Operation Brain Trauma Therapy: 2016 Update

Transcription

1 MILITARY MEDICINE, 183, 3/4:303, 2018 Operation Brain Trauma Therapy: 2016 Update Patrick M. Kochanek, MD, MCCM*; Helen M. Bramlett, PhD ; C. Edward Dixon, PhD ; W. Dalton Dietrich, PhD ; Stefania Mondello, MD, MPH, PhD ; Kevin K.W. Wang, PhD ; Ronald L. Hayes, PhD**; Audrey Lafrenaye, PhD ; John T. Povlishock, PhD ; Frank C. Tortella, ST, PhD ; Samuel M. Poloyac, PharmD, PhD ; Philip Empey, PharmD, PhD ; Deborah A. Shear, PhD ABSTRACT Operation brain trauma therapy (OBTT) is a multi-center, pre-clinical drug and biomarker screening consortium for traumatic brain injury (TBI). Therapies are screened across three rat models (parasagittal fluid percussion injury, controlled cortical impact [CCI], and penetrating ballistic-like brain injury). Operation brain trauma therapy seeks to define therapies that show efficacy across models that should have the best chance in randomized clinical trials (RCTs) and/or to define model-dependent therapeutic effects, including TBI protein biomarker responses, to guide precision medicine based clinical trials in targeted pathologies. The results of the first five therapies tested by OBTT (nicotinamide, erythropoietin, cyclosporine [CsA], simvastatin, and levetiracetam) were published in the Journal of Neurotrauma. Operation brain trauma therapy now describes preliminary results on four additional therapies (glibenclamide, kollidon-va64, AER-271, and amantadine). To date, levetiracetam was beneficial on cognitive outcome, histology, and/or biomarkers in two models. The second most successful drug, glibenclamide, improved motor function and histology in CCI. Other therapies showed model-dependent effects (amantadine and CsA). Critically, glial fibrillary acidic protein levels predicted treatment effects. Operation brain trauma therapy suggests that levetiracetam merits additional pre-clinical and clinical evaluation and that glibenclamide and amantadine merit testing in specific TBI phenotypes. Operation brain trauma therapy has established that rigorous, multi-center consortia could revolutionize TBI therapy and biomarker development. *Safar Center for Resuscitation Research, Department of Critical Care Medicine, University of Pittsburgh School of Medicine, 4401 Penn Avenue, Pittsburgh, PA Department of Neurological Surgery, University of Miami Miller School of Medicine, 1095 NW 14th Terrace, Miami, FL Safar Center for Resuscitation Research, Department of Neurological Surgery, University of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA Department of Neurological Surgery, University of Miami Miller School of Medicine, 1095 NW 14th Terrace, Miami, FL Department of Neurosciences, University of Messina, Via Consolare Valeria 1, Messina 98125, Italy. Program for Neuroproteomics and Biomarkers Research, Departments of Psychiatry, Neuroscience, and Chemistry, University of Florida, P.O. Box , Gainesville, FL **Center for Innovative Research, Center for Neuroproteomics and Biomarkers Research, Banyan Biomarkers, Inc., Progress Blvd., Alachua, FL Department of Anatomy and Neurobiology, Virginia Commonwealth University, 1101 East Marshall Street, Richmond, VA Department of the Army, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD Department of Pharmacy and Therapeutics, Center for Clinical Pharmaceutical Sciences, University of Pittsburgh, 3501 Terrace St., Pittsburgh, PA Department of Pharmacy and Therapeutics, Center for Clinical Pharmaceutical Sciences and the Clinical Translational Science Institute, University of Pittsburgh, 3501 Terrace St., Pittsburgh, PA Brain Trauma Neuroprotection and Neurorestoration Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD This material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting true views of the Department of the Army or Department of Defense. INTRODUCTION Despite a number of single and multi-center clinical trials in the field of traumatic brain injury (TBI), particularly trials targeting severe and/or moderate injury levels, investigators have failed to identify any new treatment to advance care beyond current guidelines-based therapy. Given that guideline-based care addresses mechanisms such as intracranial hypertension, clinical and subclinical seizure activity, and aspects of supportive care such as optimization of hemodynamics, ventilation, and nutrition, much of the search for new therapies has focused on other aspects of secondary injury, namely, the cellular and molecular pathophysiological sequelae of TBI set into motion by the primary injury that contribute to the ultimate damage and/or dysfunction observed, and the search for a neuroprotective magic bullet. 1,2 Pre-clinical research in a variety of models of TBI has identified many highly promising candidates; however, those tested within the rigors of clinical trials have not successfully translated to improved outcomes. Failures have included corticosteroids, therapeutic hypothermia, progesterone, and erythropoietin (EPO), and others. 3 9 Many explanations for the failure of these clinical trials have been postulated including the heterogeneity of background care across sites, inappropriate selection of the study populations, the use of insufficiently sensitive outcome assessment tools (the Glasgow Outcome Scale), obligate delays in administering new therapies in clinical trials, challenges in doi: /milmed/usx184 Association of Military Surgeons of the United States All rights reserved. For permissions, please journals.permissions@oup.com. MILITARY MEDICINE, Vol. 183, March/April Supplement

