Patrick D. Lyden - US grants

Stroke is the third leading cause of death in the United States and the primary cause of neurologic morbidity. Vascular causes of dementia are the second most common cause of dementia in elderly Americans. No specific therapy currently exists for either stroke or cerebral vascular dementia. The failure to develop effective therapy for these diseases may be partially the result of the absence of a reproducible animal model of this type of cerebral vascular disease. Recently, we have developed new animal models that allow for the rapid and inexpensive testing of new drugs that may be effective in the treatment of cerebral vascular disease. We propose to extend our previous observations by refining a method for the quantitative analysis of the histopathologic consequences of multifocal cerebral ischemia. We will use the quantitative methods to assess the effect of drug treatment on histopathologic consequences of cerebral ischemia. Finally, we propose to quantify the effect of multifocal cerebral ischemia on behavior (learning). We hypothesize that learning behavior will change in a measurable way depending on the topographic distributions of lesions following ischemia and that these observations will be altered by pharmacologic therapy. These methods will allow us to screen new drugs for their potential benefit in the treatment of victims of stroke or vascular dementia, and will provide insights into the mechanisms underlying the dementia apparent in many patients with vascular lesions.

The specific objectives of this contract are to establish the validity of current documentation of observed early initial response treatment of acute ischemic stroke with recombinant tissue plasminogen activator (rt- PA) and to establish time limits for delivering such treatment. The objectives will be accomplished by completing a Phase II-B randomized controlled clinical study of rt-PA for acute ischemic stroke.

Replicate findings of a previous phase II trial in which stroke patients treated with citicoline for six weeks appeared to attain independent living status more frequently than patients treated with placebo.

human therapy evaluation; stroke; cardiovascular disorder chemotherapy; drug tolerance; drug screening /evaluation; drug adverse effect; intravenous administration; acute disease /disorder; pharmacokinetics; clinical research; human subject;

Determine safety, tolerability and efficacy of aptiganel hydrochloride. Compare the efficacy of 2 doses of intravenous aptiganel hydrochloride with that of placebo in patients with acute ischemic stroke and to characterize pharmacokinetic parameters of aptiganel hydrochloride.

Study safety and tolerability of GV150526 as treatment for stroke by evaluating clinical assessments, adverse events, and laboratory and pharmacokinetic tests.

Study the safety and tolerability of GV150526 as treatment for stroke by evaluating clinical assessments, adverse events, and laboratory and pharmacokinetic tests.

Among putative neuroprotection strategies, hypothermia has long been recognized as the most potent. Recent insights in understanding ischemia and reperfusion suggest that hypothermia may be an ideal modality for extending the stroke therapy time window. Although difficulties cooling patients limited testing of this potentially effective therapy, recent developments in technology allow us to mount a Phase 1 clinical trial of intravascular cooling for patients presenting beyond 3 hours. The purpose of this study is to confirm the performance of an endovascular cooling device and to establish the parameters for a larger clinical study, such as sample size and indices of measurement. Preliminary effectiveness of hypothermia treatment in combination with thrombolysis for stroke will also be evaluated in this limited patient population. To do this, we will address 2 specific aims: 1) Characterize the safety profile of hypothermia in stroke patients who initiate cooling between 3 and 6 hours after stroke. Substantial pre-clinical data supports the hypothesis that the window for effective hypothermia may be longer that 3 hours. We will establish safety of the cooling device, and collect outcome data to be used for estimating sample size in larger, Phase 2 trials. 2) Establish the safety of thrombolysis and hypothermia used together between 3 to 6 hours after stroke onset. Thrombolysis trials failed to demonstrate benefit to patients treated later that 3 hours following stroke onset. In each trial, however, there was a trend toward benefit, and meta-analyses suggest that there is a positive, but small effect in later treated patients. We hypothesize that hypothermia, combined with thrombolysis, may prove effective in such patients. We will establish the safety of invasive, endovascular-cooling catheter in combination with thrombolysis. We will collect safety and outcomes data to aid in the design of larger, controlled Phase 2 trial.

