Enhancing Enrollment in Acute Stroke Trials: Current State and Consensus Recommendations.

The Stroke Treatment Academic Industry Roundtable (STAIR) convened a session and workshop regarding enrollment in acute stroke trials during the STAIR XII meeting on March 22, 2023. This forum brought together stroke physicians and researchers, members of the National Institute of Neurological Disorders and Stroke, industry representatives, and members of the US Food and Drug Administration to discuss the current status and opportunities for improving enrollment in acute stroke trials. The workshop identified the most relevant issues impacting enrollment in acute stroke trials and addressed potential action items for each. Focus areas included emergency consent in the United States and other countries; careful consideration of eligibility criteria to maximize enrollment and representativeness; investigator, study coordinator, and pharmacist availability outside of business hours; trial enthusiasm/equipoise; site start-up including contractual issues; site champions; incorporation of study procedures into standard workflow as much as possible; centralized enrollment at remote sites by study teams using telemedicine; global trials; and coenrollment in trials when feasible. In conclusion, enrollment of participants is the lifeblood of acute stroke trials and is the rate-limiting step for testing an exciting array of new approaches to improve patient outcomes. In particular, efforts should be undertaken to broaden the medical community's understanding and implementation of emergency consent procedures and to adopt designs and processes that are easily incorporated into standard workflow and that improve trials' efficiencies and execution. Research and actions to improve enrollment in ongoing and future trials will improve stroke outcomes more broadly than any single therapy under consideration.

Defining a Target Population to Effectively Test a Neuroprotective Drug.

BACKGROUND AND PURPOSE: We aim to identify the subgroup of acute ischemic stroke patients with higher probabilities of benefiting from a potential neuroprotective drug using baseline outcome predictors and test whether different selection criteria strategies can improve detected treatment effect. METHODS: We analyzed the association between final infarct volume (FIV), measured on 24- to 72-hour computed tomography, and National Institutes of Health Stroke Scale at discharge/day 5 of acute stroke patients who underwent endovascular treatment. Models were adjusted for age, sex, and affected hemisphere. We analyzed the impact of absolute (5-15 mL) and relative (33%) FIV reductions in the National Institutes of Health Stroke Scale in the whole population and in different subsets of patients selected according to baseline imaging criteria using computed tomography perfusion. RESULTS: We analyzed 627 patients; association between FIV and 5-day National Institutes of Health Stroke Scale was best described with a quadratic function, with a regression coefficient ß=1.56 ([95% CI, 1.45-1.67] P<0.001) in the adjusted analysis. In the models considering a fixed absolute (5/15 mL) FIV reduction, treatment effect was highest when patients with predicted larger FIV were excluded, whereas in a 33% FIV reduction model, treatment effect increased with the exclusion of patients with expected excellent outcomes. CONCLUSIONS: Patients either with excellent outcomes after endovascular thrombectomy or with large infarcts may dilute the treatment effect in stroke neuroprotective drug trials. Computed tomography perfusion on admission may help selecting adequate patients according to expected drug effect profile.

Pharmacological Modulation of Neutrophil Extracellular Traps Reverses Thrombotic Stroke tPA (Tissue-Type Plasminogen Activator) Resistance.

Background and Purpose- Recanalization of the occluded artery is a primary goal in stroke treatment. Unfortunately, endovascular treatment is not always available, and tPA (tissue-type plasminogen activator) therapy is limited by its narrow therapeutic window; importantly, the rate of early arterial recanalization after tPA administration is low, especially for platelet-rich thrombi. The mechanisms for this tPA resistance are not well known. Since neutrophil extracellular traps (NETs) have been implicated in this setting, our aim was to study whether NET pharmacological modulation can reverse tPA resistance and the role of TLR4 (Toll-like receptor 4), previously related to NET formation, in thrombosis. Methods- To this goal, we have used a mouse photothrombotic stroke model, which produces a fibrin-free thrombus composed primarily of aggregated platelets and thrombi obtained from human stroke patients. Results- Our results demonstrate that (1) administration of DNase-I, which promotes NETs lysis, but not of tPA, recanalizes the occluded vessel improving photothrombotic stroke outcome; (2) a preventive treatment with Cl-amidine, impeding NET formation, completely precludes thrombotic occlusion; (3) platelet TLR4 mediates NET formation after photothrombotic stroke; and (4) ex vivo fresh platelet-rich thrombi from ischemic stroke patients are effectively lysed by DNase-I. Conclusions- Hence, our data open new avenues for recanalization of platelet-rich thrombi after stroke, especially to overcome tPA resistance.

