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Keywords:

  • acute stroke therapy;
  • ischaemic cascade;
  • ischaemic stroke;
  • neuroprotection;
  • STAIR;
  • translation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction to neuroprotection
  4. How to define and measure neuroprotection
  5. The current status of neuroprotection
  6. Difficulties in translation into clinical practice
  7. Conclusions – the future of neuroprotection in ischaemic stroke
  8. Acknowledgements
  9. References
  10. Supporting Information

Neuroprotection seeks to restrict injury to the brain parenchyma following an ischaemic insult by preventing salvageable 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 of methodological 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 fulfilled 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 haematopoietic growth factors, and inhibitors of the nicotinamide adenine dinucleotide phosphate oxidases, a source of free radical production which is a key step in the pathophysiology 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.


Introduction to neuroprotection

  1. Top of page
  2. Abstract
  3. Introduction to neuroprotection
  4. How to define and measure neuroprotection
  5. The current status of neuroprotection
  6. Difficulties in translation into clinical practice
  7. Conclusions – the future of neuroprotection in ischaemic stroke
  8. Acknowledgements
  9. References
  10. Supporting Information

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 experimental neuroprotectants being trialled preclinically and clinically. Where thrombolysis aims to break down the occluding clot to restore blood flow to the ischaemic brain, neuroprotection seeks to limit ischaemic injury by preventing the salvageable neurons in the penumbra that surrounds the core from dying. Rapid restoration of oxygen and glucose by thrombolysis will always provide the most effective neuroprotection, but directly targeting the brain parenchyma to confer neuroprotection 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 neuroprotection [1]. Neuroprotective agents aim to salvage ischaemic tissue, limit infarct size, prolong the time window for thrombolytic 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 successful translation to clinical practice so far [3].

figure

Figure 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, α-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; PLA2, phospholipase A2.

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This review attempts to define neuroprotection and outline the current status of the neuroprotection field both preclinically and clinically, while identifying the problems associated with translating neuroprotection from the bench to bedside. It also describes some promising neuroprotectants, the use of neuroprotection alongside thrombolysis, and finally some concerns with the current criteria for preclinical neuroprotection studies.

How to define and measure neuroprotection

  1. Top of page
  2. Abstract
  3. Introduction to neuroprotection
  4. How to define and measure neuroprotection
  5. The current status of neuroprotection
  6. Difficulties in translation into clinical practice
  7. Conclusions – the future of neuroprotection in ischaemic stroke
  8. Acknowledgements
  9. References
  10. Supporting Information

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 strategies, 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 neuroprotection). 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 parenchyma itself. Nevertheless, given the wide array of biochemical pathways that have been elucidated to play a role in ischaemic 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 neuroprotection achieved. Some agents produced substantial protection 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 Roundtable (STAIR) criteria [5, 6], many neuroprotective studies exhibit low methodological quality [2] with a wide heterogeneity in the methodology used. This has led to increasingly variable results meaning many neuroprotective agents proceeded to clinical investigation with only weak preclinical evidence 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].

Table 1. Reasons for translational failure of neuroprotective agents from pre-clinical to clinical studies
Animal modelsHuman studies
Highly controlled, homogeneous populationVariable, heterogeneous population
Younger animalsOlder patients
Limited comorbiditiesNumerous comorbidities
Induced onset of strokeSpontaneous onset of stroke
Uniform aetiologyVariable aetiologies
Ischaemic territory usually from middle cerebral arteryIschaemic 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 durationVariable occlusion duration
Adequate sample sizeInadequate sample size
Wide scope for dose optimizationReduced scope for dose optimization
Multiple routes of administrationLimited routes of administration
Rapid availability of the drugs to the target areaSlow availability of the drugs to the target area
Infarct volume as outcomeFunction as outcome

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 neuroprotection instead of directly targeting the neuron [10]. Interestingly, the same compound also raised CBF in the ischaemic region which may have contributed to its neuroprotective effects [11]. The concept of neuroprotection produced by enhancing CBF rather than inhibiting the ischaemic cascade has recently been discussed [12]. Sophisticated imaging tools might help to delineate genuine neuroprotection 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]. Unfortunately, meaningful functional testing in small laboratory animals, especially mice, is frequently hampered by stroke severity and the limited correlation with higher brain functioning in humans, and so representative tests must be carefully selected.