The Stroke Team is the engine of the UCSD Stroke Center and the SPOTRIAS proposal, organized to respond immediately to acute stroke. The UCSD Stroke Team is recognized intemationally: we consistently lead recruitment into acute intervention trials. We propose to expand the Team's availability and to show that patients can be treated within 2 hours of symptoms onset. This Core has 3 specific aims: 1) To respond to code stroke 24 hours per day, 7 days per week. To do this, we will continue our current team management, enhanced with SPOTRIAS sponsored research projects. 2) To treat 12 patients per year within 2 hours of stroke onset. Prior data collected here and elsewhere shows that patient enrollment times tend to cluster near the stated upper limit for time to treatment. By moving the accepted 3-hour time window up to two hours, we should obtain even better response rates than expected from typical t-PA use. Further, the procedures required to treat patients within a 2-hour window will have collateral benefits to other patients and SPOTRIAS research trials. 3) To Collect, Review, and use QA data to improve delivery. Our pilot data shows that we can document several important Stroke Code indicators, including door-to-physician, door-to CT scan, door-to-Neurologist, and door-to-needle times. We will use the QA data collected at UCSD and at other hospitals in our network to identify barriers to 60 minute door-to-needle times. This will be coordinated with othere SPOTRIAS centers nationally to assure coherence.

[unreadable] DESCRIPTION (provided by applicant): Although peptide angiogenic signals may promote new vessel formation, may offer neuroprotection, and may facilitate neuronal migration during recovery, our pilot data suggests a different function after ischemia: the brain may utilize angiogenic signaling only to subserve removal of necrotic debris, i.e., "clean-up". Our central hypothesis is that after ischemia, angiogenic growth factors are secreted to open blood brain barrier, stimulate macrophage infiltration, and to create vascular channels for removal of necrotic debris. Our aims are: we will measure capillary and neuronal density 30 days after focal brain ischemia to establish whether ischemia stimulates persisting microvessels, preserves neurons, or both. Then we will determine if microvessel or neuronal densities can be augmented with intra-arterial infusions of VEGF or bFGF or both. Do Macrophages Influence the Growth of New Microvessls? We will block macrophage entry into the brain by depleting them (whole body irradiation) or inhibiting them (colchicine/chloroquine), and expect to see a marked reduction of both macrophages and microvessels near the ischemic zone. We will increase macrophage entry into the ischemic zone with tissue necrosis factor, macrophage inflammatory protein-I, or monocyte chemoattractant protein-1 and expect to find more microvessels and macrophages. We will inhibit VEGF activity immediately after ischemia with anti-VEGFR antibody, a VEGFR-Fc fusion protein, and a tyrosine kinase inhibitor specific for VEGFR. Does Angiopoietin-2 Signal Microvessel Degradation? We will provide VEGF beginning 10 days after stroke and continuing to 17 days after stroke, to blunt the degradation signal; we predict that microvessels will persist. We will administer a TIE-2 receptor-Fc binding protein from Day 10 to 17 after stroke to bind and remove TIE-2 ligands (especially Ang-2), again predicting that microvessels will persist. Does angiogenic signaling ameliorate cognitive deficit after stroke? Using a bioassay suited to studying pharmacological synergism, we will study protective effects of VEGF, bFGF, or both using a bioassay and a spatial navigation test.

