new perspectives on developing acute stroke therapy

New Perspectives on Developing AcuteStroke Therapy

Marc Fisher, MD,1 and Rajiv Ratan, MD, PhD2

The development of additional acute stroke therapies to complement and supplement intravenous recombinant tissue-type plasminogen activator within the first 3 hours after stroke onset remains an important and pressing need. Muchhas been learned about the presumed target of acute stroke therapy, the ischemic penumbra, and clinically availableimaging modalities such as magnetic resonance imaging and computed tomography hold great promise for at leastpartially identifying this region of potentially salvageable ischemic tissue. Understanding the biology of ischemia-related cell injury has also evolved rapidly. New treatment approaches to improve outcome after focal brain ischemiawill likely be derived by looking at naturally occurring adaptive mechanisms such as those related to ischemic pre-conditioning and hibernation. Many clinical trials previously performed with a variety of neuroprotective and throm-bolytic drugs provide many lessons that will help to guide future acute stroke therapy trials and enhance the likeli-hood of success in future trials. Combining knowledge from these three areas provides optimism that additional acutestroke therapies can be developed to maximize beneficial functional outcome in the greatest proportion of acute strokepatients possible.

Ann Neurol 2003;53:10–20

Information about the cellular and molecular patho-physiology of tissue injury after focal brain ischemiahas increased drastically over the past 5 years. Addi-tionally, major advances occurred in the ability ofpreclinical and clinical researchers to rapidly andcomprehensively image acute ischemic stroke shortlyafter onset. Despite these important advances, the de-velopment of additional treatments for acute ischemicstroke has not progressed much. The use of intrave-nous recombinant tissue-type plasminogen activator(rt-PA) was approved in the United States in 1996and more recently in a few other countries based onthe results of the National Institute of NeurologicalDisorders and Stroke (NINDS) rt-PA trial.1 The onlyother acute stroke treatment trials that can be consid-ered to have been positive were the 3-hour time win-dow trial of the defibrinogenating agent, ancrod, andthe 6-hour intraarterial trial of prourokinase, athrombolytic drug related to urokinase.2,3 There havebeen many other negative acute stroke interventiontrials, primarily with neuroprotective drugs, but also afew with thrombolytic drugs.4 These negative trialsall had a drug-initiation time window of at least 5 to6 hours or longer. The development of additionaltherapies for acute ischemic stroke is imperative be-cause the incidence is increasing in many countries,

and intravenous rt-PA is used in a low percentage ofpatients because of the restrictive 3-hour time win-dow.

The large unmet need for the development of addi-tional acute stroke therapies implies that continuingthe development process is vital. Several key areas areapparent for directing and enhancing the process ofacute stroke therapy development: using imaging to de-tect the target of therapy, exploring novel therapeuticapproaches based on new scientific information andconcepts, and, lastly, improving clinical trials for eval-uating therapies and expanding the therapeutic timewindow.

Identifying the Target of Acute Stroke TherapyThe primary goal of acute stroke therapy is to preventischemic brain tissue not yet irreversibly damaged fromproceeding towards infarction, that is, to reduce ulti-mate infarct size.5 It is widely presumed that reducinginfarct size will translate into improved neurologicaland functional outcome because, in general, smaller in-farcts should have greater amounts of preserved, func-tional brain. The reduction of infarction and improve-ment of outcome is predicated on the assumption thatat early time points after stroke onset a reasonable per-

From the 1Department of Neurology, University of MassachusettsMedical School, Worcester; and 2Department of Neurology (Neu-roscience), Harvard Medical School and Beth Israel Deaconess Med-ical Center, Boston, MA.

Received May 6, 2002, and in revised form Aug 8. Accepted forpublication Aug 15, 2002.

Address correspondence to Dr Fisher, Department of Neurology,University of Massachusetts Medical School, Memorial Healthcare,119 Belmont Street, Worcester, MA 01605.E-mail: [email protected]

NEUROLOGICAL PROGRESS

10 © 2002 Wiley-Liss, Inc.

centage of the ischemic lesion is potentially salvageablewith timely and appropriate therapy.6 This idea of po-tentially salvageable ischemic tissue is directly related tothe concept of the ischemic penumbra and the exis-tence of a therapeutic time window after stroke onset.Many definitions of the ischemic penumbra have beenproposed, but the most relevant one for therapeuticconsiderations is that suggested by Hakim, that is, isch-emic tissue that is fundamentally reversible.7

Several imaging modalities provide evidence sup-porting the existence of an ischemic penumbra instroke patients. Positron emission tomography (PET)studies were the first type of imaging to provide con-firmatory information implying that an ischemicpenumbra may persist for hours after stroke onset.Initial PET studies were performed in cat and pri-mate stroke models, and the studies then were ex-tended into stroke patients.8,9 PET studies performedin stroke patients up to 18 hours after onset demon-strated the existence of substantial volumes of isch-emic tissue with cerebral metabolic rate of oxygenconsumption values above 1.4ml/100gm/min thatthen diminish below this threshold of presumed tissueviability on delayed scans performed several days lat-er.10 Another presumed marker of the ischemic pen-umbra on PET studies is ischemic tissue with an in-creased oxygen extraction fraction and a reduction ofcerebral blood flow to the range of 7 to 17ml/100gm/min.11 Ischemic tissue with such characteristics onPET were observed in 10 of 11 untreated patientsstudied up to 18 hours after stroke onset, and por-tions of this presumed penumbral tissue were ob-served to be infracted on subsequent computed to-mography (CT) scans.

