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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., (© 2024 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

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.

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., Competing Interests: Disclosures Dr Broderick receives monies from National Institute of Neurological Disorders and Stroke (NINDS) for research grants, receives study medication and monies for temperature monitoring and after-business hour enrollments from Novo Nordisk for NINDS-funded FASTEST Trial (rFVIIa for Acute Hemorrhagic Stroke Administered at Earliest Time Trial). The Department of Neurology receives monies to an educational/research fund from Genentech for his role on Executive Committee of Timeless Trial; from Roche Pharmaceuticals for consulting, from BrainsGate Ltd for consulting; from Basking Biosciences, from Kroger Prescription Plans Inc’s Pharmacy &Therapeutics Committee as voting member of committee. Dr Silva reports funding from the Brazilian Ministry of Health for the Resilient and OPTIMAL trials; consulting and speaker fees from AstraZeneca, Biogen, Boehringer Ingelheim, and Pfizer; consulting fees from IschemaView and Bayer, and speaker fees from Abbott. Dr Selim receives grant funding from the NINDS and NIA (related to and outside of the current work), and royalties from Up to Date and Cambridge University Press and is on the Advisory Board of MedRhythms Inc. Dr Kasner has received grant funding (to institution) from Genentech, Diamedica, Bristol-Myers Squibb, Bayer, Stryker, and Medtronic; consulting fees from Shionogi, Abbvie, Artivion, AstraZeneca, and NeuExcell; and royalties from UpToDate and Elsevier. Dr Adeoye receives funding from NINDS and has equity in and is the Chief Medical Officer for Sense Diagnostics, Inc and receives compensation from NICO Corporation for data and safety monitoring services. Dr Hill reports grants from MicroVention Inc; employment by University of Calgary; grants from Medtronic; grants from Boehringer Ingelheim; grants from Medtronic; grants from Medtronic; grants from Canadian Institutes of Health Research; grants from Biogen Inc; compensation from Brainsgate Ltd for consultant services; and grants from NoNO Inc. Dr Mistry receives funding from National Institutes of Health/NINDS and Patient-Centered Outcomes Research Institute (PCORI) and receives monies for consulting for RAPID AI, Abbvie, and the American Heart Association. Dr Smith is an employee of AbbVie Inc, and holds AbbVie stock and stock options. All inputs to this article are based on personal experiences and opinions, and do not necessarily reflect AbbVie’s views. J. Sutherland is the Clinical Operations Manager for Revalesio. Dr Lyden receives royalty Income for “Thrombolytic Therapy for Acute Stroke” Third Edition Springer Press, is the PI for NINDS grants U24 NS13006000 and UG3 NS119199, and monies for consulting for Apex Innovations. Dr Jauch receives monies for consulting for RapidAI, and reports compensation from Schultz & Pogue, LLP, and McLeod Law Group for expert witness services. Dr Hernandez-Jimenez works for aptaTargets Dr Mocco receives grant funding from Stryker Neurovascular, Penumbra, and Microvention, as well as a research award from PCORI. Dr Mocco reports stock holdings in Endostream; stock holdings in Viz.ai; stock options in Neuroradial Technologies; stock options in Echovate; compensation from CVAid for consultant services; compensation from Penumbra Inc for other services; stock options in Whisper; compensation from RIST Neurovascular, Inc, for consultant services; stock options in Neurotechnology Investors, LLC, Managers; stock holdings in Q’Apel; grants from PCORI; compensation from Stryker for other services; compensation from MicroVention Inc for other services; stock holdings in Vastrax; stock holdings in Imperative Care Inc; compensation from Synchron for consultant services; stock holdings in Blinktbi; stock holdings in Cerebrotech; stock options in Sim&Cure; stock holdings in Spinaker; stock options in Spinaker; compensation from CVAid for consultant services; stock options in Songbird; stock options in E8; compensation from Perflow for consultant services; employment by Mount Sinai Health System; stock options in Tulavi; stock options in Borvo; stock holdings in Viseon Inc; stock options in Radical; and stock options in Neurolutions. E. Deljkich is Vice President, Clinical Affairs, Imperative Care. Dr Kamel is the PI for the ARCADIA trial (Atrial Cardiopathy and Antithrombotic Drugs in Prevention After Cryptogenic Stroke; NIH/NINDS U01NS095869), which received in-kind study drug from the BMS-Pfizer Alliance for Eliquis and ancillary study support from Roche Diagnostics; other funding from NIH (R01HL144541, R01NS123576, and U01NS106513); Deputy Editor for JAMA Neurology; clinical trial steering/executive committees for Medtronic, Janssen, and Javelin Medical; end point adjudication committees for AstraZeneca, Novo Nordisk, and Boehringer Ingelheim; and household ownership interests in TETMedical, Spectrum Plastics Group, and Burke Porter Group.