We propose a Specialized Program of Translational Research in Acute Stroke (SPOTRIAS) at the University of California, San Diego, Stroke Center (UCSD). The Core facilities and projects will meet SPOTRIAS target goals, increasing the number of thrombolytic-treated patients, particularly those treated within 2 hours following stroke. To achieve this, we offer 4 inter-related projects. 1) To find a simple, serum marker of acute stroke, we will characterize the free fatty acid (FFA) response in acute stroke patients, including the time course. We will determine whether FFA levels correlate with various stroke outcomes, and attempt to distinguish ischemia from common mimics. We will characterize the FFA response associated with effective thrombolysis. 2) To use hypotherrnia to prolong the time window for thrombolysis in a Phase 1 trial, by studying thrombolysis beginning 3 to 6 hours following stroke onset, combined with 24 hours of intravascular cooling. We hypothesize that hypothermia will be safe in such patients. We will proceed to a Phase 2 trial to show that hypothermia reduces hemorrhage rates and improves outcomes in 3 to 6 hour thrombolysis patients. 3) To use a non-invasive, bedside test---contrast enhanced transcranial ultrasoundmto identify vascular occlusion and perfusion defects before and after thrombolysis. We will test whether ultrasound may separate stroke from mimics, and identify potential treatment candidates from among patients with mild or fluctuating deficits presenting within 2 hours. 4) To determine whether novel broadband wireless Internet technology will allow UCSD Stroke Team faculty to direct thrombolysis at remote hospitals by placing audio/video consultation capability at remote Emergency Departments in San Diego. Acute consults may increase the number of 2-hour thrombolytic treatments. These projects are built on the core facilities of the UCSD Stroke Center, which include a nationally recognized stroke team, a training program, a data collection and statistical analysis unit, and a specimen collection and storage facility. All of the needed teams, collaborations, and facilities are in place. The SPOTRIAS program will allow us to enhance and initiate translational research that ultimately will benefit stroke patients by treating more patients under 2 hours, and finding ways to treat additional patients later.

DESCRIPTION (provided by applicant): Although peptide angiogenic signals may promote new vessel formation, may offer neuroprotection, and may facilitate neuronal migration during recovery, our pilot data suggests a different function after ischemia: the brain may utilize angiogenic signaling only to subserve removal of necrotic debris, i.e., "clean-up". Our central hypothesis is that after ischemia, angiogenic growth factors are secreted to open blood brain barrier, stimulate macrophage infiltration, and to create vascular channels for removal of necrotic debris. Our aims are: we will measure capillary and neuronal density 30 days after focal brain ischemia to establish whether ischemia stimulates persisting microvessels, preserves neurons, or both. Then we will determine if microvessel or neuronal densities can be augmented with intra-arterial infusions of VEGF or bFGF or both. Do Macrophages Influence the Growth of New Microvessls? We will block macrophage entry into the brain by depleting them (whole body irradiation) or inhibiting them (colchicine/chloroquine), and expect to see a marked reduction of both macrophages and microvessels near the ischemic zone. We will increase macrophage entry into the ischemic zone with tissue necrosis factor, macrophage inflammatory protein-I, or monocyte chemoattractant protein-1 and expect to find more microvessels and macrophages. We will inhibit VEGF activity immediately after ischemia with anti-VEGFR antibody, a VEGFR-Fc fusion protein, and a tyrosine kinase inhibitor specific for VEGFR. Does Angiopoietin-2 Signal Microvessel Degradation? We will provide VEGF beginning 10 days after stroke and continuing to 17 days after stroke, to blunt the degradation signal; we predict that microvessels will persist. We will administer a TIE-2 receptor-Fc binding protein from Day 10 to 17 after stroke to bind and remove TIE-2 ligands (especially Ang-2), again predicting that microvessels will persist. Does angiogenic signaling ameliorate cognitive deficit after stroke? Using a bioassay suited to studying pharmacological synergism, we will study protective effects of VEGF, bFGF, or both using a bioassay and a spatial navigation test.