Diffusion-perfusion magnetic resonance imaging(MRI) studies currently are the most widely used im-aging modality that supports the existence of an isch-emic penumbra hours after stroke onset. Diffusion-weighted MRI (DWI) abnormalities in focal ischemiaidentify brain regions with evidence of high-energymetabolism failure, leading to increased intracellularwater accumulation.12 Perfusion-weighted MRI(PWI) provides information about the status of mi-crovascular perfusion and in the clinical setting is anindex of cerebral blood flow.12 The two studies canprovide volumetric data and be performed consecu-tively over a few minutes. The volume of the perfu-sion abnormality is commonly greater than the vol-ume of the diffusion abnormality initially after strokeonset. Over time, the percentage of stroke patientswith at least a 20% volume discrepancy of perfusiongreater than diffusion lesion volume diminishes.13 Itis currently estimated that up to 6 hours after strokeonset greater than 50% of stroke patients will havesuch a mismatch.13 The region of perfusion abnor-mality without a diffusion abnormality is thought to

represent an approximation of the ischemic penum-bra.14 Patients demonstrating such a DWI-PWI mis-match appear to respond more favorably to intrave-nous thrombolysis when it is initiated beyond the3-hour window than patients without a mis-match.15,16 This observation supports the hypothesisthat the presence of a DWI-PWI mismatch helps toidentify patients with potentially salvageable ischemictissue who therefore are more amenable to therapeuticintervention. The concept of the DWI-PWI mis-match is a useful first step in using clinically available,rapid MRI techniques to identify the existence of apotential ischemic penumbra. Several caveats must beconsidered before suggesting that the DWI-PWI mis-match represents the ischemic penumbra. The firstconcern is that the mismatch is directly dependent onhow the two MRI techniques are performed and an-alyzed. DWI data should be acquired as an averagevalue from at least the three main orthogonalplanes.17 The DWI image then is derived from theapparent diffusion coefficient (ADC) map based onthe mean value from the three planes. The DWI im-age depicts ischemia as an easily identified region ofhyperintensity. Tracing the border of the ischemic,hyperintense lesion and then integrating the lesionarea by the slice thickness provides the ischemic le-sion volume. Threshold ADC values are not widelyused to define abnormality in human DWI studies asthey are in animal studies. The potential for observerbias in the interpretation of human DWI studies isapparent.

For human PWI studies, several PWI parameterscan be used to define the abnormal region.17 Thethree parameters usually obtained with PWI are cere-bral blood volume (CBV), mean transit time (MTT),and an index of cerebral blood flow (CBF) based onthe other two parameters.18 Recently, a more quanti-tative depiction of CBF has become possible with theavailability of more precise information about the ar-terial input function.19 Quantitative CBF mapping isnot, however, widely used in clinical practice. Themost common method for quantitative PWI mappingis to use MTT and associated time to peak (TTP)values on the ischemic side as compared with the nor-mal hemisphere.12 The size of the PWI lesion thenwill depend on the side-to-side threshold chosen todefine the abnormal volume in the ischemic hemi-sphere. A small difference threshold will lead to amuch larger abnormal perfusion volume than a largedifference threshold.20 The DWI-PWI mismatch ob-viously is directly dependent on how large the PWIlesion volume is. Recent information from severalgroups suggests that a mean MTT or TTP delay of 4to 6 seconds compared with the normal hemisphereprovide PWI maps that are most useful, althoughthese values will need to be validated in a larger sam-

Fisher and Ratan: Stroke Therapy Trial 11

ple and compared with other PWI parameters.20,21

Another important concern with the DWI-PWI mis-match concept is that it presumes that the DWI le-sion derived from abnormal ADC values representsirreversibly injured ischemic tissue at all time pointsafter stroke onset. Recent studies in both animals andpatients have demonstrated that DWI abnormalitiescan be reversed by early reperfusion.22,23 The DWIlesion therefore is not synonymous with irreversibleinjury. It is likely that absolute ADC values will pro-vide a more accurate representation of irreversible ver-sus potentially reversible ischemic injury early afterstroke onset, although even low ADC values may bepotentially reversible.24 The DWI-PWI mismatchconcept therefore will have to be refined to allow dif-fusion and perfusion MRI to better characterize theischemic penumbra early after stroke onset. The isch-emic penumbra will likely be represented more accu-rately by coregistering absolute ADC and CBF val-ues that change their predictability of reversible versusirreversible injury over time. Deriving this informa-tion will require successful human treatment experi-ments.

