ApTOLL molecules for treatment of ischemic stroke and intracranial hemorrhage

Abstract

APRIL research shows that in a selected stroke patient, infusion of 0.2 mg / kg of ApTOLL (SEQ ID NO: 1) in combination with intravascular treatment is safe, and the death rate of 90 days is reduced. ApTOLL has proved to limit intracranial hemorrhage and reperfusion injury which may occur after recanalization, and also improve early nerve injury and long-term disability. ApTOLL molecules are provided for reducing the risk of intracranial or secondary bleeding after acute ischemic stroke, reducing nerve injury, and improving nerve recovery in a subject.

Description

Technical Field

[0001] The present invention relates to the field of oligonucleotides and therapeutic aptamers for human health, and is particularly suitable for treating intracranial hemorrhage and improving neurological recovery in patients with thrombotic diseases, especially acute ischemic stroke. Background Art

[0002] Over the past two decades, reperfusion therapy has significantly improved the care of acute ischemic stroke (AIS). Since 1995, tissue plasminogen activator (tPA) has been the only approved specific treatment for AIS and has had a positive impact on the lives of many stroke patients. However, its benefits are severely limited by its limited therapeutic window of <4.5 hours after stroke onset and its limited efficacy. It is estimated that tPA is only used in approximately 5% of AIS patients.

[0003] Until 2015, most clinical guidelines recommended mechanical thrombectomy as first-line treatment for ischemic stroke caused by large vessel occlusion (LVO). In recent years, the number of patients who benefit from endovascular thrombectomy (EVT) has rapidly increased worldwide, and its indications are gradually expanding to patients with a large infarct core or in the later time window within 24 hours of symptom onset.

[0004] However, these treatments are by no means completely complication-free. The greatest concern with the use of recanalization therapies such as tPA or EVT is the increased risk of bleeding, often referred to as hemorrhagic transformation (HT), which refers to the transformation of the stroke into an area of bleeding. Other bleeding associated with ischemic stroke may also occur; this is the case with intracranial hemorrhage (ICH), which refers to any bleeding within the intracranial vault, including the brain parenchyma and surrounding meningeal spaces. Therefore, both types of bleeding (hereafter referred to as ICH in this document) occur after an ischemic stroke and may also be referred to as secondary hemorrhage and may cause new symptoms or worsening of existing symptoms. Patients who suffer from secondary hemorrhage may suffer greater neurological damage and more difficult neurological recovery, or even die. Several trials have concluded that ICH is considered the leading cause of neurological deterioration (NIHSS score ≥4).

[0005] Furthermore, despite its proven efficacy, tPA has limited efficacy in recanalizing LVO. Indeed, the larger the occluding clot, meaning a more proximal occlusion, the lower the early recanalization rate after thrombolysis. Several studies have described recanalization rates as low as 10% for terminal internal carotid artery (TICA) occlusions or 30% for M1-MCA (M1 segment of the middle cerebral artery) occlusions within hours of intravenous (iv) tPA treatment.

[0006] Endovascular reperfusion therapy is currently considered fully implemented, meaning that acute stroke patients now consistently undergo complete vascular evaluation (computed tomography angiography and CT perfusion [CTA / CTP]), and those presenting with LVO undergo EVT, which ensures recanalization rates as high as 85–90%. Furthermore, several studies have shown that EVT has a wider therapeutic window (up to 24 hours after stroke onset in selected cases) compared with 4.5 hours with tPA. However, although EVT consistently achieves substantial recanalization in 85–90% of cases, more than 50% of treated patients will develop moderate to severe disability.

[0007] Despite the known inherent risks of reperfusion therapy, the benefit-risk ratio favors its use. In this context, it is of great interest to identify new drugs with potential neuroprotective effects to improve stroke outcomes while reducing intracranial hemorrhage, in parallel with reperfusion therapy.

[0008] ApTOLL is an anti-inflammatory drug with proven neuroprotective effects in preclinical models. It is an aptamer targeting the extracellular domain of TLR-4, a receptor involved in the innate immune response. It also responds to tissue damage-associated molecular patterns (DAMPs) and is directly involved in a large number of diseases such as ischemic stroke. In particular, the inflammatory component triggered by the acute phase of stroke is considered an attractive target for promoting patient recovery, and ApTOLL has a potential high therapeutic effect in this regard. WO2015197706A1 (AptaTargets SL) describes nucleic acid aptamers with the ability to specifically bind to and inhibit TLR-4, in particular ApTOLL, and their uses (including stroke).

[0009] In fact, the efficacy of ApTOLL has been confirmed in preclinical experimental models of cerebral and myocardial ischemia. In particular, WO2020230108A1 (AptaTargets SL) discloses methods and compositions for treating ischemic stroke and demonstrates the efficacy of ApTOLL in a rodent stroke model (Example 2). In addition, as described in Example 3.1 of WO2020230108A1 and in Hernández Jiménez M et al. in 2022, first-in-human studies have demonstrated the safety of ApTOLL in healthy subjects and established its pharmacokinetics. Summary of the Invention

[0010] One problem addressed by the present invention is to provide novel uses and methods for improving functional outcomes in patients, for example, reducing neurological damage while reducing bleeding complications associated with ischemic stroke and reperfusion techniques.

[0011] The solution is based on administering the ApTOLL molecule (eg, 0.2 mg / kg of ApTOLL, SEQ ID NO: 1) to a subject in need thereof for reducing neurological damage and intracranial hemorrhage, as well as other effects related to ischemic stroke and reperfusion techniques as explained below.

[0012] In this context, the inventors designed a Phase Ib / IIa clinical study, codenamed APRIL (hereinafter referred to as the APRIL study), to evaluate the safety of ApTOLL and its biological effects in patients with AIS who were eligible to receive EVT with or without intravenous thrombolysis. The protocol for this clinical study is described in Example 3.2 of WO2020230108A1 (AptaTargets SL), but the efficacy is still unclear.

[0013] The APRIL study was divided into two parts:

[0014] (1) Phase Ib (n=32 AIS patients): ApTOLL administered as a single intravenous injection (30-minute infusion), dose escalation across 4 single-dose levels (8 patients / level), randomization (1:3); and

[0015] (2) Phase IIa (n=119 patients): Single dose intravenous administration (30-minute infusion), parallel (3 groups, placebo: ApTOLL dose A: ApTOLL dose B), randomized (√2:1:1).

[0016] After Phase Ib was completed, the Data Safety Monitoring Board (DSMB) conducted an unblinded review of the study groups based on preliminary safety results and selected two doses (A and B) for testing in Phase IIa. Patients who received placebo, dose A, or dose B in Phase Ib were further analyzed along with patients enrolled in Phase IIa (total number of patients: 151).

[0017] The primary objective of the APRIL study was to evaluate whether different doses of ApTOLL administered intravenously were safe and well-tolerated compared with placebo when administered in conjunction with endovascular therapy and intravenous fibrinolysis (if indicated). The APRIL study was unable to draw conclusions regarding the effectiveness of ApTOLL in improving outcomes in patients with acute stroke.

[0018] Surprisingly, in addition to demonstrating the safety of ApTOLL, the inventors also found that, compared to placebo, administration of 0.2 mg / kg of ApTOLL within 6 hours of onset, combined with EVT, was associated with a meaningful clinical effect of significantly reducing 90-day mortality and disability. ApTOLL was able to improve survival in human patients with AIS, reduce intracranial hemorrhage, reduce cerebral edema, and improve neurological recovery. The working examples herein provide results from the APRIL study, with detailed experimental data demonstrating the aforementioned effects of ApTOLL (e.g., Example 1, Sections 1.13-1.15).