DESCRIPTION (provided by applicant): Blood brain barrier breakdown is a common element in many neurological diseases; an extensive literature demonstrates temporal and/or spatial associations between the inflammatory processes that underly blood brain barrier breakdown and subsequent death of neural tissue (PAS-04-072). Further, recent longitudinal neuro-imaging studies in stroke patients indicate that early post-ischemic blood brain barrier breakdown correlates with an increased likelihood for hemorrhagic transformation and poor clinical outcome (Latour et al, Ann Neurol, 2004, in press). However, the temporal and spatial patterns of blood brain barrier break down are poorly characterized, and the underlying mechanisms that trigger these processes remain poorly understood. We propose to develop a new experimental method to quantify blood brain barrier breakdown after experimental stroke in the adult rat. The major goal of these studies is to develop quantitative methods to measure blood brain barrier breakdown and also preserve tissue morphology for correlative and quantitative immunocytochemical studies of tissue hypoxia, thrombosis and low blood flow. In this R21 developmental project, we will use a rat stroke model of reversible middle cerebral artery occlusion (MCAo) to develop measures of ischemic heterogeneity and test potential mechanisms underlying that heterogeneity. 1. Develop methods for the quantitation of local microvascular blood brain barrier break down. 2. Validate that areas of FITC-dextran blood brain barrier breakdown correlate with ischemic pathology by co-staining for markers of tissue hypoxia, vessel reactivity, and mircrothrombosis. 3. Validate that FITC-dextran blood brain barrier breakdown correlates with in vivo measures of low blood flow in microvessels.

The Stroke Team is the engine of the UCSD SPOTRIAS Coordinating Center, organized to respond immediately to acute stroke and identify candidates for all of the Center's translational research studies in acute stroke diagnosis and treatment. The Core has 3 specific aims: (1) to respond to code stroke 24 hours per day, 7 days per week; (2) to treat a minimum of 12 patients per year within 2 hours of stroke onset, on the premise that by moving the NINDS guidelines 3-hour time window for thrombolytic treatment up to 2 hours, even better patient response rates than expected from typical rt-PA use can be achieved; and (3) to collect, review, and use quality assurance data (e.g., door-to-physician, door-to-CT scan, door-to-neurologist, and door-to-needle times) to identify and overcome barriers to 60-minute door-to-needle times and thereby improve acute stroke care through expedited delivery. Key features of the Expedited Code Stroke Protocol developed and followed by the Stroke Team include a 2-hour onset-to-treatment benchmark, in-person triage of all Code Stroke calls, unmixed rt-PA at the Emergency Department bedside, proceeding without coagulation test results and chest X-ray unless specifically indicated, no delay for formal CT interpretation, and no delay for written consent. In addition to the study of expanded availability to and expedited evaluation and treatment of acute stroke patients, the Stroke Team collects and coordinates the deposit of blood specimens (Core B) from UCSD Stroke Center clinical sites to the NINDS Coriell DNA Blood Repository and into the SPOTRIAS national repository, a centralized project geared toward eventual exploration of blood markers for stroke, transient ischemic attack, and mimics. In this capacity, the Stroke Team is facilitating and participating in vital research on whether serum biomarkers can be used to triage acutely presenting patients into low-risk and high-risk stratification groups and to predict likelihood of certain outcomes after stroke. As both a Core resource of patient access and a study of expanded and expedited response to acute stroke in the neurocritical context, the Stroke Team is a daily contributor to translational research studies with the common, overarching goal of significantly reducing the staggering economic and social toll of acute stoke in the United States and worldwide through the development and testing of new, improved diagnostic tools and innovative treatments.