Other CT-based imaging modalities also have somepromise for identifying the existence of the ischemicpenumbra. With xenon-CT, reasonably quantifiablevolumetric measurements of CBF are rapidly possi-ble.25 Recently, multislice perfusion CT has becomeavailable that also can provide estimates of perfusionin ischemic tissue.26 Initial studies suggest that CBFand CBV maps on perfusion CT may provide esti-mates of irreversible ischemic injury and the ischemicpenumbra.27,28 It is also apparent that high-qualityCT imaging of the brain parenchyma can demon-strate early, subtle changes in ischemic tissue that re-flect the development of vasogenic edema in irrevers-ibly injured ischemic tissue.29 Using xenon-CT orperfusion CT with standard CT brain imaging cantherefore likely identify hypoperfused brain regionsnot yet infracted. However, as with diffusion-perfusionMRI, many concerns remain. The precise quantificationof CBF with xenon-CT as compared with PET mea-surements remains to be established. Perfusion CT isin its infancy, and quantified whole-brain maps of per-fusion are not yet widely available. Early ischemicchanges on high-quality parenchymal CT scans are of-ten subtle, difficult to reproducibly identify, and noteasily quantifiable. Thus, CT probably will provideuseful information about the extent of the CBF distur-bance but may not precisely identify hypoperfused re-gions without irreversible injury. Are just perfusion ab-normalities enough for identifying the ischemicpenumbra? CT-based techniques probably will be ofgreatest utility for identification of the ischemic pen-umbra early after stroke onset when perfusion abnor-

malities are easily identifiable but irreversible brain in-jury is of modest extent.

Acute Stroke Therapy: Clarifying BiologicalObjectivesSuperficially, the pathophysiology of acute, ischemicstroke appears straightforward; decreased blood supplyleads to a limited supply of nutrients required for cellmetabolism and viability. However, closer inspectionshows a dauntingly complex array of variables thatcould influence cellular outcome: the extent of isch-emia, duration of ischemia, presence or absence of ath-erosclerosis, extent and rapidity of reperfusion, damageor loss to neurons and glia, in particular, oligodendro-cytes, inflammation, and hemorrhage; all of these pro-cesses superimposed on the variability of the aging pro-cess and the enormous metabolic demands of neuronsto execute their critical functions make it unlikely thata single therapy would preserve many of the brain’seloquent functions. A realistic conclusion is that a sin-gle cellular salvage approach to therapy may not befound to achieve the goals of widespread ischemic cellprotection and improved functional recovery. Whatnovel approaches might facilitate the identification ofmore effective neuroprotective agents for treatingstroke?

The Concept of Homeostasis: The WayForward for Neuronal Protection fromIschemia?As Flemin and Walter30 discuss, Walter Cannon pub-lished an important book in the 1930s titled The Wis-dom of the Body, coining the term, homeostasis. Ho-meostasis, derived from the Greek word for “steady” or“same,” refers to the concept that organisms, includinghumans, will react spontaneously to a stress by engag-ing pathways to return to a set point that is consistentwith survival. The concept, while intuitively appealing,was met by skepticism by some because of the absenceof well-defined pathways by which cells compensate fora host of stresses including hypoxia-ischemia (Fig).Two major strategies have evolved for identifying adap-tive homeostatic mechanisms potentially relevant tostroke therapy ischemic preconditioning31 and thestudy of species-specific (turtles) or state-specific isch-emic tolerance (hibernation).32–35

Ischemic preconditioning refers to the phenomenonwhereby brain tissue exposed briefly to sublethal levelsof ischemia becomes somewhat resistant to a subse-quent, more severe and sustained ischemic insult. Tol-erance is achieved by activating existing cellular pro-teins and by de novo synthesis of protective proteins.36

The aggregate effect of these transcriptional and post-transcriptional changes is to protect the brain from the

12 Annals of Neurology Vol 53 No 1 January 2003

deleterious effects of ischemia. New gene expressionduring ischemic preconditioning generated substantialinterest by stroke biologists as these changes are re-sponsible for inducing tolerance for extended time pe-riods after the preischemic conditioning stimulus asopposed to the short-lived protection induced by post-transcriptional activation of existing proteins. Manygenes were identified that may account for tolerance,including glutamate receptor subunits, heat shock pro-teins, antioxidant proteins, and antiapoptotic pro-teins.37,38

Cassettes of “tolerance” genes that are upregulatedby ischemic preconditioning and that work coordi-nately to counteract hypoxia and ischemia were identi-fied. This orchestrated gene regulation stimulated in-terest in identifying small molecules that can targetupstream signaling pathways involved in activation ofcoordinated gene cassettes. For example, erythropoietin(Epo) is protective in models of cerebral ischemia.39,40

