Novel therapies in development for the treatment of traumatic brain injury

Review

Ashley Publicationswww.ashley-pub.com

1. Introduction

2. Secondary injury

3. Attenuating secondary injury

4. Conclusion

5. Expert opinion

Monthly Focus: Central & Peripheral Nervous System

Novel therapies in development for the treatment of traumatic brain injuryRobert Vink1† and Alan J Nimmo2

1†Department of Pathology, The University of Adelaide, Australia & 2School of Pharmacy and Molecular Sciences, James Cook University, Australia

In industrialised countries, the mean per capita incidence of traumatic braininjury (TBI) that results in a hospital presentation is 250 per 100,000. InEurope and North America alone, this translates to > 2 million TBI presenta-tions annually. Approximately 25% of these presentations are admitted forhospitalisation. Despite the significance of these figures, there is no singleinterventional pharmacotherapy that has shown efficacy in the treatmentof clinical TBI. This lack of efficacy in clinical trials may be due, in part, to theinherent heterogeneity of the traumatic brain injury population. However,it is the multifactorial nature of secondary injury that also poses a majorhurdle, particularly for those therapies that have been designed to specifi-cally target an individual injury factor. It is now becoming increasingly rec-ognised that any successful TBI therapy may have to simultaneously affectmultiple injury factors, somewhat analogous to other broad spectrum inter-ventions. Recent efforts in experimental TBI have therefore focussed ondeveloping novel pharmacotherapies that may affect multiple injury factorsand thus improve the likelihood of a successful outcome. While a number ofinterventions are noteworthy in this regard, this review will focus on threenovel compounds that show particular promise: magnesium, substance Pantagonists and cyclosporin A.

Keywords: cyclosporin A, head injury, magnesium, neuropeptides, neuroprotection, neurotrauma, substance P

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1. Introduction

Traumatic brain injury (TBI) is the biggest killer of individuals under 44 years of age.Those individuals who survive TBI are often left with permanent neurological defi-cits that adversely affect their quality of life and, in many instances, prevent theirreturn to the workforce. It is now recognised that neuronal cell death resulting fromTBI is caused through both primary and secondary injury mechanisms. Primaryinjury occurs at the time of the traumatic event and may generally be considered tobe the mechanical disruption caused by TBI, including, amongst others, laceration,contusion, shearing and axonal stretching. By its nature, primary injury can only beprevented and tremendous advances have been made in this regard with the use ofsafety devices to prevent TBI, including seatbelts, airbags and helmets. Secondaryinjury, on the other hand, is the delayed biochemical and physiological events thatare initiated by the primary traumatic event, but manifest over the ensuing minutesto days afterward. It is this delayed injury cascade that is thought to be associatedwith the development of many of the neurological deficits observed after TBI. Sincethis secondary injury is delayed, there exists a window of opportunity to identify thefactors that make up the secondary injury cascade and treat with ‘antifactors’ that

2002 © Ashley Publications Ltd ISSN 1354-3784 1375

Novel therapies in development for the treatment of traumatic brain injury

could prevent, or at least attenuate, the injury process andresult in significant improvement in functional outcome [1].

Despite the identification of several promising candi-dates in experimental animal studies of TBI, the clinicaltrials that have attempted to pharmacologically inhibitindividual secondary injury factors have met with very lim-ited success. Interested readers are referred to some excel-lent analyses of these trials that have been reviewedelsewhere [2-4]. Various factors have been proposed toaccount for the lack of efficacy of these compounds anddiscussions of the inherent limitations of the experimentalanimal models, inadequate preclinical testing and hetero-geneity of the clinical TBI population can be found else-where [4,5]. However, it is the complexity of the secondaryinjury process and its role in the development of irreversi-ble brain injury that is also largely responsible for thisapparent failure to develop effective pharmacotherapies.Indeed, while a number of individual secondary injury fac-tors have been identified, their relationship to functionaloutcome and their inter-relationships with one another areyet to be fully characterised. Moreover, it is becomingincreasingly recognised that secondary injury is a multifac-torial process with different injury factors contributing tothe injury process at different time-points following theinsult. For example, it has long been recognised that theneuronal cell death that occurs in the first 24 h after TBIoccurs through a process of necrosis involving swelling ofmitochondria and other organelles and subsequent mem-brane degeneration [6]. Inhibition of necrosis was, there-fore, envisioned as being the aim of neuroprotectivetherapies. However, it has since been recognised that neu-ronal cell death after TBI can continue for days after TBIthrough a process known as apoptosis [7], whose identifyingfeatures include DNA condensation and fragmentation,cell shrinkage and the ultimate formation of apoptoticbodies [6]. What further complicates matters is the observa-tion that inhibition of necrosis has the potential to exacer-bate apoptosis and vice versa [8,9].