[unreadable] DESCRIPTION (provided by applicant): The Specialized Program of Translational Research in Acute Stroke (SPOTRIAS) at the University of California San Diego (UCSD) Stroke Center currently applies the resources of 4 Cores to conduct 3 interrelated clinical trials designed to meet the overarching SPOTRIAS goal of increasing the number of thrombolytic-treated patients. Over the course of the 5-year renewal period, we propose to move this agenda forward with 6 new clinical trials: (1) Intravascular Cooling in the Treatment of Stroke-Caffeinol (ICTuS-C), which builds on the ongoing hypothermia research track of ICTuS-L, evolving from demonstration of the feasibility and safety of intravascular cooling in combination with thrombolysis to collaborative assessment, with Houston SPOTRIAS, of the safety, feasibility, and early efficacy of the endovascular induction of hypothermia and caffeinol in acute ischemic stroke patients presenting within the 3-hour window; (2) Transcranial Ultrasound in Acute Stroke, which builds on the ongoing ultrasound imaging research track to study the potential of diagnostic ultrasound in combination with rt-PA and/or ultrasound contrast agent microbubbles on image-guided transcranial thrombolysis; (3) Stroke Team Remote Evaluation Using a Digital Observation Camera - Optimizing Practitioners' Use in Stroke (STRokE DOC OPUS), which builds on the ongoing audio-visual telemedicine research track to explore the utility of remote telemedicine consultations for acute stroke by determining whether our pioneering hub-and-spoke model can be replicated in other communities and whether next-generation, optimized technology will increase use of the STRokE DOC system in areas with limited access to Internet-based technology; (4) Counterpulsation to Upgrade Forward Flow in Stroke (CUFFS), a new pilot assessment of the tolerability, feasibility, and safety of noninvasive external counterpulsation (ECP) in acute ischemic stroke patients within 0-48 hours of symptom onset; (5) Safety of tPA plus Transcranial Emission of Low-Energy Lasers for Acute Stroke Recovery (StELLAR), the first combination study of the NeuroThera Laser System with rt-PA; and (6) Spot Sign for Predicting and Treating ICH Growth (STOP IT), a study of acute intracerebral hemorrhages to determine the sensitivity and specificity of CT angiography for the prediction of hematoma growth and to test the feasibility of CT angiography for the guidance of hemostatic therapy. Altogether, our SPOTRIAS proposal for the renewal period aims to integrate and synthesize further the considerable strengths of the UCSD Stroke Center in conducting translational research that ultimately benefits stroke victims within our geographical practice range and worldwide as we treat more patients under 2 hours and explore new ways to safely and effectively treat other, guideline-ineligible patients outside the 3-hour rt-PA window, including through improved patient selection and stratification and additional treatments combined with and potentially augmenting thrombolysis. [unreadable]

DESCRIPTION (provided by applicant): Although thrombolytic therapy for acute stroke continues to gain wider acceptance and usage, there remains a compelling need for neuroprotective therapy and new treatment to reduce thrombolytic complications. Thrombin is a serine protease that plays a critical role in the coagulation cascade. Thrombin induces protection at low doses (thrombin preconditioning) but acts as a neurotoxin at high doses, killing cells via the protease activated receptors (PARs). We now propose to test the hypothesis that thrombin partially mediates edema and cell death during stroke via the PAR-1 receptor signaling pathway. Using a protease-cleavable cell-penetrating probe developed by Dr. Roger Tsien, we will determine directly whether thrombin activation mediates edema and tissue injury. Using a highly reproducible and quantifiable model of ischemic edema and tissue injury, we will test a variety of pharmacological manipulations of coagulation or PAR-1 and its presumed signaling pathways. Using lentiviral mediated RNA interference, we will reduce gene expression of PAR-1 in focal areas of the parietal cortex of adult mice prior to standard MCAo and then determine the effect of PAR-1 knock-down on tissue injury. Using knock-out mice deficient in PAR-1, PAR-2, PAR-3, or PAR-4 subjected to MCAo, we will determine whether each or all PAR receptors influence vascular disruption and tissue injury after MCAo. Finally, we will extend our previous studies to include behavioral measures of cerebral injury and seek to determine whether thrombin exacerbates-and whether argatroban ameliorates-behavioral deficits after MCAo. We will use intra-arterial mutant thrombin designed to activate PAR-1 receptor but not affect coagulation during ischemia. We will also test neuroprotective drugs such as mutant APC or others screened for effect on stroke outcome. Taken together, these studies will reveal whether thrombin cytotoxicity plays a role in stroke and whether agents active at PAR-1 signaling pathway represent an effective therapy. Relevance The search for effective neuroprotectants is hampered by many factors: we continue to lack a full understanding of the molecular mechanisms of ischemic cell death and vascular disruption, as well as the patho-anatomic mechanisms of ischemic edema, infarction, and behavioral impairment. Fundamental exploration of molecular and patho- anatomic mechanisms AND translational studies of therapeutics remain highly significant and critical to understanding and treating stroke, the most common cause of adult disability in the world.