[0019] In particular, the inventors found that ApTOLL reduced intracranial hemorrhage and significantly reduced symptomatic intracranial hemorrhage by 34.25% compared to placebo.

[0020] Furthermore, they found that ApTOLL reduced the number of deaths: Death from any cause occurred in 10 patients assigned to placebo (18.2%) and in two patients assigned to ApTOLL 0.2 mg / kg (4.8%; absolute difference from placebo -13%; 95% CI: -25% to -1%), for a 73% reduction in deaths.

[0021] The final infarct volume was 44 mL in patients assigned to placebo and 23.5 mL in patients assigned to ApTOLL 0.2 mg / kg (log-transformed mean difference in final infarct volume from placebo, -42%; 95% CI: -66% to 1%), resulting in a 46.59% reduction in final infarct volume.

[0022] The NIHSS score (National Institute of Health Stroke Scale) assessed at 72 hours was 7 in patients assigned to placebo and 3 in patients assigned to ApTOLL 0.2 mg / kg (the mean difference in logarithmic transformed 72-hour NIHSS from placebo was -45%; 95% CI: -67% to -10%). This represents a 57.14% reduction in NIHSS score compared to placebo.

[0023] The proportion of patients assigned to placebo who achieved an mRs score (modified Rankin Scale) of 0-2 (asymptomatic to mildly disabled) at day 90 was 47.1% and 64.3% of patients assigned to ApTOLL 0.2 mg / kg, representing a 36.29% increase (i.e., improvement) in the mRs score of 0-2 compared to placebo.

[0024] In addition, ApTOLL 0.2 mg / kg reduced brain edema by 67.40% compared with placebo.

[0025] Subgroup analysis showed that patients who received ApTOLL in the early time window (<3 hours after symptom onset) and those who received ApTOLL in the later window (3-6 hours) had similar treatment effects, suggesting that ApTOLL is suitable for different clinical scenarios.

[0026] These observed effects of ApTOLL are surprising given the severity of the disease studied in the clinical trial, in which patients suffered from AIS, a very severe and acute disease, and the limited number of participants (151). These effects are clinically meaningful and have significant implications for patients with AIS, as they represent an improvement in survival and a reduction in functional impairment and disability, which represents an unmet medical need. They also represent an improvement over widely used reperfusion techniques such as EVT and thrombolysis. This is critical because these patients currently have limited treatment options, and our findings highlight the potential significance of ApTOLL in addressing this long-standing unmet medical need.

[0027] As mentioned above, WO2020230108A1 demonstrated the efficacy of ApTOLL in a rodent stroke model (Example 2). Compared to the vehicle, ApTOLL induced a reduction in infarct size, revealing its potential protective effect. Furthermore, the protective effect was maintained in mice for 21 days after stroke. WO2020230108A1 and Hernández-Jiménez M et al. also described the results of the first human study of ApTOLL in 2022. This study aimed to determine the safety and pharmacokinetics of ApTOLL in healthy subjects.

[0028] Therefore, the results of the APRIL study are the first to demonstrate the efficacy of ApTOLL in human subjects with AIS. The observed effects are novel relative to the prior art, and the effects described experimentally herein would not have been foreseen by a person of ordinary skill in the art from the prior art. In addition to the fact that the human subjects had AIS, whose biochemical / clinical profile differed from that of healthy subjects, the conditions, requirements, doses, and inclusion criteria in the APRIL study also differed from those described in the prior art; for example, EVT and fibrinolytic techniques were performed in the APRIL study, which could further compromise the patients and lead to, for example, intracranial hemorrhage. Furthermore, most of the observed effects were associated with an ApTOLL dose of 0.2 mg / kg.

[0029] The present invention thus involves completely unexpected novel effects, in particular a reduction in the risk of intracranial hemorrhage. The reasons why ApTOLL triggers these effects are still unclear, suggesting that ApTOLL may have the ability to interact with receptors other than TLR-4, for example, to prevent the development of intracranial hemorrhage after AIS.

[0030] Improved neurological recovery is also a significant result, one that cannot be inferred from prior art, as this effect was not necessarily present in previous ApTOLL studies, given the context of clinical trials. For example, the working examples herein specifically demonstrate reductions in infarct volume, NIHSS scores, and mRS scores, values that indicate significant neurological improvement in AIS patients. Furthermore, the results demonstrate that this improvement is not only reflected in mobility but also in cognitive abilities.

[0031] Therefore, the effects observed in the APRIL study, i.e., reduction in ICH, reduced neurological damage, and improved neurological recovery, can be considered as unexpected and different technical effects. Different technical effects can represent additional medical indications or new uses of known substances, for example, in the case of the EPO guideline.

[0032] Working Example 2 compares the effects of nerinetide and ApTOLL in the ESCAPE-NA1 and APRIL studies, respectively. ESCAPE-NA1 was a Phase III clinical trial with 1,105 patients, but the results were very limited because efficacy was only observed in patients who did not receive tPA treatment. Therefore, the applicability of nerinetide in future clinical practice will be limited to the above-mentioned types of patients. The results for patients who did not receive tPA treatment were a 19% increase in 0-2mRS and a 37% reduction in mortality. In contrast, the APRIL study was conducted on only 151 patients, and the results showed that compared with patients who received or did not receive tPA treatment, 0-2mRS (ie, mRS score decreased to 0-2mRS) increased by 36%, and mortality decreased by 72%. Example 2 indicates that the effects of the APRIL study are positive and applicable to a wider range of patients and medical scenarios.

[0033] As described above, despite the APRIL regimen described in Example 3.2 of WO2020230108A1, the results were not expected relative to the defined objectives and endpoints. It is noteworthy that the above results were unexpected and have significant implications for AIS patients, such that the results exceeded any possible expectations of the skilled clinicians involved in the study.

[0034] None of these surprising effects could be derived from the study by García-Culebras A et al., 2017. This paper describes TLR - / - Late-stage administration of tPA in mice does not induce TLR-4 + / + However, the study had some limitations, which are also cited in the document:

[0035] (1) Delayed administration is set at a time when it is still safe for humans;

[0036] (2) The protective effects of TLR-4 deficiency need to be investigated in future long-term outcome trials; and

[0037] (3) The involvement of TLR-4 in thrombectomy-induced HT remains to be studied.

[0038] In short, this study was performed in a mouse model with a complete knockout of the TLR-4 receptor, so the mechanism is different from the normal situation, for example, when the stroke is triggered, these mice do not develop an inflammatory response in the TLR-4 pathway. In contrast, the APRIL study used ApTOLL in patients with AIS: therefore, firstly, the aptamer partially inhibits TLR-4, which is different from the effect of a complete loss of TLR-4 in all tissues; and secondly, in contrast to the mouse model of García-Culebras A et al., 2017, patients develop inflammation in the TLR-4 pathway in the brain and suffer from multiple complications in clinical practice.

[0039] Thus, a first aspect of the present invention relates to an ApTOLL molecule for reducing the risk of intracranial or secondary bleeding (or preventing intracranial bleeding or reducing the occurrence of intracranial bleeding) in a subject after acute ischemic stroke, wherein the ApTOLL molecule is administered after acute ischemic stroke (i.e., after the stroke has occurred).

[0040] Another aspect relates to an ApTOLL molecule for reducing the risk of intracranial or secondary bleeding following a thrombotic disorder (or preventing intracranial bleeding or reducing the occurrence of intracranial bleeding) in a subject, wherein the ApTOLL molecule is administered after the thrombotic disorder has occurred.

[0041] Aspects of the present invention relate to ApTOLL molecules for use in reducing neural damage and improving neural recovery following acute ischemic stroke in a subject, wherein the ApTOLL molecules are administered following acute ischemic stroke.