The current status of neuroprotection

  1. Top of page
  2. Abstract
  3. Introduction to neuroprotection
  4. How to define and measure neuroprotection
  5. The current status of neuroprotection
  6. Difficulties in translation into clinical practice
  7. Conclusions – the future of neuroprotection in ischaemic stroke
  8. Acknowledgements
  9. References
  10. Supporting Information

Even after 30 years of neuroprotection research, no neuroprotective 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.

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 mechanisms in stroke pathophysiology. Up to now, numerous drugs targeting 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 activities [2]. Overall, 1026 candidate stroke drugs have been identified 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 cerebral 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 neuroprotective 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 different animal studies for meta-analysis. This method, originally applied for clinical trials in humans, was recently used to obtain 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, minocycline, and statins as potential approaches to neuroprotection in the clinical setting. The ongoing clinical trials investigating neuroprotection for acute ischaemic stroke have been outlined in Table 2. A list of neuroprotective strategies that have undergone completed studies for acute ischaemic stroke and that have all failed to show neuroprotection in the clinic is shown in Supporting Information Table S1.

Table 2. Neuroprotective compounds currently undergoing clinical trials*
CategoryName(s)MechanismClinical phaseManufacturer
  1. *Information gathered from Stroke Trials Registry [3], O'Collins et al. [2], and Cochrane Clinical Trials Database [20].

AntioxidantEbselenFree radical scavengerIIIDaiichi Pharmaceutical Co., LTD
Edaravone (MCI-186)Free radical scavengerIIIMitsubishi Pharma Corporation
Anti-apoptotic/regenerationAX200 (filgrastim, G-CSF analogue)Growth factorIISygnis Bioscience GmbH & Co KG
Human Chorionic Gonadotropin (hCG)/Erythropoietin (NTx-265)Growth factors, oxygen deliveryIIStem Cell Therapeutics Corp.
ExcitotoxicityMagnesium sulphateNMDA ion channel blockerIIIMany manufacturers (Abbott Laboratories for FAST-MAG)
Fluid regulatorsAlbuminHaemodiluting agentIIIBaxter Bioscience
OthersCiticoline (CDP choline)Membrane stabilizerIIIFerrer Grupo
Deferoxamine mesylateIron chelatorIINovartis Pharma
DP-b99Metal ion chelatorIIID-Pharm Ltd
HemicraniectomyReduce cerebral oedema and intracranial pressureIIINone
HypothermiaReduce cerebral oxygen metabolism, synaptic inhibitorIIINone
InsulinReduce glucose and brain damageIIIEli Lilly
LovastatinHMG CoA reductase inhibitor, antioxidantIIMany manufacturers
MinocyclineAntibiotic, pleiotropic protective effectsIIIWyeth
SimvastatinHMG CoA reductase inhibitor, antioxidantIIIMany manufacturers
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 neuroprotective through several mechanisms including decreasing excitatory 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 Controlled 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, electrolyte 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 molecules in the blood, plays a key role in restricting fluid leaking 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, maintaining vascular patency, and preventing re-occlusion after successful thrombolysis [4].

The pilot study Albumin in Acute Stroke (ALIAS) demonstrated 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 contribute 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 neuroprotection 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 paramedics 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 ischaemic neurons, powerful effects of this naturally occurring electrolyte on stroke outcome in clinical practice may be surprising, especially when given as a monotherapy. A combinatory approach with other neuroprotective or clot-breaking agents 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 cerebral 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 could reduce infections such as pneumonia or urinary tract infections resulting from stroke-induced immunosuppression [41].

In an open-label evaluator study, minocycline administration led to a significantly better outcome in acute stroke patients compared to placebo [40]. Similarly, in Minocycline to Improve Neurological Outcome in Stroke, minocycline was safe 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 minocycline 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, widespread 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 lovastatin 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 with interferon-β 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 improvements 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 following acute ischaemic stroke [3].

