledgements

The authors were supported by Fondation Leducq (BAS and

AMB), Medical Research Council UK (AMB), the National

Institute for Health Research Biomedical Research Centre

(JSB, FA and AMB), the Deutsche Forschungsgemeinschaft

(SFB 688, TPA13 and KL 2323/4-1, CK), the Wilhelm-Sander

Stiftung (2009·017·1, CK), the European Union (Seventh

Framework Programme FP7, HEALTH-F2-2009-241778,

CK), and the Bundesministerium für Bildung und Forschung

(BMBF, 01GN0980, JM).

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Table S1. Neuroprotective strategies that have completed

trials for acute ischaemic stroke.1 All neuroprotective strate-

gies have thus far failed to show an improvement in clinical

benefit following acute ischaemic stroke.

Please note: Wiley-Blackwell are not responsible for the

content or functionality of any supporting materials supplied

by the authors. Any queries (other than missing material)

should be directed to the corresponding author for the article.


© 2012 The Authors.418 Vol 7 July 2012 407–418

3. Neuroprotection for Ischaemic Stroke Translation From the Bench to the Bedside

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