[0042] In another aspect, the invention relates to an ApTOLL molecule for use in increasing survival and / or reducing brain edema in a subject after acute ischemic stroke, wherein the ApTOLL molecule is administered after acute ischemic stroke.

[0043] Other aspects of the present invention relate to ApTOLL molecules for use in improving anxiety / depression complications, daily activities, mobility and / or self-care in a subject after acute ischemic stroke, wherein the ApTOLL molecules are administered after acute ischemic stroke.

[0044] Throughout the specification and claims, the term "comprise" and its variations are not intended to exclude other technical features, additives, components or steps. Other objects, advantages and features of the present invention will become apparent to those of ordinary skill in the art after reading the specification or can be learned by practicing the present invention. In addition, the present invention covers all possible combinations of the specific and preferred embodiments described herein. The following examples and drawings are provided herein for illustrative purposes and are not intended to limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The APRIL study flow chart is shown (Pbo: placebo; DSMB: data safety monitoring board; AIS: acute ischemic stroke).

[0046] Figure 2 Figure 5. Median and interquartile range of baseline and 72-hour NIHSS scores according to treatment assignment, as well as median and interquartile range of baseline predicted infarct core on CT perfusion and final infarct volume (magnetic resonance imaging at 72 hours) according to treatment assignment. NIHSS: National Institutes of Health Stroke Scale.

[0047] Figure 3 The distribution of global disability at 90 days according to treatment assignment is shown. The stacked bar chart shows the distribution of modified Rankin scale (0 to 6 points) at 90 days according to treatment assignment.

[0048] Figure 4 Figure 1 is a graphic representation of the quality of life assessment of patients treated with ApTOLL 0.05 mg / kg, 0.02 mg / kg, or placebo. The quality of life assessment included the following criteria: mobility, self-care, usual activities, pain / discomfort, and anxiety / depression.

[0049] Figure 5 A detailed flowchart of the procedures in the APRIL study is presented. LVO: large vessel occlusion. TICA: terminal internal carotid artery. mRS: modified Rankin Scale. NIHSS: National Institutes of Health Stroke Scale. CT / CTA / CTP: computed tomography / computed tomography angiography / computed tomography perfusion. MRI: magnetic resonance imaging. ASPECTS: Alberta Stroke Project Early CT Score. CBF: cerebral blood flow. DWI: diffusion-weighted imaging. rt-PA: recombinant tissue plasminogen activator. EVT: endovascular thrombectomy.

[0050] Figure 6 Shown are the primary, secondary, and tertiary structures of ApTOLL (SEQ ID NO: 1), also known as ApTLR#4FT.

[0051] Figure 7 Presented are comparisons of mRS and survival outcomes after administration of 0.2 mg / kg ApTOLL in the APRIL study and 2.6 mg / kg nerinetide (NA-1) in the ESCAPE-NA1 study. mRS: modified Rankin Scale. tPA: tissue plasminogen activator. EVT: endovascular thrombectomy.

[0052] Figure 8 ApTOLL (SEQ ID NO: 1) and its variants ApTOLL-Mut 1-6 (SEQ ID NO: 17-22) were assayed for antagonist TLR4 activity. TLR4 receptor activity was expressed as a percentage relative to the control LPS-Ek up. The antagonistic activity of the aptamer was identified by a reduction in the percentage of activation of LPS-Ek up.

[0053] Figure 9 Competition assay for the TLR4 receptor representing ApTOLL (SEQ ID NO: 1) and its variants ApTOLL-Mut 1-6 (SEQ ID NO: 17-22). Mutants competing with the ApTOLL sequence for the same binding site in the TLR4 receptor were identified by reducing the percent binding of the ApTOLL sequence (control) relative to ApTOLL in each mixture of ApTOLL / ApTOLL-Mut (1-6). DETAILED DESCRIPTION

[0054] The present invention relates to the use and methods of ApTOLL molecules (e.g., SEQ ID NO: 1) in reducing the risk of intracranial hemorrhage in a subject after AIS; the use and methods of ApTOLL molecules in reducing the risk of intracranial hemorrhage in a subject after thrombotic disease; the use and methods of ApTOLL molecules in reducing neurological damage and improving neurological recovery in a subject after AIS (e.g., reduction in infarct volume and NIHSS score and improvement in mRS score); and the use and methods of ApTOLL molecules in improving survival and / or reducing cerebral edema in a subject after AIS, wherein the ApTOLL molecules are administered after AIS (or after thrombotic disease). Also provided are administration procedures and dosages (e.g., 0.2 mg / kg) of the ApTOLL molecules; arterial recanalization techniques (e.g., EVT or thrombolysis); subject characteristics (e.g., pre-stroke mRS score); and nucleic acid aptamers, variants, derivatives, chemically modified aptamers, pharmaceutical compositions, and formulations thereof.

[0055] Before describing the present invention in more detail, it should be understood that this disclosure is not limited to the specific compositions or process steps described, which may, of course, vary. It will be clear to one of ordinary skill in the art after reading this specification that each individual aspect described and illustrated herein has its own components and features, which can be easily separated or combined with the features of any of the other aspects without departing from the scope or spirit of this specification. Any enumerated method can be performed in the order of events enumerated or in any other order that is logically possible.

[0056] The headings provided herein are not limitations of the various aspects of the specification, which can be defined by reference to the entire specification. It should also be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting, as the scope of this specification will be limited only by the appended claims.

[0057] Accordingly, the terms defined below are more fully defined by reference to the specification as a whole.

[0058] definition

[0059] To facilitate understanding of this specification, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0060] Treat: As used herein, the terms "treat," "treatment," and "therapy" refer to clinical intervention that prevents (e.g., inhibits or stops) a disease or condition (e.g., intracranial hemorrhage); cures a disease or condition; delays the onset of a disease or condition; reduces the severity or extent of a disease or condition (e.g., reduces the extent of intracranial hemorrhage); ameliorates or eliminates one or more symptoms or sequelae associated with a disease or condition; or provides a beneficial effect to a subject suffering from a disease or condition, without necessarily curing the disease or condition.

[0061] In some embodiments, the term refers to a clinical intervention that ameliorates one or more symptoms; ameliorates one or more sequelae; prevents (e.g., inhibits, arrests, or delays) one or more symptoms; prevents (e.g., inhibits, arrests, or delays) one or more sequelae; delays one or more symptoms; delays one or more sequelae; ameliorate one or more symptoms; ameliorate one or more sequelae; shortens the duration of one or more symptoms; shortens the duration of one or more sequelae; reduces the frequency of one or more symptoms; reduces the frequency of one or more sequelae; reduces the severity of one or more symptoms; reduces the severity of one or more sequelae; improves quality of life; improves survival; prevents (e.g., inhibits, arrests, or delays) the recurrence of a disease or condition; delays the recurrence of a disease or condition; reduces the severity of a disease (e.g., reduces the extent of an intracranial hemorrhage); or any combination thereof, e.g., relative to what would be expected without treatment with an ApTOLL molecule of the invention.

[0062] The term "treatment" also includes prophylaxis or prevention (e.g., inhibiting, stopping, or delaying) of a disease or condition or its symptoms or sequelae. Prophylaxis refers to a treatment or course of action designed to prevent, stop, inhibit, reduce the risk of, reduce the occurrence of, or delay the onset of a disease or condition (e.g., intracranial hemorrhage), or to prevent, stop, inhibit, or delay symptoms associated with a disease or condition.

[0063] In some embodiments, the disease or condition is intracranial hemorrhage following a thrombotic disorder, particularly an ischemic stroke. In some embodiments, the ApTOLL molecule is used to treat intracranial hemorrhage, or to prevent intracranial hemorrhage, or to reduce the occurrence of intracranial hemorrhage, or to reduce the extent of intracranial hemorrhage following a thrombotic disorder (e.g., an ischemic stroke).