TLR4-Binding DNA Aptamers Show a Protective Effect against Acute Stroke in Animal Models.

Since Toll-like receptor 4 (TLR4) mediates brain damage after stroke, development of TLR4 antagonists is a promising therapeutic strategy for this disease. Our aim was to generate TLR4-blocking DNA aptamers to be used for stroke treatment. From a random oligonucleotide pool, we identified two aptamers (ApTLR#1R, ApTLR#4F) with high affinity for human TLR4 by systematic evolution of ligands by exponential enrichment (SELEX). Optimized truncated forms (ApTLR#1RT, ApTLR#4FT) were obtained. Our data demonstrate specific binding of both aptamers to human TLR4 as well as a TLR4 antagonistic effect. ApTLR#4F and ApTLR#4FT showed a long-lasting protective effect against brain injury induced by middle cerebral artery occlusion (MCAO), an effect that was absent in TLR4-deficient mice. Similar effects were obtained in other MCAO models, including in rat. Additionally, efficacy of ApTLR#4FT in a model of brain ischemia-reperfusion in rat supports the use of this aptamer in patients undergoing artery recanalization induced by pharmacological or mechanical interventions. The absence of major toxicology aspects and the good safety profile of the aptamers further encourage their future clinical positioning for stroke therapy and possibly other diseases in which TLR4 plays a deleterious role.

Seladin-1/DHCR24 Is Neuroprotective by Associating EAAT2 Glutamate Transporter to Lipid Rafts in Experimental Stroke.

BACKGROUND AND PURPOSE: 3ß-Hydroxysteroid-Δ24 reductase (DHCR24) or selective alzheimer disease indicator 1 (seladin-1), an enzyme of cholesterol biosynthetic pathway, has been implicated in neuroprotection, oxidative stress, and inflammation. However, its role in ischemic stroke remains unexplored. The aim of this study was to characterize the effect of seladin-1/DHCR24 using an experimental stroke model in mice. METHODS: Dhcr24(+/-) and wild-type (WT) mice were subjected to permanent middle cerebral artery occlusion. In another set of experiments, WT mice were treated intraperitoneally either with vehicle or U18666A (seladin-1/DHCR24 inhibitor, 10 mg/kg) 30 minutes after middle cerebral artery occlusion. Brains were removed 48 h after middle cerebral artery occlusion for infarct volume determination. For protein expression determination, peri-infarct region was obtained 24 h after ischemia, and Western blot or cytometric bead array was performed. RESULTS: Dhcr24(+/-) mice displayed larger infarct volumes after middle cerebral artery occlusion than their WT littermates. Treatment of WT mice with the seladin-1/DHCR24 inhibitor U18666A also increased ischemic lesion. Inflammation-related mediators were increased after ischemia in Dhcr24(+/-) mice compared with WT counterparts. Consistent with a role of cholesterol in proper function of glutamate transporter EAAT2 in membrane lipid rafts, we found a decreased association of EAAT2 with lipid rafts after ischemia when DHCR24 is genetically deleted or pharmacologically inhibited. Accordingly, treatment with U18666A decreases [(3)H]-glutamate uptake in cultured astrocytes. CONCLUSIONS: These results support the idea that lipid raft integrity, ensured by seladin-1/DHCR24, plays a crucial protective role in the ischemic brain by guaranteeing EAAT2-mediated uptake of glutamate excess.

Aging increases microglial proliferation, delays cell migration, and decreases cortical neurogenesis after focal cerebral ischemia.