2 extrapolating dosing from animals to humans, inadequate brain pharmacokinetic and pharmacodynamic information, and use of pleiotropic drugs for which the specific mechanism(s) of actionthatneedtobeinhibitedremainunclear Another explanation for these translational failures is the possibility that patient heterogeneity precludes the ability to detect beneficial effects in the clinical trials. In addition to the role of genetic and epigenetic differences between patients, along with other confounders, the broad spectrum of anatomic TBI phenotypes such as contusion, diffuse axonal injury, extracranial or intracranial hematomas, and so on may mandate either the identification of an extremely powerful therapy that has broad neuroprotective properties across multiple TBI models or precision medicine-based approaches that target each phenotype with a specific new therapy. Finally, it has also been suggested that the discontinuity between pre-clinical and clinical trials in TBI reflects the possibility that the published pre-clinical literature is over-inflated regarding therapeutic efficacy, a suggestion confirmed in cancer research. 14,15 To identify new candidate therapies in TBI with the best chance for successful clinical translation, we established operation brain trauma therapy (OBTT) in This is a multicenter, pre-clinical therapy and biomarker screening consortium for the field of TBI focused largely on moderate to severe injury levels. Figure 1 outlines the components and overall goal of the OBTT consortium. The two central hypotheses of OBTT are that (1) a therapy that shows efficacy across multiple models should have the best chance for clinical translation in a conventional randomized clinical trial (RCT) in TBI, and (2) therapeutic success may be highly dependent on model and could inform clinical trial design and help guide future precision medicine based clinical trials focused on targeted pathologies. The OBTT performs primary screening of therapies in rats across three models, namely parasagittal fluid percussion injury (FPI) at the University of Miami, controlled cortical impact (CCI) at the University of Pittsburgh, and penetrating ballistic-like brain injury (PBBI) at the Walter Reed Army Institute of Research (WRAIR). Secondary screening is carried out at Virginia Commonwealth University using a midline FPI model in micro pigs. The OBTT has been screening two types of therapies: (1) agents considered low-hanging fruit, namely, therapies that are approved by Food and Drug Administration (FDA) for other indications that, based on the existing literature support, should have a high probability for success and thus could be rapidly advanced to clinical trials, and (2) agents considered to be high-risk, high-reward, namely, therapies that are novel and/or target a unique secondary injury mechanism, but have limited pre-clinical evidence. 16,17,24 With regard to blood-based biomarker testing in OBTT, although several novel biomarkers are being evaluated in our ongoing studies, a centerpiece of our work has been the assessment of two clinically relevant serum-detectable proteins (glial fibrillary acidic protein [GFAP] and ubiquitin carboxy-terminal hydrolase-l1 [UCH-L1]). Both of these biomarkers have been studied extensively in clinical investigations, including testing FIGURE 1. Overview of OBTT. Therapy selection is based on suggestions from the principal investigator (PI), the site PIs, programs, an oversight committee, and externally. Selection is made by secret ballot of the site PIs and finalized at the annual meeting of the National Neurotrauma Society. Primary therapy screening is carried out in three established rat TBI models. Secondary screening is carried out in a fluid percussion injury model in micro pigs. Blood-based biomarkers are assessed in all studies. Our goal is to carry out highly rigorous studies to inform clinical trials with regard to therapies and biomarkers. We seek to identify therapies that show either robust efficacy across models or highly model-dependent benefit to help guide either conventional clinical trials or precision medicine based clinical trials on targeted pathologies. For biomarkers, we are testing the utility of both conventional biomarkers and novel target engagement biomarkers once again to inform clinical trials. Fluid percussion injury (FPI). 304 MILITARY MEDICINE, Vol. 183, March/April Supplement 2018

3 in combat theater, are being used in current clinical trials and are under consideration for FDA approval for diagnostic or prognostic use in clinical TBI. 23,25 28 Thus, they provide a special opportunity in OBTT both to compare findings across models and link our pre-clinical findings to published and ongoing clinical investigations. These serum biomarkers are alsobeingusedinobtttoassesstheirtheranosticutilityfor each treatment that is evaluated. A synopsis of the initial biomarker work in OBTT, which addresses issues such as crossmodel comparisons, correlation of serum biomarkers with conventional outcomes (histology and function), and model stability across studies, has been published. 