Difficulties in translation into clinical practice

  1. Top of page
  2. Abstract
  3. Introduction to neuroprotection
  4. How to define and measure neuroprotection
  5. The current status of neuroprotection
  6. Difficulties in translation into clinical practice
  7. Conclusions – the future of neuroprotection in ischaemic stroke
  8. Acknowledgements
  9. References
  10. Supporting Information

Examples of neuroprotective therapies from preclinical 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 experiments, 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 proceeded 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 suggests 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 protection, 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 investigating tirilazad in acute stroke patients. Patients were not thrombolysed, and so were not stratified between transient and permanent ischaemia, although preclinical evidence suggested 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 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 primates (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 used in 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, randomized, placebo-controlled trial that revealed a small but significant 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 disappointingly 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 frequency 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 neuroprotective action of NXY-059 may be mediated in the endothelium 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 publication 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 supposedly neuroprotective by scavenging free radicals and preventing 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. To date, all clinical attempts to scavenge reactive oxygen or nitrogen 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 enzymatic 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 neuroprotection assessment outlined the problems associated with translation. 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 associated with significant structural and functional changes in the brain, which affects outcome and the ability to recover after an ischaemic event [72].

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 clinical 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 events in 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 recanalization 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 transformation. 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 scavengers [75, 76], NMDA receptor antagonists [77], α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonists [78], antioxidant agents [79], anti-inflammatory agents [80], and antiplatelet agents [80-83].

The concept of thrombolysis combined with neuroprotective 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 clomethiazole (γ-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 strategy 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 thrombolytic, 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 be difficult 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 neuroprotective agents that have shown promising effects.

Haematopoietic growth factors

Haematopoietic growth factors are a group of regulatory molecules that are responsible for the mobilization, proliferation, 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-CSF and 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 assessment, was considered for analysis, the efficacy of EPO was lowered, suggesting that the benefit of EPO might be overestimated [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 outcomes 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 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]. Exploratory 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 stroke patients (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 production. Many other enzymes are able to form ROS, e.g. xanthine oxidase, 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 experimental stroke research [5], we could demonstrate that NOX4-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 neuronal apoptosis, after both transient and permanent cerebral ischaemia. This was independent of gender and age as elderly mice were equally protected. Interestingly from a translational perspective, application of the only validated pharmacological 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 significantly 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 neuroprotection in ischaemic stroke

  1. Top of page
  2. Abstract
  3. Introduction to neuroprotection
  4. How to define and measure neuroprotection
  5. The current status of neuroprotection
  6. Difficulties in translation into clinical practice
  7. Conclusions – the future of neuroprotection in ischaemic stroke
  8. Acknowledgements
  9. References
  10. Supporting Information

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 hypothesis. 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 revolutionized 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 provided important mechanistic insights into the complex pathophysiology of ischaemic stroke which will pave the way for upcoming 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, for example, 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 evidence, it is in most cases neither necessary nor feasible to plan and perform basic stroke studies like large controlled randomized 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 neurological diseases like multiple sclerosis should teach the stroke community that even imperfect animal models can serve as a basis for hypothesis-driven research that ultimately facilitates the development of new drugs.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction to neuroprotection
  4. How to define and measure neuroprotection
  5. The current status of neuroprotection
  6. Difficulties in translation into clinical practice
  7. Conclusions – the future of neuroprotection in ischaemic stroke
  8. Acknowledgements
  9. References
  10. Supporting Information

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).

References

  1. Top of page
  2. Abstract
  3. Introduction to neuroprotection
  4. How to define and measure neuroprotection
  5. The current status of neuroprotection
  6. Difficulties in translation into clinical practice
  7. Conclusions – the future of neuroprotection in ischaemic stroke
  8. Acknowledgements
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction to neuroprotection
  4. How to define and measure neuroprotection
  5. The current status of neuroprotection
  6. Difficulties in translation into clinical practice
  7. Conclusions – the future of neuroprotection in ischaemic stroke
  8. Acknowledgements
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
ijs770-sup-001-tblS1.docx86KTable S1. Neuroprotective strategies that have completed trials for acute ischaemic stroke.1 All neuroprotective strategies have thus far failed to show an improvement in clinical benefit following acute ischaemic stroke.

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