BACKGROUND: Aging is not just a risk factor of stroke, but it has also been associated with poor recovery. It is known that stroke-induced neurogenesis is reduced but maintained in the aged brain. However, there is no consensus on how neurogenesis is affected after stroke in aged animals. Our objective is to determine the role of aging on the process of neurogenesis after stroke. METHODS: We have studied neurogenesis by analyzing proliferation, migration, and formation of new neurons, as well as inflammatory parameters, in a model of cerebral ischemia induced by permanent occlusion of the middle cerebral artery in young- (2 to 3 months) and middle-aged mice (13 to 14 months). RESULTS: Aging increased both microglial proliferation, as shown by a higher number of BrdU(+) cells and BrdU/Iba1(+) cells in the ischemic boundary and neutrophil infiltration. Interestingly, aging increased the number of M1 monocytes and N1 neutrophils, consistent with pro-inflammatory phenotypes when compared with the alternative M2 and N2 phenotypes. Aging also inhibited (subventricular zone) SVZ cell proliferation by decreasing both the number of astrocyte-like type-B (prominin-1(+)/epidermal growth factor receptor (EGFR)(+)/nestin(+)/glial fibrillary acidic protein (GFAP)(+) cells) and type-C cells (prominin-1(+)/EGFR(+)/nestin(-)/Mash1(+) cells), and not affecting apoptosis, 1 day after stroke. Aging also inhibited migration of neuroblasts (DCX(+) cells), as indicated by an accumulation of neuroblasts at migratory zones 14 days after injury; consistently, aged mice presented a smaller number of differentiated interneurons (NeuN(+)/BrdU(+) and GAD67(+) cells) in the peri-infarct cortical area 14 days after stroke. CONCLUSIONS: Our data confirm that stroke-induced neurogenesis is maintained but reduced in aged animals. Importantly, we now demonstrate that aging not only inhibits proliferation of specific SVZ cell subtypes but also blocks migration of neuroblasts to the damaged area and decreases the number of new interneurons in the cortical peri-infarct area. Thus, our results highlight the importance of using aged animals for translation to clinical studies.

Toll-like receptor 4 modulates cell migration and cortical neurogenesis after focal cerebral ischemia.

Toll-like receptor 4 (TLR4) mediates brain damage after stroke. Now our objective is to determine TLR4 involvement in stroke-induced neurogenesis. Stroke was induced by permanent middle cerebral artery occlusion in wild-type and TLR4-deficient mice. Stereological and densitometric analysis of immunofluorescence-labeled brain sections and FACS analysis of cell suspensions were performed. Our results show that subventricular zone (SVZ) cell proliferation after stroke depends on infarct size. Second, when comparing brains with similar lesions, TLR4 attenuated SVZ proliferation, as shown by a decrease in prominin-1(+)/EGFR(+)/nestin(-) cells (type-C cells) at 1-2 d, and in BrdU(+) cells at 7 d, in TLR4(+/+) vs. TLR4(-/-) mice. Interestingly, 7 d after the infarct, neuroblasts in TLR4(+/+) mice migrated farther distances, reaching areas closer to the lesion than those in TLR4-deficient mice. However, at 14 d, TLR4-deficient mice presented a higher number of neuroblasts in all migratory zones than the TLR4(+/+) counterparts, which suggests that TLR4 deficiency delays neuroblast migration. Consistently, TLR4(+/+) mice showed an increased number of interneurons (NeuN(+)/BrdU(+)/GAD67(+) cells) in peri-infarct cortex 14-28 d after stroke. Our data indicate that, despite a negative effect on SVZ cell proliferation, TLR4 plays an important role in stroke-induced neurogenesis by promoting neuroblasts migration and increasing the number of new cortical neurons after stroke.

Cerebroprotective Effects of the TLR4-Binding DNA Aptamer ApTOLL in a Rat Model of Ischemic Stroke and Thrombectomy Recanalization.

ApTOLL, a TLR4 modulator aptamer, has demonstrated cerebroprotective effects in a permanent ischemic stroke mouse model, as well as safety and efficacy in early phase clinical trials. We carried out reverse translation research according to STAIR recommendations to further characterize the effects and mechanisms of ApTOLL after transient ischemic stroke in rats and to better inform the design of pivotal clinical trials. Adult male rats subjected to transient middle cerebral artery occlusion were treated either with ApTOLL or the vehicle intravenously at different doses and time-points. ApTOLL was compared with TAK-242 (a TLR4 inhibitor). Female rats were also studied. After neurofunctional evaluation, brains were removed for infarct/edema volume, hemorrhagic transformation, and histologic determinations. Peripheral leukocyte populations were assessed via flow cytometry. ApTOLL showed U-shaped dose-dependent cerebroprotective effects. The maximum effective dose (0.45 mg/kg) was cerebroprotective when given both before reperfusion and up to 12 h after reperfusion and reduced the hemorrhagic risk. Similar effects occurred in female rats. Both research and clinical ApTOLL batches induced slightly superior cerebroprotection when compared with TAK-242. Finally, ApTOLL modulated circulating leukocyte levels, reached the brain ischemic tissue to bind resident and infiltrated cell types, and reduced the neutrophil density. These results show the cerebroprotective effects of ApTOLL in ischemic stroke by reducing the infarct/edema volume, neurofunctional impairment, and hemorrhagic risk, as well as the peripheral and local immune response. They provide information about ApTOLL dose effects and its therapeutic time window and target population, as well as its mode of action, which should be considered in the design of pivotal clinical trials.