It is therefore unlikely that a single injury factor isresponsible for all of the observed functional deficits fol-lowing TBI. Rather, an intervention that simultaneouslyinhibits or at least attenuates a number of secondary injuryfactors is more likely to achieve a successful outcome. Fewinterventions have been identified that have effects on adiverse range of secondary injury factors. Nonetheless,three compounds with multifactorial effects have beenidentified in experimental brain injury studies and haveeither entered or are about to enter clinical trials in TBI.This review will briefly summarise some of the more rele-vant secondary injury mechanisms associated with thedevelopment of neuronal cell death following TBI. Theproperties that characterise magnesium, substance P antag-onists and cyclosporin A (CsA) as novel ‘broad spectrum’interventional pharmacotherapies for use in TBI, will thenbe discussed.

2. Secondary injury

2.1 ExcitotoxicityOne of the first recorded events after TBI is the release of exci-tatory neurotransmitters such as glutamate. Glutamate is foundin almost every brain region and in almost half of all brain syn-apses. Its release has been noted as early as minutes after experi-mental brain injury [10] with a maximum value being achieved10 min after injury. While experimental studies suggest that theglutamate concentration falls rapidly after this peak, increasedlevels during the first few days after severe clinical injury havebeen reported [11]. These sustained high levels of glutamateseem to be correlated more to structural damage rather thanvesicular release. Once released, glutamate can bind to anumber of different receptor subtypes, with the NMDA (N-methyl D-aspartate) receptor widely shown to be associatedwith neurotoxicity [12]. With significant quantities of glutamatereleased immediately after the traumatic event, binding to theNMDA receptor is known to promote substantial calciuminflux with resultant calcium overload. Not only does this causedepolarisation, the high intracellular calcium concentration isknown to activate a plethora of calcium-dependent enzymes,including proteases, lipases and endonucleases, whose uncon-trolled activation form part of an autodestructive cascade.Glutamate excitotoxicity is, however, not only mediated by theNMDA receptor, but also by the non-NMDA glutamate recep-tors [8]. Thus, glutamate excitotoxicity may involve a number ofdifferent mechanisms, each dependent on a different receptor.Such complexity makes effective pharmacological blockade ofglutamate excitotoxicity problematic.

2.2 Calcium-mediated eventsAccumulation of excessive levels of calcium has long beenconsidered part of the final common pathway of neuronal celldeath [13]. This has been largely due to the recognition thatcalcium is a ubiquitous second messenger whose uncontrolledaccumulation can initiate a series of destructive enzymaticreactions. Entry of calcium is via one of three voltage sensitivecalcium channels and by ligand gated calcium channels. All ofthese calcium channels have their own specific antagonists.Furthermore, there are Na+/Ca2+ antiporters that normallyextrude calcium from the cell, but at high internal Na+ con-centration, have been shown to translocate Ca2+ into the cell[14]. Add to this the presence of intracellular Ca2+ stores thatcan release their ion stores upon stimulation by second mes-sengers [15] activated by TBI and we have a highly complex,integrated system for the regulation of intracellular Ca2+ con-centration. It is, therefore, perhaps not unexpected that phar-macotherapies directed at inhibiting one of these processes inan effort to attenuate intracellular Ca2+ accumulation havedemonstrated limited success.

2.3 MagnesiumA role for magnesium in the pathophysiology of TBI was firstproposed in the late 1980s, following the observation that

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brain intracellular free magnesium concentration declines aftera traumatic event and that this decline is correlated to func-tional outcome [16]. This observation has now been replicatedby a number of research groups in a number of different exper-imental models that simulate features of clinical TBI [17-19]. Assuch, free magnesium decline seems to be a ubiquitous featureof injury to the central nervous system (CNS). What is partic-ularly relevant to TBI is that magnesium is involved in the reg-ulation of a number of physiological and biochemicalprocesses within the cell, each of which plays an important rolein normal cell function. For example, magnesium is essentialfor normal cellular bioenergetics, including both substratelevel and oxidative phosphorylation [20]. Indeed, magnesium isa mandatory cofactor in all energy producing and consumingreactions involving carbohydrate, lipid, nucleic acid and pro-tein metabolism. Over 300 of the enzymes involved in theseprocesses are magnesium dependent and have a direct require-ment for optimum magnesium levels [21]. For example, the for-mation of the initiation complex in protein synthesis dependson magnesium ions [22] and any decline in magnesium willinhibit protein synthesis. Indeed, reducing levels of magne-sium to < 0.25 mM have been shown to inhibit both RNAaggregation and DNA synthesis [23]. Magnesium is also man-datory for ATPase function. Therefore, reducing magnesiumlevels will inhibit ion homeostasis maintained by the Na+/K+