In ischemic preconditioning, Epo expression is upregu-

lated primarily in astrocytes, and the target of this li-gand, the erythropoietin receptor, is upregulated pri-marily on neurons.41,42 Thus, upregulation of thereceptor and its associated ligand occurs in two distinctcell types, and it is likely that increases in one of thesegenes but not both would result in a submaximal pro-tective response. The coordination of genetic programsin ischemic preconditioning likely occurs betweenbrain parenchyma and the vascular system. These ge-netic responses, reflecting adaptation at cellular, local,and systemic levels, form a functional “neurovascularunit” in which compensatory responses are engaged fortwo main purposes in mind: neuronal survival underconditions of hypoxia-ischemia and compensatory in-creases in blood flow and oxygen delivery to minimizethe duration or extent of hypoxia-ischemia.43–45

If a complex and coordinated genetic program in-volving tens or possibly hundreds of genes in neurons,glia, and vascular endothelial cells must be executed toachieve optimal ischemic preconditioning in the “neu-

Fig. Homeostasis and hypoxia-ischemia. In response to hypoxia and ischemia in the brain, the fate of the tissue is determined, inpart, by the severity of the initial insult. In the case of mild or short periods of ischemia, cellular compensatory mechanisms (“ho-meostatic mechanisms”) are engaged. These mechanisms include the activation or inhibition of pre-existing proteins and invariablynew gene expression. The net result is to maintain tissue viability during hypoxia-ischemia and to facilitate the restoration of bloodflow. In the case of moderate ischemia, adaptive homeostasis is again engaged. However, only partial tissue salvage occurs. In neu-rons that sustained irreparable harm from the initial insult, apoptosis is triggered. This apoptosis is considered homeostatic because itinvolves deleting dysfunctional neurons. Preservation of dysfunctional neurons could lead to absent or aberrant (eg, epilepsy or pain)responses, so inhibition of apoptosis is not always a desired therapeutic goal. The case of severe hypoxia-ischemia likely refers to thecore of the infarct. Tissue dies by necrosis because homeostatic pathways involved in protection or apoptosis have been obliterated.The model asserts that therapeutic approaches that augment native homeostatic mechanisms or restore homeostatic machinery will beneuroprotective and will optimize the survival of tissue capable of performing its assigned functions.


rovascular” unit, how could such a complex responsebe mimicked with a single drug? One possible solutionis to identify transcription factors specific to hypoxia-ischemia that, when activated, regulate common DNAregulatory element(s) in the promoter regions of thegenes that are upregulated in distinct cell types by isch-emic preconditioning.46 There may be common se-quences in promoters of many genes that are upregu-lated by ischemic preconditioning. Identification ofthese sequences as well the proteins that bind themcould represent targets for drug discovery. Small mol-ecules capable of activating transcription factors thatare activated by hypoxia-ischemia could augment pro-tective responses and induce tolerance. Such small mol-ecules would be potential candidates for acute stroketherapy.

Hypoxia-Ischemia Tolerance in Turtles andHibernating Squirrels: What Can They Teachabout Postischemic Treatment?In addition to ischemic preconditioning, other para-digms are used to understand mechanisms that areengaged by the neurons, in particular, and the brain,in general, to provide resistance to hypoxia andhypoxia-ischemia. Anoxia-tolerant species are instruc-tive about the physiological adaptations that occur inthe central nervous system for it to survive an anoxicor hypoxic-ischemic insult.32–35 Apparently, loss ofcritical energy substrates (eg, oxygen, glucose) inducesthe severe downregulation of energy turnover as wellas enhanced efficiency of the existing ATP-producingpathways rather than engagement of alternative en-ergy substrates. Suppression of energy turnover ap-pears to provide the greatest protection under condi-tions of hypoxia and ischemia. Major sinks of energyunder normoxic conditions have been identified: (1)protein synthesis46; (2) protein degradation; (3)Na�/K� pumping; (4) urea biosynthesis; and (5) glu-cose biosynthesis. Under conditions of anoxia/isch-emia in turtle brain cells, ATP demand decreasesdrastically. Specifically, protein synthesis and degrada-tion decrease to 10% of normoxic rates, and urea andglucose biosynthesis (which may occur in some celltypes in the brain) decrease to zero. Decreases in theenergy demands of the Na�/K� ATPase are less thanfor overall ATP turnover. Thus, during periods ofhypoxia-ischemia, in some cell types, the Na�/K�

ATPase becomes the cell’s dominant energy sink, ac-counting for up to 75% of the ATP demand of thecell.47 Interestingly, in brain, where under normoxicconditions Na�/K� ATPase function is required tomaintain ionic gradients and membrane potential,compensation for hypoxia includes not only a de-crease in Na�/K� ATPase function, but also a de-crease in membrane ion permeability.48 These

changes in membrane ion permeability, termed chan-nel arrest by Hochachka and Lutz32 andHochachka,35 permit the anoxic turtle brain to main-tain an electrochemical potential while decreasing theenergy demands on the Na�/K� ATPase.