23 For additional details on the infrastructure of the OBTT consortium, the approach used for therapy selection, our manual of operations, the many approaches used across the consortium to maximize rigor in all of the investigations, the various outcomes used across the TBI models in primary and secondary screening, and the outcome scoring matrix used to rank therapies, please see our previous overview articles on OBTT along with articles published in a special issue of the Journal of Neurotrauma, in March This article updates our findings of the primary screening that has been carried out to date. Much of the work presented reflects the content of a presentation made by OBTT in a panel session at the 2016 Military Health System Research Symposium. This article addresses the current state of our research now where, in addition to the completed work on the first five therapies, testing has been completed on four additional therapies. Please recognize that for therapies 6 9, the results presented in this report are not final, but they provide an initial and unprecedented comparison of nine therapies across three TBI models. METHODS In primary therapy screening by OBTT sites, each of the centers uses their established model at a single injury level without major modifications. This includes use of outcomes that are also established at each site. Detailed specific methods for each model and each outcome used at each of the screening sites have been published. 17,18 This methods section represents a brief summary of our approach. All sites used adult male Sprague Dawley rats ( g), cared for in accordance with each site s Institutional Animal Care and Use Committee, the United States Army (ACURO), and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats were housed in temperaturecontrolled rooms (22 C) with a 12-h light/dark cycle and given access to food and water ad libitum, except as noted in the methods. Animal Models FPI Model University of Miami School of Medicine Rats were anesthetized (70% N 2 O/30% O 2,1 3% isoflurane) 24 h before injury and surgically prepared for parasagittal FPI. A right craniotomy was performed and a plastic injury tube was placed over the exposed dura. The scalp was sutured closed and rats returned to their cages. After fasting overnight, rats were anesthetized, and tail artery and jugular vein catheters were placed. Rats were then intubated and subjected to a moderate FPI. Blood gas levels were measured from arterial samples 15 min before and 30 min after FPI. Fluid percussion injury served as our model to assess the effects of therapies on hemodynamics and blood gases. These early time points for assessment provided baseline information and data describing the impact of TBI, along with data on the acute effect of therapies, depending on the details of the timing of drug administration which varied across therapies (in an attempt to mimic the most promising published studies for each therapy). After TBI, the rats were returned to their cages with food and water ad libitum. Sham rats underwent all procedures except for the FPI. CCI Model University of Pittsburgh School of Medicine Rats were anesthetized (2 4% isoflurane in 2:1 N 2 O:O 2 ), intubated, and placed in a stereotaxic frame. A parasagittal craniotomy was performed and rats were impacted with the CCI device (Pittsburgh Precision Instruments, Inc.) at a depth of 2.6 mm at 4 m/s. The scalp was sutured closed and rats were returned to their cages. Shams underwent all procedures except for the CCI. PBBI Model WRAIR PBBI was performed as previously described. 18 Isofluraneanesthetized rats were placed in a stereotaxic device for insertion of the PBBI probe into the right frontal cortex at a depth of 1.2 cm. The pulse generator was activated and the elliptical balloon was inflated to a volume equal to 10% of the total brain volume. After probe withdrawal, the craniotomy was sealed with sterile bone wax and wounds were closed. Sham rats underwent all procedures except for the PBBI probe insertion. Biomarker Serum Sample Preparation Blood samples (0.7 ml) were collected at 4 and 24 h after TBI and on day 21 before perfusion for histological analysis. Blood withdrawals for the FPI and PBBI models were taken from an indwelling jugular catheter at 4 and 24 h after TBI and via tail vein at identical time points after CCI. At the terminal endpoint (21 d), blood samples were taken via cardiac puncture. Blood was prepared for serum in FPI and PBBI and plasma in CCI. 18,23 Samples were shipped via FedEx overnight (on dry ice) to Banyan Biomarkers, Inc., for analysis of biomarker levels. Outcomes The approaches to outcome testing, scoring, and specific outcome methods and metrics have been previously described ,23 MILITARY MEDICINE, Vol. 183, March/April Supplement

4 Our outcomes include (1) sensorimotor, (2) cognition, (3) neuropathology, and (4) biomarkers. Sensorimotor Methods FPI Model. The spontaneous forelimb or cylinder test was used to determine forelimb asymmetry. The gridwalk task was used as well to determine fore- and hind limb sensorimotor integration. Assessments occurred on post-injury day 7. CCI Model. Two sensorimotor tests, the beam balance and the beam-walking tasks, were used on the first five consecutive days after CCI. PBBI Model. A modified neuro exam was employed to evaluate rats at 15 min, 1, 7, 14, and 21 d post-injury. Further motor coordination and balance assessments utilized the fixed-speed rotarod task on days 7 and 10 post-injury. Cognitive Testing All sites used the Morris water maze (MWM) to assess cognition. Spatial learning was assessed over approximately d post-injury. Primary outcomes included path latency (all sites), pathlength (only FPI), and thigmotaxis (only PBBI). Probe trial was performed at all sites to gauge retention of platform location after its removal. The Miami site also tested working memory on days 20 and 21, and both the Pittsburgh and WRAIR sites employed a visible platform task on days Detailed descriptions of cognitive testing were previously provided. 