and Ca2+ ATPase complexes. In addition to direct effects onenzymes, magnesium also has effects on plasma membraneintegrity [24] and ion channel activity. Particularly notable isthe fact that magnesium is an endogenous antagonist of Ca2+

channels [25], including the glutamate NMDA channel [26].Moreover, by blocking presynaptic Ca2+ channels, magnesiuminhibits glutamate release [27]. Finally, magnesium is known toblock the mitochondrial permeability transition pore impli-cated in apoptosis [28].

Clearly, there are numerous avenues by which magnesiumcan regulate normal physiology, thus making it a particularlyattractive target for pharmacologies aimed at attenuating mul-tiple secondary injury factors.

2.4 Free radicalsFree radicals are highly reactive molecules produced as a nor-mal by-product of oxidative metabolism. Under normal con-ditions their concentration is tightly controlled byendogenous antioxidant mechanisms, including glutathioneand the enzyme superoxide dismutase. However, traumaticinjury profoundly increases the production of free radicals,particularly when iron released from haemoglobin is present[29-31]. At high concentrations, these reactive oxygen species(ROS) cause considerable damage to proteins, lipids andDNA, with rapid cell death likely if the damage continuesunabated. Even if cell death is not rapid, there is evidence thatcontinued exposure to ROS opens the mitochondrial transi-tion pore, thus promoting cell death via apoptosis [32].

Administration of antioxidants has been shown to be effec-tive in experimental models of traumatic injury to the CNS

[33], particularly when there is an ischaemic or haemorrhagiccomponent to the insult. However, on their own, they havenot shown efficacy in clinical trials of TBI.

2.5 Mitochondrial damageMitochondrial integrity is critical to cell survival, given thehigh energy requirements of neurons. While most studieshave examined mitochondrial function in ischaemia, thereare now a number of studies that have focussed on TBI [34].Initial studies have shown that TBI induces impairments inthe rate of respiration, with mild-to-moderate injury pro-ducing slight but insignificant changes in respiratory rateand in coupling between electron transport and ATP synthe-sis [35], while more severe injuries produce significantimpairments of mitochondrial respiration and respiratorycoupling [36]. These alterations in mitochondrial respirationwere associated with alterations in respiratory chain cyto-chrome oxidase expression and activity [37]. In addition to animpaired ability of mitochondria to carry out oxidativephosphorylation, TBI profoundly impairs the ability ofmitochondria to actively transport calcium [38]. Not onlydoes this impact on the cytosolic Ca2+ concentration and theassociated Ca2+ induced cell death, it also promotes the per-meability changes of the mitochondrial permeability transi-tion pore [39]. This pore is integrally involved in apoptoticcell death as well as with uncoupling and inhibition of oxi-dative phosphorylation and stimulating the generation ofmitochondrial ROS [34].

2.6 OedemaIn a recent clinical study, oedema was found to be responsi-ble for 50% of all deaths recorded in young victims of braininjury [40]. This, in itself, emphasises the importance ofbrain oedema in determining outcome following TBI.There has been a considerable amount of research done inan effort to understand the mechanisms associated with theformation of oedema, and how this relates to functionaloutcome following TBI [41]. There are primarily two formsof oedema initiated after TBI. The first is vasogenicoedema, which is apparent very early after the traumaticevent and is associated with an increased permeability of theblood–brain barrier (BBB). Water is found to accumulate inthe extracellular space as osmotic substances escape fromthe vasculature. The second form of oedema is cytotoxicoedema, which develops when osmotic pressure in theintracellular space increases to beyond that observed in thesurrounding space. This form of oedema is usually associ-ated with some form of neurotoxic event and, although itdevelops at later time points than vasogenic oedema, it hasbeen reported to account for most of the brain swellingafter TBI [42]. Nonetheless, it is the initial vasogenic oedemathat is thought to be permissive for the subsequent develop-ment of cytotoxic oedema [43]. Clearly, inhibition of oedemais a highly desirable objective in the pharmacological man-agement of TBI.