Paradoxical Increases in ATP Consumption inMitochondria during Hypoxia and IschemiaWhat physiological changes to hypoxia-ischemia areadaptive and how could engaging these mechanismsearly or more robustly using small molecules result inneuroprotection48? Maladaptive homeostasis is ob-served. Understanding how tolerant species deal withthese maladaptive responses might be instructive. It iswell established that under conditions of normoxia,glucose is taken up into a cell and broken down topyruvate. Pyruvate is transported into the mitochon-drial matrix where it is metabolized to generate elec-tron equivalents in the form of NADH and FADH2.Electrons from these substances are passed from com-plex I to complex IV in the mitochondrial electrontransport chain. The transfer of electrons results inpumping of protons out of the inner mitochondrialmatrix to set up a proton gradient. The flow of pro-tons down their gradient back into the mitochondrialmatrix through the F1/F0 ATPase results in ATP gen-eration.49 Paradoxically, in the absence of oxygen,The F1/F0 ATPase system functions in reverse tomaintain the mitochondrial membrane potential andthereby turns the mitochondria from an ATP pro-ducer to and ATP consumer. Interestingly, as anaer-obic glycolysis takes over, cellular pH decreases, lead-ing to the binding of an inhibitory subunit of F1/F0

ATPase (IF1) and thus prevents hydrolysis of ATP bythis membrane protein complex. The inhibitory sub-unit of the F1/F0 ATPase I (IF1) is upregulated inanoxic tolerant species independent of lactic acidosis,suggesting that understanding the functions and reg-ulation of this protein may be important for treatinghypoxia and ischemia.

From the study of anoxia-tolerant species, a coherentview of cell survival under conditions of limited energysubstrates has begun to emerge. During the early stagesof hypoxia, hypoxia sensors are engaged that trigger acascade of hypoxia-defense processes as outlined in Ta-ble 1.32–35 Further understanding how hypoxia istransduced into the adaptive changes in anoxic tolerantspecies could facilitate the development of small mole-cules that engage these pathways in hypoxia intolerantspecies such as humans to render them more hypoxiatolerant.


Apoptosis: Adaptive or MaladaptiveHomeostasis?A discussion of homeostatic pathways in the preventionof neuronal loss after cerebral ischemia would be in-complete without mentioning the biological process ofapoptosis. Classically, apoptosis is defined by its mor-phological characteristics and refers to the cytoplasmicshrinkage and chromatin condensation and fragmenta-tion that occurs after a host of pathological and phys-iological death stimuli. This type of cell death is often,but not always, associated with fragmentation of DNAinto 185bp ladders.50,51 Initially, it was believed thatidentifying cell death as apoptotic would define a par-ticular type of cell death (eg, caspase-dependent, bcl-2–sensitive death). It is now clearer that many mecha-nisms can lead to apoptosis, and thus the concept hasless direct therapeutic relevance. The mechanism of celldeath should be defined as a way of identifying a spe-cific intervention that would limit the process; apopto-sis or necrosis per se does not imply a specific celldeath mechanism.

Whether inhibiting apoptosis ultimately will bebeneficial for stroke patients remains unresolved. Af-ter ischemia, it remains unclear whether apoptosis isengaged in neurons that have critical levels of damageor whether it is activated inappropriately by dysregu-lated second messengers. The two outcomes have verydistinct therapeutic implications. If apoptosis is acti-vated because the cell has sustained a critical level ofdamage to proteins, lipids, or DNA, then abrogatingthe death pathway without repairing the damagecould lead to preservation of “dysfunctional” neuronsand be deleterious for functional recovery. Con-versely, if the apoptotic program is aberrantly acti-vated by calcium or free radicals in a cell that hassustained no damage, then inhibiting the apoptoticprogram could be beneficial. Apoptosis that occurs asa result of cell damage can be considered “homeostat-ic” or appropriate, whereas apoptosis that may be in-appropriately activated can be labeled “pathological”or maladaptive apoptosis. Because therapeutic ap-proaches must be targeted at restoring and preserving

neurological function, it may not always be desirableto inhibit apoptotic cell death after stroke.

Developing Therapies for Acute IschemicStroke: Past, Present, and FutureAs mentioned initially, only three acute stroke trialscan be considered to have been positive in that theyachieved a statistically significant outcome for the pre-specified outcome measure. Only one of the trials, theNINDS rt-PA trial led to drug approval and the clin-ical use of intravenous rt-PA within 3 hours of strokeonset.1 Many other phase 3 trials were performed witha variety of purported neuroprotective drugs and alsothrombolytic trials beyond 3 hours.4 All of these trialsexcept for the prourokinase trial must be considerednegative because they did not improve the primaryoutcome measure. These trials have provided a wealthof information concerning how to do improve futureacute stroke therapy trials. Exploring why acute stroketherapy trials may have failed is useful, and a list ofpotential explanations is provided in Table 2. Forthrombolytic trials beyond 3 hours after stroke onset,three primary reasons for unsuccessful trials are appar-ent. The first is safety. For streptokinase trials, an ex-cess rate of hemorrhagic side effects occurred, and thisdevastating side effect outweighed any potential bene-fit.52 A second important issue for thrombolytic trialsperformed with a later time window is patient selec-tion. The presumed target for thrombolysis is patientswith a persistent vascular occlusion who have still havepotentially salvageable ischemic tissue. Only thePROACT-2 trial of intraarterial prourokinase at-tempted to identify in part the existence of these con-ditions by randomizing patients with an angiographi-