18 Histopathology After behavioral testing, rats were anesthetized and perfused with 4% paraformaldehyde (FPI and PBBI) or 10% phosphate buffered formalin (CCI). Brains were processed for paraffin embedding or frozen sectioning. Coronal slices were stained with hematoxylin and eosin (H&E) for lesion volume (all sites) and cortical (FPI) or hemispheric (CCI and PBBI) tissue volume as previously described. 18 Both lesion volume and tissue volume loss were expressed as a percent of the contralateral ( non-injured ) hemisphere (CCI and PBBI) or as a percent of the contralateral cortex (FPI). In FPI, lesion volume and tissue volume loss were expressed as a percent of the contralateral cortex given the small lesion size and established standard protocol in Miami. Biomarker Assessments Blood levels of neuronal and glial biomarkers UCH-L1 and GFAP were measured by enzyme-linked immunosorbent assay (ELISA) at 4 and 24h after injury. 17,23 Primary outcome metrics for the biomarkers consisted of evaluating (1) the effect of drug treatment on blood biomarker levels at 24 h post-injury and (2) the effect of drug treatment on the difference between 24 and 4 h (delta 24 4 h) levels. We chose these two outcomes for specific reasons: 24 h post-injury represents an optimal time window for evaluating any substantial effects of a drug on biomarker levels. On the other hand, the delta 24 4 h accounts for the initial severity of injury while allowing each rat to serve as its own control. GFAP and UCH-L1 levels at 1 h after TBI were also assessed as an exploratory method (based on prior work by the OBTT consortium) to determine if the performance of UCH-L1 was further optimized with earlier sampling given its short half-life in blood. The results of these exploratory 1-h sampling assessments are not part of the OBTT scoring matrix, were carried out for future potential investigations, and are thus not reported in this manuscript. Similarly, the results of the 21-d sampling for biomarkers represent findings targeting study of the potential use of biomarkers to identify chronic markers for TBI and define long-term therapeutic effects and are outside of the scope of this report. OBTT Outcome Scoring Matrix To determine therapeutic efficacy across models, a scoring matrix summarizing all of the primary outcome metrics (sensorimotor, cognition, neuropathology [lesion volume and tissue volume], and biomarker [24 h and delta 24 4h] assessments) was developed. A maximum of 22 points for each model could be achieved (Table I). Given its importance to recovery of independent function in humans, cognitive outcome was weighted the highest in each of the models. Details of the OBTT scoring matrix are published. 17,18 Therapies Selected and Treatment Regimens Table II outlines the first nine therapies selected for testing by OBTT. The approach to therapy selection was previously reported 16,17,24 but involves a detailed review of the literature for promising therapies, a secret ballot vote by the site PIs, and final selection at an annual meeting of OBTT investigators. Table II also provides a brief description of the dose, route of administration, and treatment protocol used for each therapy. The treatment regimen used was identical across all sites, was based on the best literature available, was modified as necessary by our team of pharmacists (SMP and PE), and when possible, utilized intravenous (IV) administration. Table II also provides a brief description of the mechanisms targeted by each therapy. Regarding therapy selection, in addition to choosing promising agents, a broad spectrum of potential mechanistic targets was sought in our initial studies to provide insight into mechanisms of secondary injury and/or repair that might deserve future testing. Statistical Analysis Physiological data, contusion and tissue volumes, and probe trial were analyzed using a one-way analysis of variance (ANOVA). One-way ANOVA or repeated measures ANOVA was used to analyze motor tasks depending on the specificities of the data collection. Repeated measures ANOVA was used to analyze data for the hidden platform and working memory tasks. Post hoc analysis, when appropriate, used the Student Newman 306 MILITARY MEDICINE, Vol. 183, March/April Supplement 2018