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3. Attenuating secondary injury

3.1 MagnesiumHaving discussed the pathophysiology of magnesium declineafter TBI above (Section 1.3), the evidence that suggests thatadministration of magnesium may be neuroprotective willnow be addressed. Initial studies administered intravenousmagnesium sulfate prior to injury in an effort to prevent themagnesium decline after trauma [16]. In addition to prevent-ing the brain free magnesium decline, it was noted that thefunctional outcome of the animals was significantly betterthan that observed in vehicle treated controls. The influenceof brain magnesium status on outcome following TBI waslater confirmed in studies demonstrating that dietary magne-sium depletion prior to injury exacerbated neurological defi-cits after injury, whereas preinjury administration ofmagnesium sulfate attenuated these deficits [44]. Since thoseearly prophylactic studies, numerous reports have since con-firmed that postinjury administration of magnesium saltsresults in a significant improvement in both motor and cogni-tive outcome after either TBI [45-49] or a lesion inducinginjury to the sensorimotor cortex [50]. While these studieshave used either the sulfate or chloride salts of magnesium,recent dose/response studies confirm that both salts areequally effective when given by intravenous or intramuscularinjection [51]. Indeed, both salts were shown to enter the brainintracellular compartment and increase free magnesium con-centration after TBI [51]. Even a bolus dose given as late as24 h after TBI had a significant effect on neurological out-come, although administration within 12 h of injury was rec-ommended for maximum efficacy [52]. It should be noted thatthere are also some contradictory reports in the literature, par-ticularly in hypoxia/ischaemia studies, suggesting that magne-sium may not be neuroprotective. However, these reportstend to use an intravenous dose between 100 – 300 mg/kg,far in excess of the optimal dose reported in the dose/responsestudies [51,53].

In addition to neurological outcome, a variety of outcomeparameters have shown improvement with magnesiumadministration. Reduction in magnesium after TBI wasshown to be associated with profound oedema formation [54].Not surprisingly, administration of magnesium after TBIreduced brain oedema [45,55], although it was only recentlydemonstrated that this might be via a direct attenuation ofBBB opening [56]. Up until now, it was generally believed thatit was the effect of magnesium on the activity of the Na+/K+

ATPase that accounted for this reduction in brain swellingafter TBI.

Recent studies have also demonstrated a reduction in post-traumatic lesion size with magnesium treatment [57] similar tothat described when magnesium is administered after ischae-mia [58]. This reduction in lesion volume with magnesiumtreatment after TBI was associated with a reduction in acutecytoskeletal alterations, a condition that the authors attributeto activation of calpain [59]. Calpain is one of the calcium-acti-

vated proteases activated after TBI, whose substrates includecytoskeletal proteins, receptor proteins, signal transductionenzymes and transcription factors. Moreover, becausecytoskeletal damage was exacerbated when injury was inducedin a magnesium deficient animal, the authors suggest that themagnesium reduces cytoskeletal injury by inhibiting calpain[59]. Certainly, a decrease in magnesium concentration canresult in an increased calcium concentration, as has beenshown in the hippocampal CA1 region [60]. Presumably thisoccurs via increased glutamate NMDA channel influx, sincetraumatic stretch injury has been shown to reduce the magne-sium blockade of the NMDA channel [61]. Thus, magnesiumcould inhibit calpain activation by both reducing Ca2+ influxand also by directly inhibiting calpain itself.

The potentially protective effects of magnesium are notlimited to calcium-mediated events, oedema formation andinhibition of neurotransmitter release and binding. Magne-sium is a known membrane stabiliser [24] and has beenreported to reduce membrane lipolysis [62]. This has anumber of positive effects on ion channels as reflected in amagnesium-induced inhibition of membrane depolarisa-tion and cortical spreading depression [63] and improve-ment in spinal somatosensory evoked potentials followingspinal cord injury [64]. By reducing lipid peroxidation [64],magnesium has also been shown to decrease hypoxia-induced increases in oxygen free radicals [65] and attenuatethe production of ROS [66,67]. These observations are con-sistent with the improvement in endogenous antioxidantlevels [68] and the reduction in malondialdehyde levels [69]

reported following post-traumatic magnesium administra-tion. The positive effect on membranes is also reflected inthe ability of magnesium to preserve mitochondrial mem-brane potential [70]. This presumably will have positiveeffects on oxidative phosphorylation and explain the reduc-tion in lactate levels observed when magnesium is adminis-tered following TBI [69]. Therefore, by improvingmembrane stability and decreasing the production of ROS,magnesium may preserve the mitochondrial capacity foroxidative phosphorylation and facilitate the synthesis of theadenine nucleotides. However, while the mitochondria areprimarily concerned with energy metabolism, their role inapoptotic cell death following nervous system insults hasnow become firmly established [34].