ApTOLL: A new therapeutic aptamer for cytoprotection and (re)myelination after multiple sclerosis.

BACKGROUND AND PURPOSE: ApTOLL is an aptamer selected to antagonize toll-like receptor 4 (TLR4), a relevant actor for innate immunity involved in inflammatory responses in multiple sclerosis (MS) and other diseases. The currently available therapeutic arsenal to treat MS is composed of immunomodulators but, to date, there are no (re)myelinating drugs available in clinics. In our present study, we studied the effect of ApTOLL on different animal models of MS. EXPERIMENTAL APPROACH: The experimental autoimmune encephalomyelitis (EAE) model was used to evaluate the effect of ApTOLL on reducing the inflammatory component. A more direct effect on oligodendroglia was studied with the cuprizone model and purified primary cultures of murine and human oligodendrocyte precursor cells (OPCs) isolated through magnetic-activated cell sorting (MACS) from samples of brain cortex. Also, we tested these effects in an ex vivo model of organotypic cultures demyelinated with lysolecithin (LPC). KEY RESULTS: ApTOLL treatment positively impacted the clinical symptomatology of mice in the EAE and cuprizone models, which was associated with better preservation plus restoration of myelin and oligodendrocytes in the demyelinated lesions of animals. Restoration was corroborated on purified cultures of rodent and human OPCs. CONCLUSION AND IMPLICATIONS: Our findings reveal a new therapeutic approach for the treatment of inflammatory and demyelinating diseases such as MS. The molecular nature of the aptamer exerts not only an anti-inflammatory effect but also neuroprotective and remyelinating effects. The excellent safety profile demonstrated by ApTOLL in animals and humans opens the door to future clinical trials in MS patients.

eIF4F complex disruption causes protein synthesis inhibition during hypoxia in nerve growth factor (NGF)-differentiated PC12 cells.

Poor oxygenation (hypoxia) influences important physiological and pathological situations, including development, ischemia, stroke and cancer. Hypoxia induces protein synthesis inhibition that is primarily regulated at the level of initiation step. This regulation generally takes place at two stages, the phosphorylation of the subunit α of the eukaryotic initiation factor (eIF) 2 and the inhibition of the eIF4F complex availability by dephosphorylation of the inhibitory protein 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1). The contribution of each of them is mainly dependent of the extent of the oxygen deprivation. We have evaluated the regulation of hypoxia-induced translation inhibition in nerve growth factor (NGF)-differentiated PC12 cells subjected to a low oxygen concentration (0.1%) at several times. Our findings indicate that protein synthesis inhibition occurs primarily by the disruption of eIF4F complex through 4E-BP1 dephosphorylation, which is produced by the inhibition of the mammalian target of rapamycin (mTOR) activity via the activation of REDD1 (regulated in development and DNA damage 1) protein in a hypoxia-inducible factor 1 (HIF1)-dependent manner, as well as the translocation of eIF4E to the nucleus. In addition, this mechanism is reinforced by the increase in 4E-BP1 levels, mainly at prolonged times of hypoxia.

Silent information regulator 1 protects the brain against cerebral ischemic damage.

BACKGROUND AND PURPOSE: Sirtuin 1 (SIRT1) is a member of NAD+-dependent protein deacetylases implicated in a wide range of cellular functions and has beneficial properties in pathologies including ischemia/reperfusion processes and neurodegeneration. However, no direct evidence has been reported on the direct implication of SIRT1 in ischemic stroke. The aim of this study was to establish the role of SIRT1 in stroke using an experimental model in mice. METHODS: Wild-type and Sirt1-/- mice were subjected to permanent focal ischemia by permanent ligature. In another set of experiments, wild-type mice were treated intraperitoneally with vehicle, activator 3 (SIRT1 activator, 10 mg/kg), or sirtinol (SIRT1 inhibitor, 10 mg/kg) for 10 minutes, 24 hours, and 40 hours after ischemia. Brains were removed 48 hours after ischemia for determining the infarct volume. Neurological outcome was evaluated using the modified neurological severity score. RESULTS: Exposure to middle cerebral artery occlusion increased SIRT1 expression in neurons of the ipsilesional mouse brain cortex. Treatment of mice with activator 3 reduced infarct volume, whereas sirtinol increased ischemic injury. Sirt1-/- mice displayed larger infarct volumes after ischemia than their wild-type counterparts. In addition, SIRT1 inhibition/deletion was concomitant with increased acetylation of p53 and nuclear factor κB (p65). CONCLUSIONS: These results support the idea that SIRT1 plays an important role in neuroprotection against brain ischemia by deacetylation and subsequent inhibition of p53-induced and nuclear factor κB-induced inflammatory and apoptotic pathways.