[0064] In other embodiments, the disease or condition is neural damage following acute ischemic stroke. In some embodiments, the ApTOLL molecule is used to treat neural damage, or to reduce neural damage, or to improve neural recovery following ischemic stroke.

[0065] Ischemic stroke: As used herein, this term refers to a type of stroke (also known as cerebrovascular disease, cerebral infarction, cerebral attack or stroke) characterized by a significant reduction in cerebral blood flow in an abnormally sudden manner, resulting in neurological deficits. In an ischemic stroke, blood perfusion is interrupted by a sudden and immediate interruption of blood flow due to occlusion of any artery perfusing the brain, which produces the appearance of an infarcted area. Arterial occlusion is usually due to atherosclerosis or emboli originating from other sites (usually the heart or other arteries) (cerebral embolism). Ischemic stroke is a pathology characterized by increased TLR-4 expression and / or increased TLR-4 activation. Given that activation of TLR-4 generates a signaling cascade leading to the release of inflammatory cytokines such as IL-1, IL-8, TNF-α, IL-6 and IL-12, and the activation and / or recruitment of inflammatory cells, resulting in inflammation and cellular damage, pathologies characterized by increased TLR-4 expression and / or increased TLR-4 activation can also be characterized by having an inflammatory component. "Acute ischemic stroke" refers to the first stage of ischemic stroke, during which the patient is treated (e.g., using the ApTOLL molecule). However, "ischemic stroke" and "acute ischemic stroke" are used interchangeably herein. Acute ischemic stroke is hereinafter referred to as AIS.

[0066] Stroke onset: This term, as used in this article, refers to the time when a stroke is triggered. In clinical practice, stroke onset corresponds to symptom onset, which is the first clinical symptom or sign of a specific condition, in this case, stroke, i.e., the time when symptoms began or the time when the person last looked normal (LTSW). Because the difference between stroke onset and symptom onset is minimal, the terms "stroke onset" and "symptom onset" are used interchangeably in this article. Some stroke symptoms include sudden numbness or weakness of the face, arm, or leg (especially on one side of the body), sudden confusion, difficulty speaking or understanding speech, sudden loss of vision in one or both eyes, sudden difficulty walking, dizziness, loss of balance or lack of coordination, sudden onset of severe headache with no known cause, etc.

[0067] In the APRIL study, several parameters regarding symptom onset, such as EVT, pharmacological thrombolysis, and inclusion criteria were defined.

[0068] In the APRIL study, several time points were defined relative to the patient's assignment to the trial (similar to baseline) rather than stroke onset. These were final infarct volume (measured 72 hours after assignment), NIHSS score (measured 72 hours after assignment), and mRS score (measured 90 days after assignment).

[0069] In some cases, patients were assigned close to symptom onset, e.g., an inclusion criterion of an infarct volume between 5 cc and 70 cc was measured before assignment, which is close to symptom onset. Therefore, in these cases, the terms "stroke onset," "symptom onset," "assignment," and "baseline" can be used interchangeably.

[0070] Intracranial hemorrhage (ICH): This term is used in this article to refer to intracerebral hemorrhage that occurs spontaneously as a complication of ischemic stroke and is usually triggered by reperfusion therapies such as thrombolysis or EVT. ICH refers to any bleeding within the intracranial vault, including the brain parenchyma and the surrounding meningeal space. Because ICH in this specification are caused by ischemic stroke and they are not the primary cause of the disease, they are also referred to as "secondary hemorrhage" in this specification. ICH can be symptomatic ICH (sICH) or asymptomatic ICH (aICH). ICH that causes new symptoms or worsening of existing symptoms is called sICH. Researchers determined that ICH was indeed associated with a significant worsening of neurological status based on a worsening of NIHSS ≥4 points.

[0071] In this specification, ICH also includes hemorrhagic transformation (HT). HT refers to the transformation of a stroke into an area of hemorrhage. HT is a common complication of ischemic stroke and is often exacerbated by reperfusion with thrombolysis or EVT. HT occurs when the blood-brain barrier (BBB) is sufficiently disrupted to allow extravasation of peripheral blood into the brain. When HT occurs, it increases stroke morbidity and mortality.

[0072] In some embodiments, the intracranial hemorrhage is symptomatic. In some embodiments, the intracranial hemorrhage is hemorrhagic transformation.

[0073] Intracranial hemorrhage can be measured using imaging procedures such as noncontrast CT, CTA, CPT, and MRI. Intracranial hemorrhage can be classified according to the Heidelberg hemorrhage classification.

[0074] Time window: This term, also called "therapeutic window", as used herein, refers to the time range between injury and treatment during which the treatment remains effective. In the present specification, treatment can be medical treatment with a fibrinolytic / thrombolytic agent (e.g., alteplase or tenecteplase), also called intravenous thrombolysis (IVT), and its time window is in accordance with the European Stroke Organization (ESO) guidelines. Berge E et al. provided recommendations on the time window for different thrombolytic agents in 2021, which is generally within 4.5 hours after stroke onset. In the present specification, treatment can also be EVT (embolectomy), which is recommended within a time window of 24 hours, in particular within 8 hours after stroke onset (Jovin TG et al., 2015). It is worth noting that the APRIL study protocol defined a 6-hour time window for EVT, with the aim of complying with the 8 hours.

[0075] Neurological damage: This term used herein refers to neurological damage or deterioration following an ischemic stroke. Neurological damage includes, but is not limited to, neuromuscular dysfunction that leads to disability, such as reduced mobility, apraxia, pain syndrome, limb spasticity, and urinary incontinence; cognitive impairment, which negatively affects the patient's cognitive abilities, ranging from memory loss to dysfunction of reasoning, speech, learning ability, language processing, and problem-solving abilities; and psychiatric disorders (emotional problems), such as depression, anxiety, emotional instability, crisis reactions, and post-stroke fatigue. This can lead to widespread disability and dependence in stroke patients in their daily activities. In the APRIL study and this specification, the damage was quantified using infarct volume, NIHSS score, and mRS score, as defined below. "Neurological damage" and "neurodamage" can be used interchangeably in this description. In one aspect, the ApTOLL molecule is used to reduce neurological damage / damage and also to improve neurological recovery after AIS in subjects.

[0076] In one embodiment, the neurological impairment comprises functional impairment and / or disability. In another embodiment, the neurological impairment comprises neuromuscular dysfunction, cognitive impairment, and / or mental disturbance.

[0077] In some embodiments, the ApTOLL molecule is used to treat nerve damage, or to reduce nerve damage, or to improve nerve recovery after ischemic stroke. In this sense, the term "neurorecovery" refers to the amelioration or improvement of nerve damage.

[0078] NIHSS score: The National Institutes of Health Stroke Scale (NIHSS) is a tool used by healthcare providers to objectively quantify the damage caused by a stroke. The NIHSS consists of 11 items, each of which scores a specific ability between 0 and 4. For each item, a score of 0 generally indicates normal function of that specific ability, while higher scores indicate some level of impairment. The individual scores for each item are added together to calculate the patient's total NIHSS score. The highest possible score is 42 and the lowest score is 0. Stroke Severity Score: 0 is no stroke symptoms; 1-4 is a mild stroke; 5-15 is a moderate stroke; 16-20 is a moderate to severe stroke; 21-42 is a severe stroke (Lyden P et al., 1994). The NIHSS can help doctors quantify the severity of a stroke in an acute setting. In an embodiment, the reduction in neurological damage and the improvement in neurological recovery are measured by the NIHSS score.