The potential role of magnesium in apoptosis hasrecently been reviewed [71] and this discussion will, there-fore, focus on aspects that are relevant to TBI. Magnesiumhas the ability to block the mitochondrial permeabilitytransition pore [67,72]. It is the increased permeability of thispore that is associated with the release of cytochrome c andinduction of the caspases that ultimately induce apoptoticcell death. The permeability transition is influenced by anumber of factors, including ROS, adenine nucleotide con-centration, calcium and the Bcl2 protein family. Magne-sium has an influence on all of these factors, some of whichhave already been mentioned.


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The evidence that it influences the Bcl2 family has onlyrecently been reported. In hypoxia, it was shown that admin-istration of magnesium reduces the concentration of pro-apoptotic Bax and increases the concentration of antiapop-totic Bcl2 [73]. It is the balance between these two proteinsthat has been shown to be associated with the mitochondrialpore transition. In addition to both directly and indirectlyaffecting the mitochondrial permeability transition pore,magnesium is also capable of preventing induction of pro-apoptotic p53 [74] and inhibiting DNA fragmentation factor[75] following TBI. The net result is that magnesium adminis-tered after injury to the brain decreases nuclear oxidative dam-age and DNA fragmentation [76].

While this discussion of magnesium treatment in TBI hasfocussed on biochemical events, magnesium also has anumber of beneficial effects on physiological parameters. Themost significant in terms of TBI are effects on the cerebrovas-culature. Administration of magnesium reverses delayed cere-brovasospasm after subarachnoid haemorrhage [77]. Moreover,it has been widely demonstrated that magnesium inhibits cer-ebral vasospasms [78-80]. These properties, in part, account forthe reversal of vasoconstriction and increased cerebral blood-flow that has been recorded in rats following magnesiumadministration [81,82]. Although it is thought that the ability ofmagnesium to block calcium channels may, in part, accountfor these effects, other mechanisms of action have not beenruled out [83].

The multifactorial nature of magnesium’s effects on thebrain has resulted in a number of ongoing Phase III clinicaltrials in various nervous system disorders, including TBI(Magnesium Sulfate for Neuroprotection after BrainTrauma), cerebral palsy (Randomised Clinical Trial of theBeneficial Effects of Antenatal Magnesium Sulfate [BEAM]),stroke (Intravenous MAGnesium Efficacy in Stroke [Images],Magnetic Resonance in Intravenous Magnesium Efficacy inStroke [MR Images], Field Administration of Stroke Ther-apy-Magnesium [Fast-Mag]), subarachnoid haemorrhage(Magnesium and Acetylsalicylic Acid in Subarachnoid Hem-orrhage [MASH]) and intracranial haemorrhage (MAGne-sium Efficacy and Safety in Treating Intra-CranialHemorrhage [MAGESTIC]).

3.2 Substance P antagonistsDespite the small number of reports that have appeared inbrain injury studies, substance P antagonists are receivingincreased attention as neuroprotective agents. This is, in part,because of their recent identification as an effective pharmaco-therapy in clinical depression, emesis and neuropathic pain[84] and the subsequent interest in their potential in otherpathologies of the nervous system. There is now considerableevidence to suggest that substance P plays a significant role intissue injury. For example, in cardiac studies, magnesium defi-ciency has been shown to promote substance P induced neu-rogenic inflammation [85]. Many in vivo actions of substanceP are mediated by neurokinin 1 (NK1) receptors, and selective