Citicoline (CDP-choline) increases Sirtuin1 expression concomitant to neuroprotection in experimental stroke.

CDP-choline has shown neuroprotective effects in cerebral ischemia. In humans, although a recent trial International Citicoline Trial on Acute Stroke (ICTUS) has shown that global recovery is similar in CDP-choline and placebo groups, CDP-choline was shown to be more beneficial in some patients, such as those with moderate stroke severity and not treated with t-PA. Several mechanisms have been proposed to explain the beneficial actions of CDP-choline. We have now studied the participation of Sirtuin1 (SIRT1) in the neuroprotective actions of CDP-choline. Fischer rats and Sirt1⁻/⁻ mice were subjected to permanent focal ischemia. CDP-choline (0.2 or 2 g/kg), sirtinol (a SIRT1 inhibitor; 10 mg/kg), and resveratrol (a SIRT1 activator; 2.5 mg/kg) were administered intraperitoneally. Brains were removed 24 and 48 h after ischemia for western blot analysis and infarct volume determination. Treatment with CDP-choline increased SIRT1 protein levels in brain concomitantly to neuroprotection. Treatment with sirtinol blocked the reduction in infarct volume caused by CDP-choline, whereas resveratrol elicited a strong synergistic neuroprotective effect with CDP-choline. CDP-choline failed to reduce infarct volume in Sirt1⁻/⁻ mice. Our present results demonstrate a robust effect of CDP-choline like SIRT1 activator by up-regulating its expression. Our findings suggest that therapeutic strategies to activate SIRT1 may be useful in the treatment of stroke. Sirtuin 1 (SIRT1) is implicated in a wide range of cellular functions. Regarding stroke, there is no direct evidence. We have demonstrated that citicoline increases SIRT1 protein levels in brain concomitantly to neuroprotection. Citicoline fails to reduce infarct volume in Sirt1⁻/⁻ mice. Our findings suggest that therapeutic strategies acting on SIRT1 may be useful in the treatment of stroke.

Defective hippocampal neurogenesis underlies cognitive impairment by carotid stenosis-induced cerebral hypoperfusion in mice.

Chronic cerebral hypoperfusion due to carotid artery stenosis is a major cause of vascular cognitive impairment and dementia (VCID). Bilateral carotid artery stenosis (BCAS) in rodents is a well-established model of VCID where most studies have focused on white matter pathology and subsequent cognitive deficit. Therefore, our aim was to study the implication of adult hippocampal neurogenesis in hypoperfusion-induced VCID in mice, and its relationship with cognitive hippocampal deficits. Mice were subjected to BCAS; 1 and 3 months later, hippocampal memory and neurogenesis/cell death were assessed, respectively, by the novel object location (NOL) and spontaneous alternation performance (SAP) tests and by immunohistology. Hypoperfusion was assessed by arterial spin labeling-magnetic resonance imaging (ASL-MRI). Hypoperfused mice displayed spatial memory deficits with decreased NOL recognition index. Along with the cognitive deficit, a reduced number of newborn neurons and their aberrant morphology indicated a remarkable impairment of the hippocampal neurogenesis. Both increased cell death in the subgranular zone (SGZ) and reduced neuroblast proliferation rate may account for newborn neurons number reduction. Our data demonstrate quantitative and qualitative impairment of adult hippocampal neurogenesis disturbances associated with cerebral hypoperfusion-cognitive deficits in mice. These findings pave the way for novel diagnostic and therapeutic targets for VCID.

APRIL: A double-blind, placebo-controlled, randomized, Phase Ib/IIa clinical study of ApTOLL for the treatment of acute ischemic stroke.