5 TABLE I. Scoring Matrix Used to Rank Therapies in Primary Screening in Operation Brain Trauma Therapy Drug Model Neuro Exam Motor Cognitive Neuropathology Serum Biomarker FPI None Cylinder (2) Gridwalk (2) Keuls or Tukey s test. Comparison of biomarker levels was performed using the Kruskal Wallis test followed by post hoc comparisons with Mann Whitney U and Bonferroni corrections. Delta 24 4 h biomarker levels in injured groups were calculated in each rat as the difference between 24 and 4 h biomarker concentrations. All statistical tests were two-tailed and p < 0.05 was considered significant. Statistical analysis was carried out using Statistical Analysis Software (SAS, version 9.2) or Sigmaplot (version 11.0; Systat Software, Inc.). RESULTS For primary screening, in general, each model used rats per group. For most drugs, four groups were studied (sham, TBI plus vehicle, TBI plus treatment dose 1, and TBI plus treatment dose 2). Three groups were studied when only one dose was tested, such as with therapies where continuous infusion was needed (e.g., glibenclamide, AER-271). This resulted in rats studied for seven of the initial nine drugs tested in primary screening across the full OBTT consortium and Hidden platform latency (2) Hidden platform pathlength (2) MWM probe (2) Working memory latency (2) Working memory pathlength (2) Lesion volume (2) Cortical volume (2) GFAP UCH-L1 FPI total N/A FPI Dose 1 Dose 2 CCI None Beam balance (2) Beam walk (2) Hidden platform latency (5) MWM probe (5) Lesion volume (2) Hemispheric volume (2) GFAP UCH-L1 CCI total N/A CCI Dose 1 Dose 2 WRAIR Neuroscore Rotarod (3) Hidden platform latency (5) MWM probe (3) Thigmotaxis (2) Lesion volume (2) Hemispheric volume (2) GFAP UCH-L1 PBBI total PBBI Dose 1 Dose 2 Grand total Dose 1 Dose 2 (), point value for each outcome within each model; MWM, Morris water maze; WRAIR, Walter Reed Army Institute of Research; GFAP, glial fibrillary acidic protein, UCH-L1, ubiquitin carboxy-terminal hydrolase-l1; Δ, delta; N/A, not applicable. rats studied each to evaluate glibenclamide and AER-271 in primary screening across the OBTT consortium. This manuscript thus reports findings on therapy screening using >1,000 rats. Figure 2 shows the results of the scoring matrix from the first nine therapies tested by OBTT. Results for the initial five therapies are final 24 whereas those from drugs 6 9 are preliminary, pending final review and biomarker data. The two most positive therapies tested thus far by OBTT are levetiracetam (which showed benefit in FPI and CCI at two different doses) andglibenclamide(which showedbenefit specifically in CCI). Details on the findings for each therapy are provided below. Nicotinamide. Overall, acute treatment with nicotinamide exhibited slightly deleterious effects at low dose (50 mg/kg, IV), generating 4 points, and slightly beneficial effects at high dose (500 mg/kg, IV), generating +4 points, with the positive points for benefit on motor function and a reduction in hemispheric tissue loss in CCI, and a reduction in serum UCH-L1 in PBBI. 18 However, overall effects were modest even in the model with the most benefit for this agent (CCI). MILITARY MEDICINE, Vol. 183, March/April Supplement

6 TABLE II. Therapy Selection in Operation Brain Trauma Therapy Therapy Treatment Regimen Mechanistic Rationale and/or Therapeutic Targets 1. Nicotinamide 50 or 500 mg/kg IV at 15 min and 24 h after injury 1. PARP 2. Oxidative stress 3. NADPH depletion 4. HMGCo-A reductase 5. Neuroinflammation 2. Erythropoietin 5000 or 10,000 IU/kg IV at 15 min after injury 1. Apoptosis 2. Inflammation 3. Neurogenesis 4. Angiogenesis 3. Cyclosporin-A 10 or 20 mg/kg IV at 15 min and 24 h after injury 1. Mitochondrial Permeability Transition 2. Calcineurin 3. Inflammation 4. Simvastatin 1 or 5 mg/kg oral gavage first dose at 3 h after injury and then daily for 14 d 1. Cell death 2. CBF 3. Trophic factors 5. Levetiracetam 54 or 170 mg/kg IV at 15 min after injury 1. Excitotoxicity 2. Seizures 3. SV2A 4. GABA potentiation 6. Glibenclamide 10 μg/kg IP at 10 min after injury followed by SQ infusion of 200 ng/h for 7 d by Alzet pump 1. Sulfonylurea receptor-1 2. Cerebral edema 3. MMP-9 4. Inflammasome 7. Kollidon-VA or 0.8 g/kg IV over 5 min at 15 min after injury 1. Cell membrane re-sealing 2. Blood brain barrier 8. AER mg/kg IV at 15 min after injury (administered over 30 min), followed immediately by a continuous IV infusion of 1 mg/kg/h for 48 h. 1. Aquaporin-4 antagonist 9. Amantadine 10 or 45 mg/kg IP daily for 21 d; dosing began on day 1 after TBI and was given daily at 15 min after testing EPO. Overall, acute treatment with EPO at low (5,000 IU/ kg, IV) dose generated somewhat deleterious effects ( 5 points) and at high (10,000 IU/kg, IV) dose generated negligible effect (a net 1 point). Deleterious effects were seen at both doses on motor function in CCI ( 2.5), and at low dose on lesion volume and GFAP levels ( 2.5) in PBBI. 19 Cyclosporin-A (CsA). Overall, acute treatment with CsA produced modest detrimental effects at both doses ( 1 at low [10 mg/kg, IV] and 5 at high [20 mg/kg, IV]). However, careful examination of the findings revealed that the effects were highly model-dependent. In the mildest model, FPI, low-dose CsA produced modest beneficial effects on motor function and lesion volume, whereas in the more severe CCI model, high-dose CsA produced deleterious effects on aspects of motor function, cognitive function, and on GFAP levels ( 4.5 points). Remarkably, CsA was toxic in the highly severe PBBI model and, surprisingly, administration either drug or vehicle were associated with ~33% mortality Dopamine augmentation 2. Partial NMDA receptor antagonist IV, intravenous; PARP, poly-adp ribose polymerase; NADPH, nicotinamide adenine dinucleotide phosphate; HMGCo-A, 3-hydroxy-3-methyl-glutarylcoenzyme A; CBF, cerebral blood flow; SV2A, synaptic vesicle protein 2A; GABA, γ-aminobutyric acid; SQ, subcutaneous; MMP-9, matrix metalloproteinase-9; AER, aeromics; NMDA, N-methyl-D-aspartate. Simvastatin. Despite a 14-d treatment regimen, based on the previous literature (reviewed in 21) and to target both acute and subacute