NK1 antagonists [86] were subsequently used to inhibit sub-stance P action and reduce the neurogenic inflammation.NK1 receptor blockade was reported to reduce in vivo pro-oxi-dant stress (including glutathione loss), prenecrotic perivascu-lar inflammatory infiltration, circulating histamine, PGE2and lipid peroxidation products [85]. These authors proposethat substance P release may, in fact, be one of the earliestpathophysiological events associated with injury, leading tostimulation of inflammatory cytokines and subsequent stimu-lation of free radical mechanisms of injury. Significant sub-stance P release has been detected in the nervous systemfollowing both peripheral nerve injury [87] and traumatic spi-nal cord injury [88], an effect that was blocked by inhibitors ofserotonin synthesis [88]. In the peripheral nervous system(PNS), NK1 receptor immunoreactivity has been shown toincrease following injury [89], an observation also demon-strated for glia following CNS injury [90,91]. This increase wasnot observed in undamaged areas. The increased expression ofthese receptors on the astrocytes after injury may, therefore, belinked to their transformation to reactive astrocytes. In brainischaemia, induction of NK1 receptors in the endotheliumwas shown to contribute to oedema formation [92], an obser-vation that has been well characterised in peripheral tissueinjury [93]. Accordingly, the NK1 receptor was proposed as apotential therapeutic target in the treatment of stroke [92].Targeting this receptor has indeed proven beneficial, withinhibition of the substance P NK1 receptor following experi-mental ischaemia reducing infarct volume and improvingfunctional outcome [94]. Similar beneficial effects of NK1antagonists have additionally been reported in intestinalischaemia/reperfusion injury [95] and in TBI [96]. In the latter,the improvement in both cognitive and motor outcome withadministration of an NKI antagonist was correlated with areduction in post-traumatic oedema formation. Since thereare high numbers of NK1 receptors in the hippocampus andstriatum [97] and the fact that NK1 antagonists improved cog-nitive outcome after TBI, it appears there may also be a rolefor substance P in learning and memory.

There are a variety of other mechanisms by which sub-stance P antagonists may confer a neuroprotective effect. Fol-lowing middle cerebral artery occlusion, there is a markedexpression of substance P and NK1 receptors in glutamater-gic pyramidal cells [92]. Such colocalisation of NMDA andNK1 receptors had previously been reported in spinal cord[98] and brain [99]. Moreover, this colocalisation may facilitateglutamate-mediated neurotoxicity, a hypothesis supported bythe observation that substance P potentiates cellularresponses to NMDA, an effect that can be blocked by sub-stance P antagonists [100] and has been implicated in themodulation of locomotor behaviour. Substance P is capableof regulating the action of other neurotransmitters, includingdopamine release [101] and acetylcholine release [102] and byopening inward cation channels [103], notably calcium, it mayalso modulate the presynaptic release and postsynapticactions of a number of other neurotransmitters. Substance P



also induces endothelial cells to produce nitric oxide [104],

which, in itself, has been implicated as a secondary injuryfactor. The priming of polymorphonuclear cells for oxidativemetabolism (superoxide production) by substance P [105] alsoprovides a source of ROS known to exacerbate the injuryprocess. Finally, the evidence showing that NK1 antagonistsreduce cFos expression [106] suggests that there are alsomolecular effects of the compounds still to be characterised.

To date, substance P antagonists have been shown to haveantidepressant, antiemetic and antinociceptive properties,together with an ability to reduce neurogenic inflammation,BBB permeability, oedema, lesion volume and improve func-tional outcome in both ischaemia and TBI. Such multifacto-rial effects make it an ideal candidate for further investigation.

3.3 Cyclosporin ACsA is a highly lipophilic member of the immunophilin familyused for over 2 decades as an immunosuppressant drug. Its useas a treatment in neurological disorders has been recentlyreviewed [107] and, as such, the following comments will specif-ically relate to its potential use in TBI.

Early studies had demonstrated that CsA was able toreduce cell death and reduce lesion volume following ischae-mia–reperfusion injury [108,109]. Although the precise mecha-nisms were unknown, CsA inhibits the mitochondrialpermeability transition pore opening [28,110] and it was thismechanism that was considered particularly relevant to out-come. In studies of TBI, administration of CsA was againshown to reduce lesion volume, along with post-traumaticcytoskeletal changes and axonal injury as identified usingamyloid precursor protein immunoreactivity [111,112]. Thecompound’s ability to preserve cholinergic neurons [113]

implied a potential benefit in some cognitive disorders, apotential supported by findings demonstrating a positiveeffect of CsA on hippocampal plasticity after TBI [114]. How-ever, studies to date have failed to illustrate a benefit on cog-nitive outcome after TBI despite a robust protective effect onmotor and sensorimotor function [115].

The ability of CsA to improve post-traumatic mitochon-drial membrane potential, as well as decreasing intramito-chondrial calcium levels and ROS [116], once more supportedthe compounds ability to reduce mitochondrial permeabilitytransition pore opening as a primary mechanism of action.However, later studies have since demonstrated that the com-pound does not always inhibit apoptosis, suggesting that neu-roprotection may also be conferred by mechanisms other thanjust inhibition of mitochondrial permeability transition poreopening [117]. This was later confirmed when FK-506,another neuroprotective member of the immunophilin family,but without the ability to block mitochondrial permeabilitytransition pore opening, was also successfully used to attenu-ate axonal damage after TBI [118]. Obviously, alternativemechanisms were responsible. Aside from well-knownantiparasitic, fungicidal and anti-inflammatory properties,CsA also has the ability to directly inhibit calcineurin, a cal-

cium activated phosphatase strongly implicated in secondaryinjury. The compound has also been reported to inhibit lipidperoxidation after spinal cord injury at least as effectively asmethylprednisolone [119]. Finally, the compound is a weaksubstance P NK1 receptor blocker [120].