[0079] mRS score: The modified Rankin Scale (mRS) is a commonly used scale used to measure the degree of disability or dependence in daily activities in patients with stroke or other causes of neurological disability. The scale ranges from 0 to 6, from complete health with no symptoms to death: 0-no symptoms; 1-no major disability, although there are some symptoms, but able to perform all daily activities; 2-mild disability, able to take care of themselves without assistance, but unable to carry out all previous activities; 3-moderate disability, requiring some assistance but able to walk independently; 4-moderately severe disability, unable to meet their own physical needs without assistance and unable to walk without assistance; 5-severe disability, requiring constant care and attention, bedridden, and incontinent; 6-death (Van Swieten JC et al., 1988). In an embodiment, the reduction of neurological damage and the improvement of neurological recovery are measured by the mRS score.

[0080] Infarct volume: Infarct volume is a direct measure of damaged brain tissue and is one of the final pathological steps leading to the clinical deficits caused by ischemic stroke. Therefore, final infarct volume derived from magnetic resonance imaging (MRI) represents an objective and validated measure of stroke consequence. In the APRIL study, all images were read by appropriately trained local clinicians. ASPECTS for patient selection were independently determined by local clinicians, and computed tomography perfusion (CTP) images with mismatch determination were read to identify the ischemic core at baseline. Final infarct volume (FIV) postoperatively (72 hours) was determined by MRI-FLAIR (MRI-fluid attenuated inversion recovery) or CT (if MRI was not available).

[0081] Cerebral or cerebral edema: This term, used in this article, refers to a serious complication of AIS, which is the cause of death in 5% of all patients with cerebral infarction. Cerebral edema is caused by endothelial dysfunction of the capillaries, leading to a breakdown of the blood-brain barrier (BBB). Edema can cause tissue displacement and increased intracranial pressure, which can lead to death. In clinical practice, edema is determined using imaging techniques, namely computed tomography or magnetic resonance imaging.

[0082] ASPECTS: The Alberta Stroke Program Early CT Score (ASPECTS) is a 10-point quantitative topographic CT scan score designed to provide a reliable and practical alternative to standard CT examinations with a reproducible grading system for assessing early ischemic changes (within 3 hours of symptom onset) on pretreatment CT studies in patients with anterior circulation AIS. This CT score is simple, reliable, and may identify stroke patients who are unlikely to recover independently despite receiving thrombolytic therapy. The score divides the middle cerebral artery (MCA) territory into 10 regions of interest. Thus, ASPECTS is a topographic scoring system that utilizes a quantitative approach and does not require physicians to estimate volumes from two-dimensional images (Pexman JH et al., 2011).

[0083] Subject: The terms "subject," "patient," and "individual," and variations thereof, are used interchangeably herein to refer to any mammalian subject, including but not limited to humans, domestic animals (e.g., dogs, cats, etc.), farm animals (e.g., cattle, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.), for whom diagnosis, treatment, or therapy is desired, particularly humans. The uses and methods described herein are applicable to both human therapy and veterinary applications. In embodiments, the subject is a human, and particularly a human having the characteristics described in the "Characterization of the Subject" section of this specification.

[0084] Identity: As used herein, the term refers to the overall monomer conservation between polymer molecules, for example, conservation between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules). The term "identical" is without any additional qualifiers, for example, nucleic acid A is identical to nucleic acid B, meaning that the sequences are 100% identical (100% sequence identity). Describing two sequences as, for example, "70% identical" is equivalent to describing them as having, for example, "70% sequence identity". Comparison of sequences and determination of the percent identity between two sequences can be accomplished using mathematical algorithms, such as BLAST, Needle, Stretcher, Water, Matcher, and Needleman-Wunsch, among many others known in the art. Sequence alignment can be performed using methods known in the art, such as MAFT, Clustal (ClustalW, ClustalX or Clustal-Omega), MUSCLE, and the like.

[0085] About: The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the listed values. In general, the term "about" can modify the numerical value above and below the specified value by 10% of the variance, either up or down (higher or lower). As used herein, when the term "about" or "at least about" is applied to a list of values or ranges, the same applies to all members of the list. Thus, "at least about 1, 2, 3, 4..." can be interchanged with "at least about 1, at least about 2, at least about 3, at least about 4..."

[0086] Clinical outcomes

[0087] Reduced risk of intracranial hemorrhage after AIS

[0088] As mentioned above, hemorrhagic complications are common after thrombolytic therapy (such as tPA) or mechanical thrombectomy (such as EVT), which may lead to worse functional prognosis and even death in stroke patients.

[0089] The APRIL study showed that the combined administration of ApTOLL (SEQ ID NO: 1) and EVT in selected stroke patients can limit intracranial hemorrhage and reperfusion injury that may occur after recanalization (Example 1). Only 4.8% of stroke patients who received a combination of ApTOLL and EVT experienced symptomatic intracranial hemorrhage, compared to 7.3% who received EVT alone (Table 4). Therefore, the risk of symptomatic intracranial hemorrhage after ischemic stroke was reduced by 34.25%. This unexpected reduction may be a major solution for patients with AIS and lead to improved survival. Table 4 also shows that 40.47% of patients who received 0.2 mg / kg ApTOLL experienced intracranial hemorrhage, while 47.27% of patients who did not receive ApTOLL (placebo) experienced intracranial hemorrhage. Therefore, the risk of intracranial hemorrhage in patients who received 0.2 mg / kg ApTOLL was reduced by 14.39%.

[0090] Therefore, the present invention provides an ApTOLL molecule for reducing the risk of intracranial or secondary bleeding (or preventing intracranial bleeding or reducing the occurrence of intracranial bleeding) in a subject after acute ischemic stroke, wherein the ApTOLL molecule is administered after acute ischemic stroke (i.e., after symptom onset or stroke onset).

[0091] It should be understood that the uses disclosed herein can alternatively be formulated as a method of reducing the risk of (suffering from) intracranial hemorrhage in a subject after acute ischemic stroke (or preventing intracranial hemorrhage or reducing the occurrence of intracranial hemorrhage), the method comprising administering an ApTOLL molecule to the subject, wherein the ApTOLL molecule is administered after acute ischemic stroke.

[0092] In one embodiment, the intracranial hemorrhage is hemorrhagic transformation. In another embodiment, the intracranial hemorrhage is symptomatic. In another embodiment, the intracranial hemorrhage is asymptomatic.

[0093] In some embodiments, the risk of intracranial hemorrhage is reduced by between 5% and 90% compared to a control condition, e.g., compared to the risk in a subject not treated with the ApTOLL molecule. In one embodiment, the risk of intracranial hemorrhage is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% relative to a subject not treated with the ApTOLL molecule. In one embodiment, the risk of intracranial hemorrhage is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% relative to a subject not treated with the ApTOLL molecule. In a specific embodiment, the risk of intracranial hemorrhage is reduced by at least about 10%, 11%, 12%, 13%, or 14%, and specifically about 14%, relative to a subject not treated with the ApTOLL molecule. In one embodiment, the ApTOLL molecule is administered at a dose of 0.2 mg / kg, and particularly the ApTOLL molecule is ApTOLL (SEQ ID NO: 1).

[0094] In some embodiments, the risk of symptomatic intracranial hemorrhage is reduced by between 5% and 90% compared to a control condition, e.g., compared to the risk in a subject not treated with the ApTOLL molecule. In one embodiment, the risk of symptomatic intracranial hemorrhage is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% relative to a subject not treated with the ApTOLL molecule. In specific embodiments, the risk of symptomatic intracranial hemorrhage is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% relative to a subject not treated with the ApTOLL molecule; specifically, the reduction is at least about 30%, 31%, 32%, 33%, or 34%, and more specifically, about 34%. In one embodiment, the ApTOLL molecule is administered at a dose of 0.2 mg / kg, and particularly the ApTOLL molecule is ApTOLL (SEQ ID NO: 1).