In the reperfusion era, a new paradigm of treating patients with endovascular treatment (EVT) and neuroprotective drugs is emerging as a promising therapeutic option for patients with acute ischemic stroke (AIS). In this context, ApTOLL, a Toll-like receptor 4 (TLR4) antagonist with proven neuroprotective effect in preclinical models of stroke and a very good pharmacokinetic and safety profile in healthy volunteers, is a promising first-in-class aptamer with the potential to address this huge unmet need. This protocol establishes the clinical trial procedures to conduct a Phase Ib/IIa clinical study (APRIL) to assess ApTOLL tolerability, safety, pharmacokinetics, and biological effect in patients with AIS who are eligible for EVT. This will be a multicenter, double-blind, randomized, placebo-controlled, Phase Ib/IIa clinical study to evaluate the administration of ApTOLL together with EVT in patients with AIS. The study population will be composed of men and non-pregnant women with confirmed AIS with a <6h window from symptoms onset to ApTOLL/placebo administration. The trial is currently being conducted and is divided into two parts: Phase Ib and Phase IIa. In Phase Ib, 32 patients will be allocated to four dose ascending levels to select, based on safety criteria, the best two doses to be administered in the following Phase IIa in which 119 patients will be randomized to three arms of treatment (dose A, dose B, and placebo). Identification of the trial: EudraCT: 2020-002059-38 and ClinicalTrials.gov Identifier: NCT04734548 https://clinicaltrials.gov/ct2/show/NCT04734548?term=ApTOLL&cond=Stroke&draw=2&rank=1.

Beneficial effect of TLR4 blockade by a specific aptamer antagonist after acute myocardial infarction.

Experimental evidence indicates that the control of the inflammatory response after myocardial infarction is a key strategy to reduce cardiac injury. Cellular damage after blood flow restoration in the heart promotes sterile inflammation through the release of molecules that activate pattern recognition receptors, among which TLR4 is the most prominent. Transient regulation of TLR4 activity has been considered one of the potential therapeutic interventions with greater projection towards the clinic. In this regard, the characterization of an aptamer (4FT) that acts as a selective antagonist for human TLR4 has been investigated in isolated macrophages from different species and in a rat model of cardiac ischemia/reperfusion (I/R). The binding kinetics and biological responses of murine and human macrophages treated with 4FT show great affinity and significant inhibition of TLR4 signaling including the NF-κB pathway and the LPS-dependent increase in the plasma membrane currents (Kv currents). In the rat model of I/R, administration of 4FT following reoxygenation shows amelioration of cardiac injury function and markers, a process that is significantly enhanced when the second dose of 4FT is administered 24 h after reperfusion of the heart. Parameters such as cardiac injury biomarkers, infiltration of circulating inflammatory cells, and the expression of genes associated with the inflammatory onset are significantly reduced. In addition, the expression of anti-inflammatory genes, such as IL-10, and pro-resolution molecules, such as resolvin D1 are enhanced after 4FT administration. These results indicate that targeting TLR4 with 4FT offers new therapeutic opportunities to prevent cardiac dysfunction after infarction.

Safety and Efficacy of ApTOLL in Patients With Ischemic Stroke Undergoing Endovascular Treatment: A Phase 1/2 Randomized Clinical Trial.