Table 1. Defensive Cellular Responses to Hypoxia

1. A 90% or greater decline in protein synthesis and pro-tein degradation

2. A complete cessation of urea and glucose biosynthesis3. A generalized decline in membrane permeability (‘‘channel

arrest’’) and neuronal firing frequency (‘‘spike arrest’’)4. Inhibition of F1/FO2 ATPase in mitochondria to pre-

vent these organelles from running in reverse and con-suming ATP

5. The upregulation of many genes that are designed torescue the cell from disrupted homeostasis as seen withischemic preconditioning models

Table 2. Potential Reasons to Explain Previously NegativeNeuroprotection Trials

1. The agents evaluated in clinical trials may not have beenadequately tested in preclinical studies to provide robustconfirmation of neuroprotective efficacy

2. Side effects precluded adequate drug assessment or didnot allow adequate neuroprotective blood levels to beachieved

3. Trials included patients not appropriate for the pur-ported mechanism of action of the drug being tested

4. Patients were included too late after stroke onset to al-low for adequate assessment of the drug’s neuroprotec-tive efficacy

5. Trials have been inadequately powered to detect modesttreatments effects

6. Trials included too many with patients with mild orvery severe deficits in which treatment effects are likelydifficult to assess with currently used outcome measures

7. The single outcome measure chosen to assess drug effi-cacy may not be sensitive enough to detect modest treat-ment effects


cally confirmed, proximal middle cerebral arteryocclusion.3 In this trial, patients with a moderately se-vere baseline strokes (National Institutes of HealthStroke Scale [NIHSS] median score of 17) with a me-dian time to treatment of 5.2 hours did show statisti-cally significant improvement with a 15% absolute dif-ference in the prourokinase group who achieved a 90-day outcome of 2 or less on the Modified RankinScale. None of the intravenous rt-PA trials beyond 3hours attempted to identify the presence of vascular oc-clusion or potentially salvageable ischemic tissue, andnone of these three trials was positive.53–55 However,the two ECASS trials did suggest possible beneficial ef-fects with intravenous rt-PA initiated within a 6-hourtime window. A third important issue for the delayedtime window thrombolytic trials is the sample size andpower of the trials. If less salvageable tissue exists atlater time points as implied by the current DWI-PWIdata, then the treatment effect of any therapy is goingto be less when that therapy is initiated later afterstroke onset. The 3 to 6-hour intravenous rt-PA trialshad approximately 300 to 400 patients per treatmentarm, and this number is too low to detect a statisticallysignificant absolute treatment effect of 5 to 7% ascompared with the approximately 12% observed withintravenous rt-PA in the 3-hour NINDS trial.

All of the phase 3 neuroprotection trials performedto date have been negative. There are many potentialexplanations as outlined in Table 2. Some of the neu-roprotective drugs tested did not undergo rigorous pre-clinical evaluation to provide conclusive evidence ofneuroprotective efficacy. A set of recommendations bya group of academic and industry participants was pub-lished recently to address concerns about the preclinicalevaluation of neuroprotective drugs for acute ischemicstroke (Table 3).56 The main components of these rec-ommendations are that neuroprotective drugs should