mechanisms, simvastatin had little effect on any outcome across the three models. Overall, low (1 mg/kg, oral gavage) or high dose (5 mg/kg, oral gavage) produced limited effects in any model ( 2.5 points for low and +1.5 points for high). Levetiracetam. Overall, single-dose administration of the anti-convulsant and anti-excitotoxic agent levetiracetam at 15 min after injury showed positive effects in all the three models, generating a net + 10 points at both low (54 mg/kg, IV) and high (170 mg/kg, IV) doses across OBTT. 22 Beneficial effects were seen at both doses in FPI (+6 and +5 in low and high, respectively) and in CCI (+3.5 and +5, respectively), whereas effects in PBBI were modest (+0.5 only at low dose). Also, all of the benefit of levetiracetam in the FPI model was related to effects on cognitive function, and of the overall 20+ points generated by levetiracetam, 13.5 resulted from benefit on cognitive function in FPI and 308 MILITARY MEDICINE, Vol. 183, March/April Supplement 2018

7 Therapy 0 Nicotinamide low 4.0 Nicotinamide high EPO low 5.0 EPO high 1.0 CsA low CsA high 5.0 Simvastatin low 2.5 Simvastatin high Levitiracetam low Levitiracetam high Glibenclamide* VA64 low* VA64 high* -2.0 AER-271* 4.0 Amantadine low* Amantadine high* 4.5 Points generated in the OBTT scoring matrix by therapies tested thus far across the three rat models Note that scores for drugs 6-9 (*) are preliminary pending final data quality control and review and biomarker findings FIGURE 2. Current status of scoring and ranking of therapies tested in primary screening in OBTT. To date, nine therapies have been studied across the consortium. Treatment groups are shown on the y-axis and points awarded (green positive, red negative) across the three models generated via the OBTT scoring matrix (see Table I) are shown on the x-axis. Asterisk designates preliminary findings. The two most positive therapies tested thus far are levetiracetam (which showed benefit in FPI and CCI at two different doses) and glibenclamide (which showed benefit specifically in CCI). Note that the data in this figure reflect an approach to ranking of the therapies for a traditional clinical trial that would include all TBI phenotypes (i.e., seeking a highly robust therapy that shows efficacy across all the three models). Failure to show benefit across models, however, does not preclude potential utility of an agent in a precision medicine trial within a specific clinical TBI phenotype. Please see text for details. See the Results section for details on each therapy. EPO, erythropoietin; CsA, cyclosporin-a; VA64, Kollidon-VA64; AER, Aeromics. CCI. Levetiracetam was also unique in that it did not generate any negative points in any model, suggesting a high degree of safety. Glibenclamide. Results on glibenclamide, a drug touted to mitigate brain swelling and other secondary injury mechanisms early after injury, 29 are preliminary, but findings to date suggest a highly model-dependent benefit in CCI. Glibenclamide is the second highest scoring drug thus far in OBTT (+5.5 points overall). Only one dose (10 μg/kg, intraperitoneal [IP] given at 10 min as a loading dose followed by a 7-d infusion [200 ng/h]) was tested given the need for an osmotic infusion pump using a regimen based on the previous literature. 30 Most of its points were generated in CCI and reflected full points (+4) for benefit on motor function and +2.0 points for histology from a reduction in contusion volume. It also exhibited minor benefit on motor function in FPI. Of the nine tested in OBTT, it is the only drug that reduced contusion volume in CCI. However, histological benefit was seen only in CCI. Biomarker results are pending low-dose (0.4 g/kg IV at 15 min after injury) treatment produced +3.5 points, whereas high-dose (0.8 g/kg, IV) treatment produced 2.0 points. Specifically, low-dose treatment produced some benefit on motor and cognitive function in CCI, whereas high-dose treatment produced modest deleterious effects on motor function in FPI. AER-271. As with glibenclamide, we tested a single continuous infusion dosing regimen for the novel aquaporin-4 antagonist AER-271. Dosing of this novel, proprietary agent was based on the assessment of drug levels generated in pilot studies. 32 A 2.5 mg/kg IV loading dose was given at 15 min after injury (over 30 min), followed immediately by a continuous IV infusion of 1 mg/kg/h for 48 h. Overall, treatment with AER-271 produced no benefit on behavior in any model ( 4.0 points in PBBI) and no effect on histology in any of the models, although a trend toward reduced contusion volume was seen in CCI (p = 0.06). The overall score was 4.0 points. Biomarker results are again pending. Kollidon-VA64. Overall, acute treatment with this novel agent, purported to have beneficial effects on blood brain barrier function and cellular integrity by a cell membrane resealing effect, 31 has produced only modest effects. Based on the assessment of behavioral and histological outcomes, Amantadine. Similar to simvastatin, given the putative benefit of amantadine used as a chronic therapy in TBI rehabilitation, and given the approaches taken in previous pre-clinical studies, we used regimens that included administration of either 10 (low dose) or 45 (high dose) mg/kg IP daily for MILITARY MEDICINE, Vol. 183, March/April Supplement