Importance: ApTOLL is a TLR4 antagonist with proven preclinical neuroprotective effect and a safe profile in healthy volunteers. Objective: To assess the safety and efficacy of ApTOLL in combination with endovascular treatment (EVT) for patients with ischemic stroke. Design, Setting, and Participants: This phase 1b/2a, double-blind, randomized, placebo-controlled study was conducted at 15 sites in Spain and France from 2020 to 2022. Participants included patients aged 18 to 90 years who had ischemic stroke due to large vessel occlusion and were seen within 6 hours after stroke onset; other criteria were an Alberta Stroke Program Early CT Score of 6 to 10, estimated infarct core volume on baseline computed tomography perfusion of 5 to 70 mL, and the intention to undergo EVT. During the study period, 4174 patients underwent EVT. Interventions: In phase 1b, 0.025, 0.05, 0.1, or 0.2 mg/kg of ApTOLL or placebo; in phase 2a, 0.05 or 0.2 mg/kg of ApTOLL or placebo; and in both phases, treatment with EVT and intravenous thrombolysis if indicated. Main Outcomes and Measures: The primary end point was the safety of ApTOLL based on death, symptomatic intracranial hemorrhage (sICH), malignant stroke, and recurrent stroke. Secondary efficacy end points included final infarct volume (via MRI at 72 hours), NIHSS score at 72 hours, and disability at 90 days (modified Rankin Scale [mRS] score). Results: In phase Ib, 32 patients were allocated evenly to the 4 dose groups. After phase 1b was completed with no safety concerns, 2 doses were selected for phase 2a; these 119 patients were randomized to receive ApTOLL, 0.05 mg/kg (n = 36); ApTOLL, 0.2 mg/kg (n = 36), or placebo (n = 47) in a 1:1:√2 ratio. The pooled population of 139 patients had a mean (SD) age of 70 (12) years, 81 patients (58%) were male, and 58 (42%) were female. The primary end point occurred in 16 of 55 patients (29%) receiving placebo (10 deaths [18.2%], 4 sICH [7.3%], 4 malignant strokes [7.3%], and 2 recurrent strokes [3.6%]); in 15 of 42 patients (36%) receiving ApTOLL, 0.05 mg/kg (11 deaths [26.2%], 3 sICH [7.2%], 2 malignant strokes [4.8%], and 2 recurrent strokes [4.8%]); and in 6 of 42 patients (14%) receiving ApTOLL, 0.2 mg/kg (2 deaths [4.8%], 2 sICH [4.8%], and 3 recurrent strokes [7.1%]). ApTOLL, 0.2 mg/kg, was associated with lower NIHSS score at 72 hours (mean difference log-transformed vs placebo, -45%; 95% CI, -67% to -10%), smaller final infarct volume (mean difference log-transformed vs placebo, -42%; 95% CI, -66% to 1%), and lower degrees of disability at 90 days (common odds ratio for a better outcome vs placebo, 2.44; 95% CI, 1.76 to 5.00). Conclusions and Relevance: In acute ischemic stroke, 0.2 mg/kg of ApTOLL administered within 6 hours of onset in combination with EVT was safe and associated with a potential meaningful clinical effect, reducing mortality and disability at 90 days compared with placebo. These preliminary findings await confirmation from larger pivotal trials. Trial Registration: ClinicalTrials.gov Identifier: NCT04734548.

Apolipoprotein-E controls adenosine triphosphate-binding cassette transporters ABCB1 and ABCC1 on cerebral microvessels after methamphetamine intoxication.

BACKGROUND AND PURPOSE: Methamphetamine is a powerful addictive, which has been associated with ischemic stroke and brain hemorrhage in humans. Whether and how methamphetamine influences the expression of tight junctions and adenosine triphosphate-binding cassette transporters, which have previously been shown to be regulated by apolipoprotein-E (ApoE) under conditions of brain ischemia, was unknown. METHODS: C57BL/6J mice received intraperitoneal injections of methamphetamine (3 times 4 mg/kg separated by 3 hours) either alone or in combination with the ApoE receptor-2 inhibitor receptor-associated protein (40 µg/kg) or the inducible nitric oxide synthase inhibitor 1400W (5 mg/kg). Animals were euthanized 3 or 24 hours after methamphetamine exposure. Tissue responses were evaluated with Western blots, immunoprecipitation, and immunohistochemistry using total brain and cerebral microvessel extracts. RESULTS: Methamphetamine induced a transient activation of stress kinases c-Jun N-terminal kinase 1/2 and p38 in the brain parenchyma and increased intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression on cerebral microvessels without inducing loss of tight junction proteins and without inducing IgG extravasation. Methamphetamine transiently increased the expression of the luminal adenosine triphosphate-binding cassette transporter ABCB1 on cerebral microvessels and reduced the expression of the abluminal transporter ABCC1. Elevated expression of ApoE was noted in the brain parenchyma by methamphetamine, activating ApoE receptor-2 on brain capillaries, deactivating c-Jun N-terminal kinase 1/2 and c-Jun, and regulating ABCB1 and ABCC1 expression. Indeed, ApoE receptor-2 and inducible nitric oxide synthase inhibition prevented the ABCB1 and ABCC1 expression changes. CONCLUSIONS: Acute exposure to methamphetamine at doses comparable to those consumed in drug addiction does not induce tight junction breakdown but differentially regulates adenosine triphosphate-binding cassette transporters through the ApoE/ApoE receptor-2/c-Jun N-terminal kinase 1/2 pathway.

First-in-human phase I clinical trial of a TLR4-binding DNA aptamer, ApTOLL: Safety and pharmacokinetics in healthy volunteers.