DESCRIPTION (provided by applicant): Stroke is the leading cause of adult disability in America, and one of the leading causes of death in the world. If patients arrive at a hospital within 3 hours of their stroke beginning, thrombolytic therapy can be very effective. Only 40 to 50% of patients respond to lytic treatment, however, creating a priority to develop new, effective treatments for stroke. The brain consists of multiple cell types: neurons, glia, and endothelial cells, among others; during stroke, the brain loses 1.2 million neurons per minute. In the past, candidate stroke treatments that failed to benefit patients were targeted only at neurons. We now propose to test a new drug that powerfully protects neurons, glia, and endothelial cells, together known as the neurovascular unit. This drug, 3K3A-APC, acts on cells partly via the PAR-1 receptor to induce protection by several mechanisms. Multiple laboratories- using neurobehavioral and histomorphometric endpoints in several animal models-have shown the drug powerfully reduces the effects of experimental stroke, even when administered up to 4 hours after the stroke begins. In human volunteers, only mild and moderate side effects were detected at clinically relevant doses. We now propose to test 3K3A-APC-for the first time-in stroke patients who arrive at the hospital very early and receive thrombolytic treatment. Very low doses will be tried at first, and then progressively higher doses will be tried. Ultimately, we wil determine the maximum tolerated dose (MTD), that is, the largest drug dose that can be given without causing severe side effects. By the end of this study, we hope to determine a dose that is safe and well tolerated by patients suffering acute stroke. Should this study succeed, the next step would be a much larger study to test for possible benefit of 3K3A-APC in stroke patients.

We propose a novel, adaptive, secured system for parallel testing of promising interventions designed to improve outcome compared to reperfusion alone with thrombolysis, thrombectomy or both. The applicant PI and collaborators together have decades of experience using preclinical animal stroke models. Also, and perhaps uniquely, the applicant PI and collaborators have led clinical trial coordinating centers for large and small multi?center and single center Phase 1, 2, and 3 clinical trials on behalf of the Federal Government and various industry partners. Thus, by combining decades of experience and expertise in animal modeling with clinical trial management this application affords NINDS an opportunity to significantly enhance the likelihood that SPAN will guide the selection of the best agent(s) to transition to future clinical trials likely to be conducted through StrokeNet. The applicant PI and collaborators are aware of, and have participated in, many reviews and symposia detailing the significant failures of previous preclinical stroke development. A plethora of putative neuroprotectants proceeded to clinical trial based on favorable preclinical assessment, only to fail in subsequent clinical trials of human stroke patients. The plethora of clinical failures has cost industry and governments hundreds of millions of dollars and wasted the time, talent, and effort of hundreds of investigators and coordinators. The recent successful development of thrombectomy for acute ischemic stroke generated considerable enthusiasm for re?testing compounds in combination with thrombectomy. Thus, SPAN is intended to screen and select highly promising candidate treatments for possible study in StrokeNet. A success in SPAN will provide a significant impetus to renew efforts toward successful clinical deployment of novel, promising neuroprotectants. In the past 5 years, two significant developments raise new hope for neuroprotection: the appearance of compounds with multiple mechanisms of action, and the promulgation of new standards for the rigorous preclinical development of stroke treatment candidates. The SPAN effort affords the highly significant opportunity to find a promising candidate treatment, test it in StrokeNet, and then back?validate the ideal preclinical testing paradigm that predicts success in clinical trials. This present application, if funded, will achieve significant improvement and advancement of preclinical development by implementing critical technical innovations. The state?of?the?art technical solution offered by our collaborator, the Laboratory of Neuroimaging (LONI) is secure, robust, reliable, and ready to implement immediately because Drs. Lyden and Toga collaborated during RHAPSODY to provide sites a turn?key solution for uploading images (www.LONI.usc.edu). We also propose the highly novel use of distributed, masked evaluation. This novel approach allows for a secure, blinded, cost efficient, with built in central quality?control.