[0095] As mentioned above, the primary criterion for candidate selection in reperfusion is the time from stroke symptom onset. Reperfusion therapy must be administered within a narrow window: within 4.5 hours of stroke onset for tPA and within 24 hours for EVT. The restriction to tPA treatment beyond 4.5 hours disqualifies most stroke patients (approximately 85%) who are admitted after this time window, significantly limiting the eligible population. The timing of treatment is important because giving a powerful blood thinner like tPA during a stroke can cause intracerebral bleeding.

[0096] In the APRIL study, the combined administration of ApTOLL and EVT has been shown to reduce intracranial hemorrhage, such as ICH and HT, meaning that tPA and EVT treatment did not cause intracranial hemorrhage due to the effects of ApTOLL. Therefore, ApTOLL can effectively reach the tissue at risk and exert a protective effect directly in the ischemic brain area, thereby potentially extending the therapeutic window for reperfusion therapy.

[0097] Thus, in one embodiment, administration of an ApTOLL molecule of the invention (eg, SEQ ID NO: 1) to a subject following ischemic stroke extends the therapeutic window for reperfusion therapy.

[0098] In one embodiment, administration of an ApTOLL molecule to a subject after ischemic stroke extends the therapeutic window for thrombolytic drugs (e.g., tPA). In a specific embodiment, the therapeutic window for thrombolytic drugs (e.g., tPA) is extended to at least about: 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours after stroke onset.

[0099] In one embodiment, administration of an ApTOLL molecule to a subject after ischemic stroke extends the therapeutic window for EVT (e.g., thrombectomy). In a specific embodiment, the therapeutic window for EVT is extended to at least about: 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, or 48 hours after stroke onset.

[0100] In addition to reducing secondary hemorrhage, the APRIL study also showed that administration of ApTOLL 0.2 mg / kg reduced mortality, decreased final infarct volume, improved early neurological impairment and long-term disability (as measured by NIHSS and mRS scores), and decreased cerebral edema.

[0101] Thus, in some embodiments, administration of an ApTOLL molecule to a subject following an ischemic stroke results in

[0102] (i) reduction in infarct volume;

[0103] (ii) decreased NIHSS score;

[0104] (iii) an increase in the mRS score of 0–2 (i.e., a decrease in the mRS score to 0–2);

[0105] (iv) improved survival rates;

[0106] (v) reduction in cerebral edema; and

[0107] (vi) any combination of the above.

[0108] The uses disclosed herein can alternatively be formulated as providing an ApTOLL molecule (e.g., SEQ ID NO: 1) for use in treatment (i.e., therapy or method) to reduce the risk of intracranial hemorrhage or secondary hemorrhage in a subject after acute ischemic stroke, wherein the treatment comprises:

[0109] (a) Select patients who have acute ischemic stroke within 6 hours of onset.

[0110] (b) administering to the patient a dose of at least 0.2 mg / kg of the aptamer, and

[0111] (c) measurement of intracranial hemorrhage or secondary hemorrhage, particularly by imaging procedures,

[0112] wherein administration of the ApTOLL molecule reduces the risk of intracranial hemorrhage or secondary hemorrhage relative to a reference value for subjects not treated with the ApTOLL molecule.

[0113] Reduce the risk of intracranial hemorrhage following thrombotic disease

[0114] As mentioned above, the reduction in intracranial hemorrhage was an unexpected result of the APRIL study. ApTOLL (SEQ ID NO: 1) is a good candidate for reducing intracranial hemorrhage in patients with ischemic stroke. In addition, the evidence provided in the APRIL study suggests that ApTOLL may also help reduce intracranial hemorrhage in similar situations, particularly thrombotic diseases. For example, fibrinolytics are also taken after myocardial infarction, which may trigger systemic (remote) hemorrhagic transformation, for example, bleeding in the digestive system or locally. Therefore, the effect of ApTOLL in reducing intracranial hemorrhage may also be applicable to thrombotic diseases other than ischemic stroke.

[0115] Therefore, the present invention also relates to ApTOLL molecules for reducing the risk of intracranial or secondary bleeding after a thrombotic disease in a subject (or preventing intracranial bleeding or reducing the occurrence of intracranial bleeding), wherein ApTOLL is administered after the thrombotic disease occurs.

[0116] Alternatively, the present invention also relates to a method for reducing the risk of (suffering from) intracranial hemorrhage (or preventing intracranial hemorrhage or reducing the occurrence of intracranial hemorrhage) in a subject after a thrombotic disease, the method comprising administering an ApTOLL molecule to the subject, wherein the ApTOLL molecule is administered after the thrombotic disease.

[0117] In one embodiment, thrombotic disease is associated with thrombotic events, i.e., thrombosis. In one embodiment, thrombotic disease is selected from the following diseases or conditions: arterial thrombosis, including atherosclerotic thrombosis, venous thrombosis, ischemic events, acute coronary syndromes, coronary thrombotic occlusions, coronary thrombosis, cerebrovascular accidents (particularly thrombosis) due to atherosclerosis, myocardial infarction (heart attack), acute cerebrovascular ischemia (ischemic stroke), percutaneous coronary intervention, stent thrombosis, restenosis, diseases of the aorta and its branches (such as aortic aneurysm, thrombosis), peripheral arterial disease, venous thrombosis, acute phlebitis and pulmonary embolism, cancer-related thrombosis (Trousseau's syndrome), inflammatory thrombosis and thrombosis associated with infection and deep vein thrombosis and other emboli.

[0118] In a specific embodiment, the thrombotic disease is selected from the group consisting of cerebral arterial disease, cerebrovascular disease or coronary artery disease.

[0119] In a specific embodiment, the thrombotic disorder is a cerebrovascular disorder (eg, stroke or ischemia). In a specific embodiment, the cerebrovascular disorder is a stroke.

[0120] In a specific embodiment, the thrombotic disease is coronary artery disease. Coronary artery disease includes, but is not limited to, for example, cardiovascular disease, including unstable angina, myocardial infarction, acute myocardial infarction, coronary artery disease, coronary revascularization, coronary restenosis, ventricular thromboembolism, atherosclerosis, coronary artery disease (e.g., arterial occlusive disease), plaque formation, cardiac ischemia, including complications associated with coronary artery surgery, such as percutaneous transluminal coronary angioplasty (balloon angioplasty).

[0121] In a specific embodiment, the thrombotic disease is venous thromboembolism, for example, a disease involving leg swelling, pain and ulcers, pulmonary embolism, abdominal venous thrombosis. In another embodiment, the thrombotic disease is thrombotic microangiopathy, vasculitis purpura, etc.

[0122] Medical conditions associated with thrombolytic drugs are arterial thrombosis, deep vein thrombosis, acute myocardial infarction, acute ischemic stroke, venous catheter thrombosis, pulmonary embolism, thrombotic / thromboembolic diseases, etc.

[0123] In one embodiment, the ApTOLL molecule is administered at a dose of 0.2 mg / kg, and particularly the ApTOLL molecule is ApTOLL (SEQ ID NO: 1).

[0124] In one embodiment, the uses and methods described herein for reducing the risk of intracranial hemorrhage following a thrombotic disease in a subject further include the use of a thrombolytic drug. Thrombolytic drugs or agents include, for example, streptokinase, alteplase, reteplase, tenecteplase, urokinase, prourokinase, anistreplase (APSAC), and the like.

[0125] Reduce nerve damage and improve nerve recovery

[0126] The APRIL study showed consistent positive results in most predefined secondary outcome measures, including final infarct volume, early neurological damage, and long-term disability (Example 1). Notably, in the APRIL study, the values of the above three indicators were improved simultaneously.