be shown to reduce infarct size and improve functionaloutcome both acutely and subacutely in multiple ani-mal models evaluated at several sites under appropri-ately controlled conditions. The dose–response rangeand time window for efficacy should be determined inwell-characterized models and side effects should beidentified. Although initial testing in rodent models ofpermanent and temporary focal ischemia was deemedappropriate, consideration for testing in gyrencephalicspecies was recommended for novel, first in classagents. Another important problem with neuroprotec-tive drugs is that plasma and central nervous systemdrug levels that are indeed neuroprotective may not beachievable in humans because of side effects and tox-icity.57 The potential efficacy of such drugs cannot beadequately tested. The time window of drug efficacy insome cases may have been too short to determine effi-cacy in stroke patients. If a drug is only effective wheninitiated within 2 to 3 hours after stroke onset, thendoing a trial with a 6-hour or longer time window withmany patients enrolled at the end of the enrollmentwindow will not appropriately evaluate drug efficacy.Additional trial design issues also may have hamperedmany neuroprotective drug trials. Past trials have in-cluded some patients with quite mild and very severeinitial neurological deficits. It is now apparent that pa-tients with mild initial deficits (NIHSS scores of 4–7)have a high rate of spontaneous improvement withouttreatment and severely affected patients (NIHSS scores�20) have a poor natural history.58,59 Including toomany mild patients in a clinical trial will increase theplacebo responder rate and reduce the power of thetrial to detect a treatment effect. Including too manysevere patients also will affect the power of the trial byreducing the probability of observing a treatment ef-fect. Another concern in clinical trial design is the in-clusion of patients with a stroke subtype unlikely torespond to the drug being tested because of its mech-anism of action. It appears that white matter ischemicinjury has some pathophysiological mechanisms thatdiffer from gray matter.60 A substantial percentage ofischemic strokes occur predominantly within whitematter, and it therefore would be unlikely that drugsthat work primarily on mechanisms of cell injury un-related to white matter injury would be effective in thisstroke subtype. Yet, some prior trials have not beendesigned to exclude enrollment of lacunar, white mat-ter stroke patients. Choosing the primary end point foran acute ischemic stroke trial is another area of con-cern. Most neuroprotective or thrombolytic trials usedone primary end point, either a functional or disabilityscale such as the Rankin Scale or Barthel Index,whereas other trials used a neurological deficit assess-ment, measuring change score from baseline or medianscore in the treated versus placebo group. Using a sin-gle measure of treatment efficacy as the primary out-

Table 3. STAIR-1 Recommendations for Preclinical Assessmentof Neuroprotective Drugs

1. Evaluate most drugs initially in rodent permanent occlu-sion models

2. Provide an adequate dose–response curve3. Explore the time window of therapeutic benefit in well-

characterized models4. All preclinical studies should be performed in a blinded,

randomized manner with adequate physiological moni-toring

5. Outcome measures should include both infarct volumeand functional measures both within a few days afterstroke and at later time points.

6. For drugs primarily directed at reperfusion injury, initialstudies can be done in temporary occlusion models, andsuch models also can be used secondarily for standardneuroprotective drugs

7. Consideration should be given to evaluating novel, first-in-class drugs in gyrencephalic species


come may lead to missing a potential treatment effectin another domain, measured by a different assessmenttool. Another problem with outcome measures hasbeen defining the most appropriate cutoff value to de-termine a successful treatment effect. For example, inthe ECASS-2 trial of intravenous rt-PA with a 6-hourenrollment window, the prespecified primary outcomewas the percentage of patients in the active treatmentand placebo groups achieving a Modified Rankin Scoreof 0 to 1.54 There was a nonsignificant trend in favorof rt-PA with this outcome cutoff. However, a posthoc analysis with an outcome cutoff of 0 to 2 yieldeda significant treatment effect with intravenous rt-PAinitiated up to 6 hours after stroke onset. The differ-ence between a score of 1 versus 2 on the ModifiedRankin Score is slight.61 In the NINDS rt-PA trial, aglobal outcome measure combining the change inNINDS score from baseline to day 90 with the Rankinand Barthel scores was used to evaluate treatment effi-cacy.1 Using a global outcome approach for acutestroke therapy trials may provide a better assessment oftreatment efficacy than relying on one primary out-come measure that only assesses a single parameter ofpatient recovery. Interestingly, the neuroprotective andrecovery-enhancing drug, citicoline, was tested in threephase 3 trials.62–64 In none of these trials was a statis-tically significant benefit observed on the prespecifiedprimary outcome measure. However, a post hoc anal-ysis using a global outcome measure in patients withmoderate to moderately severe strokes did detect amodest, but statistically significant treatment effect thatwill need to be confirmed in another citicoline trial.65

A second series of recommendations recently was pub-lished by the aforementioned group of academic andindustry participants, focusing on how to best performnew 2 and phase 3 trials in acute ischemic stroke.66

The recommendations incorporated many of the les-sons from previous trials and made suggestions that wehope will enhance the chances for successful trials inthe future.

The current status of acute stroke therapy is thatonly one therapy is of proven value, intravenous rt-PAgiven within 3 hours of stroke onset. Estimates and anational chart review suggest that this treatment isgiven to less than 3% of acute ischemic stroke patientsin the United States and Canada.67,68 However, in afew cities, the percentage of treated patients is higher,approaching 10 to 15% in some centers.69,70 Duringthe last 30 minutes of the 3-hour time window, thebenefits of intravenous rt-PA diminishes and are not asrobust as with earlier treatment.71 Some clinicians useintravenous rt-PA beyond 3 hours in selected patients,although postmarketing reports of intravenous rt-PAuse demonstrated that protocol violations such as thedelayed use of this treatment increase the risk for hem-orrhagic complications.72 In many centers, intraarterial