8 21 d. 33,34 Treatment began on day 1 after TBI and was given at 15 min after testing each day to minimize any impact on performance of the behavioral tasks. Overall, at low dose, amantadine treatment produced 0 net points and at high dose produced 4.5 points. However, similar to CsA and glibenclamide, amantadine produced remarkably model-dependent effects across OBTT, although with a unique distribution of effects across models. Despite deleterious effects on cognitive function in FPI at high dose ( 6 points), high-dose amantadine produced +1.5 points in PBBI, with benefit on motor function and a trend (p = 0.089) toward improved histology. DISCUSSION The work of OBTT has been highly informative to the field of TBI and its findings have relevance to both the pre-clinical and the clinical arenas. First, our groundbreaking approach comparingtbimodelshasrevealedthatmodelsroutinelyusedtotest therapies in pre-clinical studies produce unique functional, histological, and biomarker profiles, and, in many cases, different responses to therapy. This may be highly desirable given the aforementioned broad clinical spectrum of TBI, particularly the wide variety of pathoanatomical phenotypes. Although none of the first nine therapies tested by OBTT produced robust benefit across models (at least using the treatment regimens that we tested), there still may be merit in attempting to identify a highly potent magic bullet to test in a conventional RCT across all phenotypes. Many additional candidates remain to be evaluated. Nevertheless, our approach has revealed important model dependence to therapeutic efficacy that suggests that a precision medicine approach to TBI trials and/or combination therapy should be strongly considered, particularly in the setting of severeandmoderatetoseveretbiasmodeledbyobtt. We identified levetiracetam, given as a single high dose early after TBI, as the most promising agent identified thus far by OBTT. 22,24 It has been advanced to secondary screening within the OBTT consortium in the aforementioned mild midline FPI model in micro pigs to determine if it shows efficacy across species and injury severity levels. Operation brain trauma therapy is convinced that levetiracetam merits additional testing in other models, and using other dosing regimens, given the fact that it both outperformed all other therapies to date and demonstrated a substantial safety profile. It was surprising that single-dose administration of this agent early after TBI produced such substantial long-term benefit; however, our approach replicated the published literature. 35 It may also be significant that levetiracetam represents a therapy that was specifically designed as a neurotherapeutic and is used by many centers as an anti-convulsant in the routine management of severe TBI. However, it has not been subjected to a clinical trial as a neuroprotective agent in TBI at any injury severity level. Given the possibility that the benefit might not simply result from anti-convulsant effects, based on our findings, clinical trials might be warranted. Neurotherapeutics useful in other diseases should be evaluated by OBTT and in multiple TBI models. However, it is clear that even levetiracetam demonstrated only moderate success in OBTT. Indeed, although the most promising agent tested, it generated only 10 of a possible 66 points at either dose. Nevertheless, defining a more successful single agent demonstrating efficacy across models remains a key goal of OBTT. Note that the data in Figure 2 reflect an approach to ranking of the therapies for a traditional clinical trial that would include all TBI phenotypes (i.e., seeking a highly robust therapy that shows efficacy across all three models). Failure to show benefit across models, however, does not preclude potential utility of an agent in a precision medicine trial within a specific clinical TBI phenotype. Specifically, our findings with three of the nine therapies suggest that a precision medicine approach to clinical trials in severe TBI may represent a logical strategy. For example, glibenclamide exhibited benefit specifically in contusion, and given both its suggested benefit on the development of brain edema in pre-clinical studies 29 and its promising performance in a clinical trial in stroke 36 and as a brain edema biomarker, 37 testing its efficacy in patients with contusion would be logical. And additional studies in contusion models are warranted. Similarly, potential benefit of amantadine in penetrating brain injury in our preliminary analysis suggests that such an approach would be a logical candidate for a clinical trial. Amantadine has shown some level of efficacy in the subacute period in facilitating the emergence of patients from coma after TBI, 38 and the setting of penetrating brain injury may represent one where rehabilitation-related strategies have special efficacy. 39,40 In any case, we believe that the findings of OBTT suggest that TBI phenotype-based clinical trials could be modeled in pre-clinical studies and that such an approach merits targeted development for future successful drug development in TBI. The findings of OBTT appear to also provide insight into the utility of the pre-clinical literature in guiding clinical trials in severe TBI. OBTT uses a highly rigorous standardized approach that likely exceeds that taken in individual laboratories. Its findings, thus, may not be surprising given the previous reports of failed reproducibility of many studies in the preclinical literature in cancer research. 