ApTOLL is an aptamer that antagonizes Toll-like receptor 4 and improves functional outcomes in models of ischemic stroke and myocardial infarction. The aim of this study was to characterize the safety and pharmacokinetics of ApTOLL in healthy volunteers. A first-in-human dose-ascending, randomized, placebo-controlled phase I clinical trial to assess safety and pharmacokinetics of ApTOLL (30-min infusion intravenously) was performed in 46 healthy adult male volunteers. The study was divided into two parts: part A included seven single ascending dose levels, and part B had one multiple dose cohort. Safety and pharmacokinetic parameters were evaluated. No serious adverse events or biochemistry alterations were detected at any dose nor at any administration pattern studied. Maximum concentration was detected at the end of the infusion and mean half-life was 9.3 h. Interestingly, exposure increased in the first four levels receiving doses from 0.7 mg to 14 mg (AUC of 2,441.26 h∗ng/mL to 23,371.11 h∗ng/mL) but remained stable thereafter (mean of 23,184.61 h∗ng/mL after 70 mg). Consequently, the multiple dose study did not show any accumulation of ApTOLL. These results show an excellent safety and adequate pharmacokinetic profile that, together with the efficacy demonstrated in nonclinical studies, provide the basis to start clinical trials in patients.

New hierarchical phosphorylation pathway of the translational repressor eIF4E-binding protein 1 (4E-BP1) in ischemia-reperfusion stress.

Eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) is a translational repressor that is characterized by its capacity to bind specifically to eIF4E and inhibit its interaction with eIF4G. Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap-dependent translation, in cell stress. This study reports a physiological study of 4E-BP1 regulation by phosphorylation using control conditions and a stress-induced translational repression condition, ischemia-reperfusion (IR) stress, in brain tissue. In control conditions, 4E-BP1 was found in four phosphorylation states that were detected by two-dimensional gel electrophoresis and Western blotting, which corresponded to Thr(69)-phosphorylated alone, Thr(69)- and Thr(36)/Thr(45)-phosphorylated, all these plus Ser(64) phosphorylation, and dephosphorylation of the sites analyzed. In control or IR conditions, no Thr(36)/Thr(45) phosphorylation alone was detected without Thr(69) phosphorylation, and neither was Ser(64) phosphorylation without Thr(36)/Thr(45)/Thr(69) phosphorylation detected. Ischemic stress induced 4E-BP1 dephosphorylation at Thr(69), Thr(36)/Thr(45), and Ser(64) residues, with 4E-BP1 remaining phosphorylated at Thr(69) alone or dephosphorylated. In the subsequent reperfusion, 4E-BP1 phosphorylation was induced at Thr(36)/Thr(45) and Ser(64), in addition to Thr(69). Changes in 4E-BP1 phosphorylation after IR were according to those found for Akt and mammalian target of rapamycin (mTOR) kinases. These results demonstrate a new hierarchical phosphorylation for 4E-BP1 regulation in which Thr(69) is phosphorylated first followed by Thr(36)/Thr(45) phosphorylation, and Ser(64) is phosphorylated last. Thr(69) phosphorylation alone allows binding to eIF4E, and subsequent Thr(36)/Thr(45) phosphorylation was sufficient to dissociate 4E-BP1 from eIF4E, which led to eIF4E-4G interaction. These data help to elucidate the physiological role of 4E-BP1 phosphorylation in controlling protein synthesis.

Influence of metabolic syndrome on post-stroke outcome, angiogenesis and vascular function in old rats determined by dynamic contrast enhanced MRI.

Stroke affects primarily aged and co-morbid people, aspects not properly considered to date. Since angiogenesis/vasculogenesis are key processes for stroke recovery, we purposed to determine how different co-morbidities affect the outcome and angiogenesis/vasculogenesis, using a rodent model of metabolic syndrome, and by dynamic enhanced-contrast imaging (DCE-MRI) to assess its non-invasive potential to determine these processes. Twenty/twenty-two month-old corpulent (JCR:LA-Cp/Cp), a model of metabolic syndrome and lean rats were used. After inducing the experimental ischemia by transient MCAO, angiogenesis was analyzed by histology, vasculogenesis by determination of endothelial progenitor cells in peripheral blood by flow cytometry and evaluating their pro-angiogenic properties in culture and the vascular function by DCE-MRI at 3, 7 and 28 days after tMCAO. Our results show an increased infarct volume, BBB damage and an impaired outcome in corpulent rats compared with their lean counterparts. Corpulent rats also displayed worse post-stroke angiogenesis/vasculogenesis, outcome that translated in an impaired vascular function determined by DCE-MRI. These data confirm that outcome and angiogenesis/vasculogenesis induced by stroke in old rats are negatively affected by the co-morbidities present in the corpulent genotype and also that DCE-MRI might be a technique useful for the non-invasive evaluation of vascular function and angiogenesis processes.