thrombolytic therapy is given beyond 3 hours to pa-tients with middle cerebral artery or basilar artery oc-clusions.23,73 This intraarterial therapy is performedwith rt-PA, using differing doses and concomitanttherapies, for example, low-dose intravenous heparinand mechanical clot dissolution. The use of intraarte-rial therapy is supported by the results of thePROACT-2 study, but rt-PA must be used, becausethe thrombolytic drug used in PROACT, prouroki-nase, is not available. Some clinicians use diffusion-perfusion MRI to choose patients for delayed intraar-terial thrombolysis, whereas others base the decision onangiographic and clinical findings. The use of intraar-terial thrombolysis beyond the 3-hour window remainscontroversial, and further trials are needed to providedefinitive information that this therapy is indeed ben-eficial. Questions remain concerning which subset ofstroke patients are benefited and with what dosing reg-imen of intraarterial rt-PA. A larger trial of prouroki-nase is planned. We hope that this will confirm theresults of PROACT-2 and lead to regulatory approvaland availability of this thrombolytic drug. No neuro-protective drugs are used to treat acute ischemic stroke.

Whither the future of acute stroke therapy? One im-portant approach is to maximize the number of pa-tients treated with intravenous rt-PA within the 3-hourtime window. Patient and physician educational ef-forts, as well as organizing delivery systems for therapid assessment and treatment of acute stroke patientswill be useful to increase the percentage of treated pa-tients. The main problem will remain the time con-straint of the 3-hour window and the concern that pa-tients treated within the last half hour of the 3-hourwindow may have a substantially lower rate of im-provement than those treated earlier. Can the 3-hourwindow for intravenous thrombolysis be extended?One approach is better patient selection by using im-aging technology to identify patients more likely to re-spond to delayed therapy. This hypothesis currently isbeing tested in an Australian intravenous rt-PA trialwith a 3 to 6-hour time window for patient enrollmentand the desmoteplase in acute stroke (DIAS) study ofintravenous desmotoplase with a beyond 3-hour enroll-ment window. Another approach is to combine intra-venous rt-PA with delayed intraarterial rt-PA in pa-tients who do not recanalize initially.74 A thirdpotential approach to extending the therapeutic timewindow for thrombolysis would be to combine neuro-protection given before or with intravenous thrombol-ysis.75 The hypothesis to be tested is that the neuro-protective drug could potentially extend the timewindow for thrombolysis by extending the time periodduring which a portion of the ischemic penumbra per-sisted. Delayed reperfusion then could effectively sal-vage this surviving penumbra and improve outcome. Afourth approach would be to more rapidly dissolve


clots by using mechanical devices or intravenous rt-PAwith ultrasound or a drug such as abciximab.

The future of neuroprotection alone is murkier, butrecent developments enhancing our understanding ofischemic pathophysiology and trial design do providehope and encouragement. The myriad number of isch-emic injury pathways implies that inhibiting only onemechanism will likely be of limited value.76 Drug com-binations targeted at several aspects of the ischemic cas-cade appear to be more rational and have a higher like-lihood of salvaging a greater portion of the ischemicpenumbra than one drug alone.77 Using neuroprotec-tive combinations without proof that any one drugalone is effective will be challenging. The drug combi-nation may interact unfavorably with each other orhave intolerable side effects. Designing and executingthe large trials needed to test combinations will be dif-ficult and expensive. It is likely that a combination trialwill have to contain at least four arms, each drug alonewith a placebo, double placebo, and the combination.Regulatory agencies will have to be convinced that twounproven therapies should be tested for more rapid de-velopment of acute stroke therapy. Another way fortargeting multiple aspects of the ischemic cascadewould be to use a single drug with multiple activities.78

This method is appealing because it avoids the prob-lems of drug interactions and simplifies trial design.Candidate drugs with multiple mechanisms of actionwill likely be identified as comprehension of the com-plexities of ischemic injury pathophysiology continueto expand. Any new neuroprotective drug or drugcombination then can be tested in well-conceived clin-ical trials that incorporate the lessons from prior trialsand recent clinical trial recommendations.

ConclusionsApproved therapy to improve outcome after acute isch-emic stroke currently is limited to intravenous rt-PAinitiated within 3 hours of onset, and this solitary ther-apy is utilized in only a very low percentage of patients.Developing additional and complimentary acute stroketherapies with a longer time window of proven efficacyremains a daunting challenge, but a vital endeavor be-cause acute ischemic stroke still is one of the mostcommon serious medical conditions with a large, un-met need for additional therapy. Much has beenlearned over the past decade about the pathophysiolog-ical mechanisms of cell injury related to focal brainischemia, the presumed brain tissue target of treat-ments likely to impede these mechanisms, and how tobetter design and conduct therapeutic trials of potentialnew treatments. It has become increasingly apparentthat multiple, concurrent, or sequential therapies areneeded to maximize salvage of ischemic brain tissue totherefore improve ultimate outcome. Developing acute

stroke therapies has proved exceedingly difficult, and inretrospect this should not be surprising because of theobvious complexity inherent with the pathophysiologyof focal brain ischemia. Now is not the time to aban-don or curtail this important endeavor, but to renewour efforts better prepared with new knowledge, in-creased enthusiasm, and guarded optimism.

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