[0127] Therefore, the present invention also provides an ApTOLL molecule for use in reducing neural damage and improving neural recovery after acute ischemic stroke in a subject, wherein the ApTOLL molecule is administered after acute ischemic stroke.

[0128] The uses disclosed herein can alternatively be formulated as a method of reducing neural damage and improving neural recovery in a subject after acute ischemic stroke, the method comprising administering an ApTOLL molecule to the subject, wherein the ApTOLL molecule is administered after acute ischemic stroke.

[0129] As described above, the inventors discovered that the reduction in intracranial hemorrhage was unexpected and subsequently led to reduced neurological damage and improved neurological recovery.

[0130] Thus, in one embodiment, the ApTOLL molecule is used to reduce neural injury and improve neural recovery after acute ischemic stroke in a subject, wherein the ApTOLL molecule is administered after acute ischemic stroke, and wherein the ApTOLL molecule reduces the risk of (or prevents) intracranial hemorrhage (e.g., HT and sICH); in other words, the reduction in neural injury and the improvement in neural recovery are mediated by reducing the risk of (or preventing) intracranial hemorrhage.

[0131] Alternatively, another embodiment is directed to a method of reducing neural injury and improving neural recovery after acute ischemic stroke in a subject, the method comprising administering an ApTOLL molecule to the subject, wherein the ApTOLL molecule is administered after acute ischemic stroke, and wherein the reduction in neural injury and improvement in neural recovery is mediated by reducing the risk of intracranial hemorrhage.

[0132] In one embodiment, the ApTOLL molecule is used to reduce neurological damage following acute ischemic stroke in a subject, wherein ApTOLL is administered after acute ischemic stroke and wherein the ApTOLL molecule reduces the risk of (or prevents) intracranial hemorrhage (e.g., HT and sICH); in other words, the reduction in neurological damage is mediated by reducing the risk of (or preventing) intracranial hemorrhage. In one embodiment, the neurological damage includes functional impairment and / or disability. In another embodiment, the neurological damage includes neuromuscular dysfunction, cognitive impairment, and / or psychiatric disorders.

[0133] In another embodiment, the ApTOLL molecule is used to improve neurological recovery after acute ischemic stroke in a subject, wherein the ApTOLL molecule is administered after acute ischemic stroke, and wherein the ApTOLL molecule reduces the risk of (or prevents) intracranial hemorrhage (e.g., HT and sICH); in other words, the improvement in neurological recovery is mediated by reducing the risk of (or preventing) intracranial hemorrhage.

[0134] In the APRIL study, reduction in neurological injury and improvement in neurological recovery were assessed by measuring several parameters at baseline, 72 hours, and / or 90 days after the subjects were assigned. These parameters were infarct volume, NIHSS score, and mRS score.

[0135] Thus, in some embodiments, reduction of nerve damage and improvement of nerve recovery is measured by:

[0136] (i) infarct volume;

[0137] (ii) NIHSS score;

[0138] (iii) mRS score; or

[0139] (iv) any combination of the above.

[0140] In a specific embodiment, nerve damage is measured by infarct volume, NIHSS score and mRS score, in particular infarct volume. In another specific embodiment, the improvement of nerve recovery is measured by infarct volume, NIHSS score and mRS score, in particular NIHSS score and mRS score, and more specifically mRS score.

[0141] In a specific embodiment, functional impairment is measured by NIHSS score. In another embodiment, disability is measured by mRS score.

[0142] The uses disclosed herein can alternatively be formulated as providing an ApTOLL molecule (e.g., SEQ ID NO: 1) for use in treatment (i.e., therapy or method) to reduce neural damage and improve neural recovery after acute ischemic stroke in a subject, wherein the treatment comprises:

[0143] (a) Select patients within about 6 hours of acute ischemic stroke onset,

[0144] (b) administering to the patient a dose of at least 0.2 mg / kg of the aptamer, and

[0145] (c) measuring infarct volume, NIHSS score, mRS score or their combination,

[0146] These include reducing nerve damage and improving nerve recovery:

[0147] (i) reduction in infarct volume,

[0148] (ii) decreased NIHSS score, and / or

[0149] (iii) mRS score decreases to 0-2,

[0150] The above are relative to reference values for subjects not treated with the ApTOLL molecule.

[0151] Infarct volume

[0152] In the APRIL study, infarct volume was measured by MRI / CTP at baseline and 72 ± 24 hours after allocation. In cases where MRI was not available at 72 ± 24 hours, CT was measured at 24 hours. In no case was the basal infarct volume considered FIV. In those cases where no images were obtained postoperatively (i.e., after ApTOLL administration), the patient was considered "missing." For patients with AIS, reduction in infarct volume was directly correlated with anatomical preservation and functional and neurological performance.

[0153] Thus, in one embodiment, reduction in neural injury and improvement in neural recovery is measured by infarct volume.

[0154] The biological effect of ApTOLL on infarct volume was assessed by the change from baseline to final infarct volume at 72 h and by comparing the final infarct volume at 72 h between study groups (placebo and ApTOLL groups).

[0155] Thus, the present invention relates to an ApTOLL molecule for use in reducing neurological damage and improving neurological recovery in a subject after acute ischemic stroke, wherein the ApTOLL molecule is administered after acute ischemic stroke, and wherein the ApTOLL molecule reduces infarct volume in the subject compared to a subject not treated with the ApTOLL molecule.

[0156] Reduction in neurological injury and improvement in neurological recovery were measured by reduction in final infarct volume compared to subjects who did not receive the ApTOLL molecule (e.g., placebo) at a specific time point (e.g., 72 h after allocation). Figure 2 The median final infarct volume at 72 h was 23.5 ml in patients who received ApTOLL 0.2 mg / kg, compared to 44 ml in patients who did not receive ApTOLL (placebo).

[0157] Thus, in one embodiment, the reduction in final infarct volume over a short period of time (e.g., 72 hours) is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% relative to a subject not treated with the ApTOLL molecule. In a specific embodiment, the reduction in final infarct volume at 72 hours is at least about 46% relative to a subject not treated with the ApTOLL molecule.

[0158] In another embodiment, the final infarct volume in the short term (eg, 72 h) is between about 10 ml and about 45 ml, more specifically between 20 ml and 25 ml.

[0159] In one embodiment, the ApTOLL molecule is administered at a dose of 0.2 mg / kg, and particularly the ApTOLL molecule is ApTOLL (SEQ ID NO: 1).

[0160] The reduction in neurological damage and improvement in neurological recovery can also be measured by a reduction in infarct volume over time (e.g., from baseline to 72 hours) compared to subjects who did not receive the ApTOLL molecule (e.g., placebo). Table 5 shows that the baseline infarct volume of patients who received 0.2 mg / kg ApTOLL was 14 ml, and the final infarct volume at 72 hours was 23.5 ml, resulting in a 67.86% increase in infarct volume due to stroke progression. In contrast, the baseline infarct volume of patients who did not receive ApTOLL (placebo) was 20.50 ml, and the final infarct volume at 72 hours was 44 ml, resulting in a 114.63% increase in infarct volume. Thus, the increase in infarct volume was milder in patients who received ApTOLL compared to those who did not receive ApTOLL. Specifically, the infarct volume of patients who did not receive ApTOLL increased by 40.80% over time compared to those who received ApTOLL.

[0161] Thus, in one embodiment, the reduction in infarct volume over time, for example, from baseline to 72 hours, is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% relative to a subject not treated with the ApTOLL molecule. In a specific embodiment, the reduction in infarct volume over time is at least about 40% relative to a subject not treated with the ApTOLL molecule. In one embodiment, the ApTOLL molecule is administered at a dose of 0.2 mg / kg, and in particular, the ApTOLL is ApTOLL (SEQ ID NO: 1).