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.

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., Competing Interests: MH-J is an employee of AptaTargets S.L. MR receives payment from Philips as co-principal investigator of the WE TRUST study and he has a consulting agreement with Medtronic, Stryker, Cerenovus, CVAid, Methinks, Anaconda Biomed, and AptaTargets S.L. JC is founder and CEO of Aldebaran Health Intelligence, S.L. MD is employed by Cureteq AG and has provided consultancy services for AptaTargets S.L. BJ is a consultant to AptaTargets S.L. and Guardian Therapeutics, Inc. FA-S has been consultant or investigator in clinical trials sponsored by the following pharmaceutical companies: Abbott, Alter, AptaTargets, Chemo, Cinfa, FAES Farma, Farmalíder, Ferrer, GlaxoSmithKline, Galenicum, Gilead, Italfarmaco, Janssen-Cilag, Kern Pharma, Normon, Novartis, Servier, Silverpharma, Teva, and Zambon. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hernández-Jiménez, Abad-Santos, Cotgreave, Gallego, Jilma, Flores, Jovin, Vivancos, Molina, Montaner, Casariego, Dalsgaard, Hernández-Pérez, Liebeskind, Cobo and Ribo.)

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., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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., Competing Interests: M.H.J. and D.P.R. are employees of aptaTargets S.L. V.M.G. is researcher from FIBio-HRC. G.F. is employee of Aptus Biotech S.L. M.Ri. receives payment from Philips as Co-principal investigator of the WE TRUST study and he has a consulting agreement with Medtronic, Stryker, Cerenovus, CVAid, Methinks, Anaconda Biomed, and aptaTargets S.L. B.J. served as consultant to aptaTargets S.L. F.A.S. and D.O. have been consultants or investigators in clinical trials sponsored by the following pharmaceutical companies: Abbott, Alter, aptaTargets, Chemo, Cinfa, FAES, Farmalíder, Ferrer, GlaxoSmithKline, Galenicum, Gilead, Italfarmaco, Janssen-Cilag, Kern, Normon, Novartis, Servier, Silverpharma, Teva, and Zambon. The information disclosed in this article is protected by the international patent application WO2015197706 and its extensions to different countries; and by the international patent WO2020/230108., (© 2022 The Authors.)

Toll-like receptor 4 (TLR4) contributes to the pathogenesis of coronary ischemia/reperfusion (IR). To test whether the new TLR4 antagonist, ApTOLL, may prevent coronary IR damage, we administered 0.078 mg/kg ApTOLL or Placebo in pigs subjected to IR, analyzing the levels of cardiac troponins, matrix metalloproteinases, pro-, and anti-inflammatory cytokines, heart function, and tissue integrity over a period of 7 days after IR. Our results show that ApTOLL reduced cardiac troponin-1 24 h after administration, improving heart function, as detected by a significant recovery of the left ventricle ejection fraction (LVEF) and the shortening fraction (FS) cardiac parameters. The extension of necrotic and fibrotic areas was also reduced, as detected by Evans blue/2,3,5-triphenyltetrazolium chloride (TTC) staining, Hematoxylin/Eosine, and Masson Trichrome staining of heart sections, together with a significant reduction in the expression of the extracellular matrix-degrading, matrix metalloproteinase 9. Finally, the expression of the following cytokines, CCL1, CCL2, MIP1-A-B, CCL5, CD40L, C5/C5A, CXCL1, CXCL10, CXCL11, CXCL12, G-CSF, GM-CSF, ICAM-1, INF-g, IL1-a, ILI-b, IL-1Ra, IL2, IL4, IL5, IL6, IL8, IL10, IL12, IL13, IL16, IL17-A, IL17- E, IL18, IL21, IL27, IL32, MIF, SERPIN-E1, TNF-a, and TREM-1, were also assayed, detecting a pronounced decrease of pro-inflammatory cytokines after 7 days of treatment with ApTOLL. Altogether, our results show that ApTOLL is a promising new tool for the treatment of acute myocardial infarction (AMI).

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., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)