Thus, CsA has a number of beneficial effects on secondaryinjury factors that may contribute to its neuroprotective prop-erties. Dose response curves in experimental studies have sup-ported a reasonable therapeutic window of effectiveness [121],and Phase III clinical trials of CsA treatment of TBI havecommenced in a collaboration between the University of Flor-ida and the Medical College of Virginia.

3.4 Other compoundsThere are a number of other multifactorial compounds thathave been proposed as potentially effective therapies for thetreatment of TBI. Some of these compounds are new andrequire further research, others have been used experimentallyfor many years, but have failed to garner widespread support,while others have primarily been used in studies of ischaemiarather than TBI. The following is a short description of someof these compounds that are of particular interest to TBI.

3.4.1 DexanabinolDexanabinol (HU-211) is a non-psychotropic analogue of tet-rahydrocannabinol that has properties as an NMDA receptorantagonist [122], a free radical scavenger, an antioxidant [123] andan inhibitor of cytokine TNF-α [124]. Although most studieshave utilised models of ischaemia to demonstrate the neuropro-tective properties of this compound, some studies have exam-ined its utility in TBI [124-126]. In summary, these studies havedemonstrated that dexanabinol reduces BBB permeability,oedema and lesion volume, while improving both motor andcognitive outcome after trauma [127]. Dexanabinol has only beenstudied under conditions of very severe TBI containing anischaemic component. It is therefore unclear whether the drugmay be efficacious in mild-to-moderate TBI that make up mostclinical trauma cases. Nonetheless, the drug is in Phase III clini-cal trial (Efficacy Assessment of Dexanabinol Treatment of TBIVictims) and it is hopeful that the treatment may be of benefitto severely injured patients.

3.4.2 AM-36AM-36 is an arylalkylpiperazine compound designed to blockthe polyamine site of the NMDA receptor and simultaneouslypossess antioxidant activity [128]. By targeting the polyaminesite, the inventors avoided the deleterious side effects associatedwith other glutamate NMDA channel blockers, such as MK-801. In addition to its ability to block NMDA channel activityand lipid peroxidation, the compound has also shown proper-ties as a sodium channel blocker and an inhibitor of apoptosis[129]. Thus, the compound is a true multifactorial drug. Whenadministered < 3 h after middle cerebral artery occlusion, AM-36 significantly attenuated infarct volume and improved motorand sensorimotor deficits [130]. The effects on functional out-


Vink & Nimmo

come correlated with histological improvement. Whilst so farlimited to studies of ischaemia, the multiple modes of action onboth necrosis and apoptosis bode well for future studies utilis-ing other injury models, including TBI.

3.4.3 Thyrotrophin releasing hormoneThe suggestion that thyrotrophin releasing hormone (TRH)and its analogues may prove useful in the treatment of injury tothe CNS has been promoted for many years [131]. Experimentalstudies have shown that TRH and the early TRH analogues,such as CG3703 and YM-14673, were able to improve motoroutcome [132]. Some of this improvement could be attributedto physiological and metabolic actions, including improvingmagnesium status [133], bioenergetic state [133] and promotingrecovery in cerebral bloodflow following brain trauma [134].However, other physiological actions of these compounds,including autonomic, analeptic and endocrine effects, wereundesirable from a clinical point of view. Accordingly, thesecompounds did not attract much support as neuroprotectiveagents and their clinical use, to date, has been limited to thepromotion of recovery from disturbances of consciousness [135].Newly developed TRH analogues have been designed to largelyeliminate the undesirable physiological actions of this class ofdrug while preserving the neuroprotective effects [136]. In doingso, the compound has become even more of a multifactorialdrug, now with nootropic actions, but without the adverse sideeffects. However, with the removal of many of the physiologicaleffects, it has become very difficult to establish how these drugsactually confer their neuroprotection and, accordingly, nomechanism of action has been proposed. Until these mecha-nisms of action have been identified, there will be a continuingreluctance to utilise these compounds.