14,15 However, we strongly believe that OBTT does not represent a bully pulpit and that its findings should not preclude further study of agents shown to lack efficacy in our trials. There are limitations to the screening approach used in OBTT including limited assessment of dose response, lack of studies of successful mechanistic targeting, lack of plasma and tissue drug concentration assessment in some cases, use of a single level of injury severity, and others. Nevertheless, the cross-model screening of therapies using an identical treatment regimen is unique and valuable. We believe that our findings should be viewed as providing clues rather than definitive answers, for the development of RCTs. Our findings also suggest that there is value to cross-model testing using the highest possible level of rigor, and reproducibility, rather than focusing solely on innovation, which arguably has 310 MILITARY MEDICINE, Vol. 183, March/April Supplement 2018

9 dominated pre-clinical research. Our findings also suggest that combination therapy should be pursued in future pre-clinical studies by OBTT, given that even the most successful individual therapies screened (levetiracetam and glibenclamide) produced only moderate or limited benefit. 41 Our data also suggest a possible new use for serum biomarkers in pre-clinical drug screening along with theranostic potential of biomarkers in clinical trials. 23,24 Although not the focus of this report, it would be remiss to overlook the fact that previous reports from OBTT have shown impressive capability of early (4 24 h) assessments of blood-based biomarker levels to serve as surrogate endpoints for predicting long-term behavioral and histological outcome along with theranostic utility in drug testing. For example, 4 and 24 h post-tbi assessment of GFAP in blood identified therapeutic benefit of levetiracetam on tissue preservation at 21 d. 22 Finally, the OBTT approach may represent a model for therapy and biomarker development well beyond its current formulation. For example, secondary insults greatly impact TBI outcomes, and TBI resuscitation may require unique therapies. A consortium testing resuscitation fluids, for example, across multiple models could help guide clinical trials in that area. Similarly, OBTT-like approaches may be meritorious for mild TBI, the use of chronic therapy to break the link between TBI and either long-term neurodegeneration or post-traumatic stress disorder, and for comparisons of therapeutic efficacy across age or sex among other confounders. Lastly, we are also testing the usefulness of two promising additional TBI biomarkers that track axonal injury (i.e., phospo-neurofilament-h [pnf-h]) and neurodegeneration/tauopathy (i.e., Tau and phospho-tau) in our multiple animal model drug screening paradigm pnf-h could serve as a target engagement biomarker that might allow blood sampling in clinical trials to provide theranostic information and help guide drug dosing, for example, for a drug specifically targeting axonal injury. The success of such an approach could have an important impact on future clinical trial in TBI. CONCLUSIONS Operation brain trauma therapy is a groundbreaking development for the field that has only scratched the surface of its potential. There is, in our opinion, a genuine need for rigorous pre-clinical multi-center studies. Consortia that represent civilian DoD partnerships, such as OBTT, could have a tremendous impact on successful translational therapy and biomarker development in the field of TBI. CONFLICT OF INTEREST Dr Hayes owns stock and is an officer of Banyan Biomarkers Inc. Dr Hayes is an employee and receives salary and stock options from Banyan Biomarkers Inc. Dr Wang is a former employee of Banyan Biomarkers Inc. and owns stock. Drs Hayes and Wang also receive royalties from licensing fees and as such all of these individuals may benefit financially as a result of the outcomes of this research or work reported in this publication. There are no other disclosures to report. ACKNOWLEDGMENTS We are grateful to the Department of Defense grants W81XWH and WH81XWH for generous support. We thank COL Dallas Hack for his support and input in helping to launch our consortium, and COL Todd Rasmussen for ongoing support. We thank Drs Tammy Crowder and Kenneth Curley for support, scientific input, and contributions to the project. We thank Dr Brenda Bart-Knauer for her support and administrative assistance. We thank Drs Ronald Poropatich and Janice Gilsdorf for helpful suggestions. We thank Linda Ryan for support with budgetary issues, Fran Mistrick for other administrative and coordinating support, Marci Provins and Natalie Nieman for assistance with manuscript preparation, and Vincent Vagni for assistance with figure preparation. We thank Rebecca Pedersen, Justin Sun, Ofelia Furones-Alonso, Milton Martinez, Juliana Sanchez- Molano, William Moreno, Ryan Treu, Jessie Truettner, Hong Q. Yan, PhD, Michelle Ma, Jeremy Henchir, and Keri Feldman for outstanding technical support in the individual TBI models across the consortium. We thank Ross Bullock, MD, PhD, Gary Fiskum, PhD, Leonard Miller, PhD, Raj Narayan, MD, David Okonkwo, MD, PhD, and Amy Wagner, MD, who have served as members of the external advisory board of OBTT, for helpful input in the development of the consortium and for initial input on therapy selection. 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