4. Conclusion

A number of experimental studies in TBI have identifiedthat magnesium, substance P antagonists and CsA haveeffects on multiple secondary injury factors. However, in aneffort to develop a designer ‘magic bullet’ drug, the unique,

broad spectrum properties of these drugs were overlooked infavour of developing designer drugs targeting a single mech-anism of injury thought to be primarily responsible for irre-versible tissue injury. It has now been accepted that a singleinjury factor is highly unlikely to be responsible for all of theinjury that occurs after trauma, particularly in view of thefact that cellular injury can occur through different mecha-nisms incorporating both necrosis and apoptosis. Accord-ingly, the focus has shifted back onto drugs that havemultifactorial effects. A great deal of evidence had alreadybeen accumulated in favour of magnesium as a neuroprotect-ant, which accounts for the large number of clinical trialsthat are currently underway using this compound. However,there is considerable effort at present to identify other suchcompounds and recent focus is on the substance P antago-nists and CsA. Despite the earlier failures in developing aneffective pharmacological intervention for TBI, it is hopedthat these new efforts will be successful.

5. Expert opinion

It is clear that TBI results in a multifactorial secondaryinjury cascade that results in neuronal cell death. It shouldalso be clear that the relative contribution of each secondaryinjury factor to the injury cascade will be largely influencedby the type of injury that has occurred. For example, verysevere injuries that have either a haemorrhagic or ischaemiccomponent would be expected to have far more free radicalproduction than those without these secondary complica-tions. It is this heterogeneity of injury combined with thecomplexity of the secondary injury cascade that is particu-larly challenging to those seeking to develop efficaciouspharmacological therapies.

Somewhat surprisingly, most, if not all, clinical trials in TBIto date have focussed on interventions that target one specificinjury factor, be it inhibition of the NMDA channel or someother individual factor. The reasons for this are somewhatunclear. However, it is encouraging to note that the more recentresearch efforts have focussed on treatment strategies that affect

Table 1. Interaction between magnesium, substance P antagonists and cyclosporin A and the various secondary injury factors and injury outcome.

Compound Injury factors Functional outcome

Lesion volume

Excitotoxicity Calcium-mediated events

Magnesium ROS Mitochondrial events

Oedema

Magnesium [26,27,60] [16,59,60] [16,51] [65-69] [28,67,70,72,73] [45,55,56] [16,44-52] [57,58]

Substance P antagonists

[100] [103] – [85,105] – [92,96] [94,96] [94]

CsA – [116] – [116,19] [28,110,116] – [115] [108,109,111,112]

CsA: Cyclosporin A: ROS: Reactive oxygen species.



multiple injury factors simultaneously. Some investigators havechosen to develop cocktails of individual drugs to achieve thisgoal and, in some cases, this has met with reasonable success[137]. However, in other cases, the combination of drugs has notbeen as effective as the individual drugs given alone [138], sug-gesting that care needs to be taken to ensure the properties ofeach drug do not counteract the other. This is one of the defi-ciencies in polypharmacy, the other being that multiple drugshave multiple side effects. For the pharmaceutical industry, thisis a most serious consideration.

The alternative to using polypharmacy is to identify poten-tial broad spectrum, therapeutic candidates that are multifac-torial in nature. While there are a number of promisingcandidates, this review has focussed on three novel com-pounds that have shown very promising results in TBI studiesto date. That is not to say that the basic research over thepotential of magnesium, substance P antagonists and CsA astherapeutic agents for use in TBI is complete. Not all of them

have been tested in multiple models of TBI, or have had com-plete dose/response studies performed or therapeutic windowof efficacy established. Nonetheless, despite these deficiencies,there is considerable excitement surrounding the prospect thatone of these agents may be the first successful pharmacother-apy to be used in TBI. All of these drugs attenuate multipleinjury factors (summarised in Table 1) and have had neuro-protective properties demonstrated in different experimentalmodels of TBI. Finally, the neuroprotection with all of thesedrugs has been demonstrated not only in terms of lesion vol-ume, but also in terms of functional outcome. It is thisapproach that will lead to the identification of a novel therapyfor the treatment of TBI.

Acknowledgement

RV and AJN are supported, in part, by the AustralianNational Health and Medical Research Council.

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AffiliationRobert Vink1† and Alan J Nimmo2

1Department of Pathology, The University of Adelaide, Adelaide, South Australia 5005, Australia2School of Pharmacy and Molecular Sciences, James Cook University, Australia†Author for correspondenceTel: +61 8 8222 3092;Fax: +61 8 8222 3093;E-mail: [email protected]


Novel therapies in development for the treatment of traumatic brain injury

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