[0162] To assess the early clinical course and long-term neurological outcome, changes from baseline in the NIHSS score and mRS scale were calculated in the APRIL study and compared between groups at the end time (72 hours for NIHSS and 90 days for mRS).

[0163] NIHSS score

[0164] NIHSS scores were measured at baseline and 72 hours after baseline to assess the early clinical course. The NIHSS assessed at 72 hours was 7 in patients assigned to placebo and 3 in patients assigned to ApTOLL 0.2 mg / kg (mean difference in log-transformed 72-hour NIHSS from placebo was -45%; 95% CI: -67% to -10%) (Table 4, Figure 2 ). This represents a 57.14% reduction compared to placebo.

[0165] Thus, in one embodiment, the reduction of nerve damage and improvement of nerve recovery is measured by NIHSS score.In a specific embodiment, the nerve damage is functional impairment.

[0166] Thus, the present invention relates to an ApTOLL molecule for use in reducing neurological damage and improving neurological recovery in a subject after acute ischemic stroke, wherein the ApTOLL molecule is administered after acute ischemic stroke, and wherein the ApTOLL molecule reduces the NIHSS score in the subject compared to a subject not treated with the ApTOLL molecule.

[0167] In one embodiment, the reduction in neurological damage and improvement in neurological recovery is measured by a reduction in the short-term (e.g., 72 hours) NIHSS score relative to subjects not treated with the ApTOLL molecule (e.g., placebo). In one embodiment, the reduction in NIHSS score is between 5% and 90% relative to subjects not treated with the ApTOLL molecule. In one embodiment, the reduction in NIHSS score is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% relative to subjects not treated with the ApTOLL molecule. In certain embodiments, the NIHSS score is reduced by at least about 40%, 45%, 50%, 55%, or 60%, specifically by at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, or 57%, and more specifically by about 57%, relative to a subject not treated with the ApTOLL molecule. In one embodiment, the ApTOLL molecule is administered at a dose of 0.2 mg / kg, and specifically the ApTOLL molecule is ApTOLL (SEQ ID NO: 1).

[0168] mRS score

[0169] In the APRIL study, the mRS scale was measured at baseline (pre-stroke mRS) and 72 hours and 90 days after baseline to assess long-term neurological outcomes. In cases where data were missing at 72 hours or 90 days, the last measurement after surgery (i.e., after ApTOLL administration) was used.

[0170] Thus, in one embodiment, the reduction of neurological damage and the improvement of neurological recovery are measured by mRS scores. In a specific embodiment, the neurological damage is disability.

[0171] Thus, the present invention relates to an ApTOLL molecule for use in reducing neurological damage and improving neurological recovery after acute ischemic stroke in a subject, wherein the ApTOLL molecule is administered after acute ischemic stroke, and wherein the ApTOLL molecule improves the subject's mRS score, i.e., reduces the mRS score by 3-6 and / or increases the mRS score by 0-2 (i.e., reduces the mRS score to 0-2), compared to a subject not treated with the ApTOLL molecule. The improvement in mRS score is reflected in a reduction in the mRS score, with the goal being a lower value between 0-2.

[0172] At a specific time point (e.g., 90 days), the reduction in neurological damage and improvement in neurological recovery is measured by a decrease in mRS score of 3-6 or an increase in mRS score of 0-2 (i.e., a decrease in mRS score to 0-2) compared to subjects not treated with the ApTOLL molecule (e.g., placebo). Figure 3 The results showed that the mRS score of 3-6 (from moderate to severe disability to death) of patients treated with ApTOLL 0.2 mg / kg was reduced by 32.40% compared with the subjects who did not receive ApTOLL treatment. In other words, the mRS score of 3-6 of patients assigned to the ApTOLL 0.2 mg / kg group was reduced by 32.40%. Figure 3 The results also showed that compared with subjects who did not receive ApTOLL treatment, patients who received ApTOLL 0.2mg / kg had an increase of 36.29% in the mRS score of 0-2 (from no symptoms, 0, to mild disability, 2). That is, 36.29% more patients with mRS scores of 0-2 were assigned to the ApTOLL 0.2mg / kg group (this means that the mRS score decreased from a higher mRS value to 0-2); in particular, compared with subjects who did not receive ApTOLL treatment, patients who received ApTOLL 0.2mg / kg had an increase of 71.23% in the mRS score of 0-1 (from no symptoms, 0, to no significant disability, 1) (this means that the mRS score decreased from a higher mRS value to 0-2). This result shows that compared with the placebo group, patients who received ApTOLL treatment within 6 hours of stroke onset had less disability in the long term (90 days), with more patients having mRS scores of 0-1 and 0-2, and fewer patients having mRS scores of 3-6.

[0173] Thus, in one embodiment, the long-term (e.g., 90-day) reduction in mRS score 3-6 is at least about 10%, 15%, 20%, 25%, 30%, or 35% compared to a subject not treated with the ApTOLL molecule. In a specific embodiment, the reduction in mRS score 3-6 at 90 days is at least about 32% relative to a subject not treated with the ApTOLL molecule.

[0174] In another embodiment, the long-term (e.g., 90 days) increase in the mRS score of 0-2 (i.e., a decrease in the mRS score to 0-2) is at least about 10%, 15%, 20%, 25%, 30%, 35%, or 40% relative to a subject not treated with the ApTOLL molecule. In a specific embodiment, the increase in the mRS score of 0-2 at 90 days is at least about 36% relative to a subject not treated with the ApTOLL molecule.

[0175] In another embodiment, the long-term (e.g., 90-day) increase in the mRS score on 0-1 (i.e., a decrease in the mRS score from 0-1) is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% relative to a subject not treated with the ApTOLL molecule. In a specific embodiment, the increase in the mRS score on 0-1 at 90 days is at least about 71% relative to a subject not treated with the ApTOLL molecule.

[0176] In one embodiment, the mRS score of 0 at day 90 (i.e., a decrease in mRS score from 0) is increased by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, or 120% relative to a subject not treated with the ApTOLL molecule. In a specific embodiment, the increase in mRS score of 0 at day 90 is at least about 120% for a subject not treated with the ApTOLL molecule.

[0177] In another embodiment, the increase in mRS score of 1 at day 90 (i.e., a decrease in mRS score to 1) is at least about 30%, 35%, 40%, 45%, or 50% relative to a subject not treated with the ApTOLL molecule. In a specific embodiment, the increase in mRS score of 1 at day 90 is at least about 50% relative to a subject not treated with the ApTOLL molecule.

[0178] In another embodiment, the reduction in mRS score of A at day 90 is at least about 10%, 15%, 20%, or 25% relative to a subject not treated with the ApTOLL molecule. In a specific embodiment, the reduction in mRS score of A at day 90 is at least about 25% relative to a subject not treated with the ApTOLL molecule.

[0179] In another embodiment, the reduction in mRS score 5-6 at day 90 is at least about 40%, 45%, 50%, 60%, 65%, 70%, 75%, or 80% relative to a subject not treated with the ApTOLL molecule. In a specific embodiment, the reduction in mRS score 5-6 at day 90 is at least about 75% relative to a subject not treated with the ApTOLL molecule.

[0180] In one embodiment, the ApTOLL molecule is administered at a dose of 0.2 mg / kg, and particularly the ApTOLL molecule is ApTOLL (SEQ ID NO: 1).

[0181] The reduction in neurological damage and improvement in neurological recovery can also be measured by an increase in the mRS score of 0-2 over time (e.g., from baseline pre-stroke mRS to 90 days) compared to subjects who did not receive the ApTOLL molecule (e.g., placebo). Table 5 shows that 100% of patients who received ApTOLL 0.2 mg / kg had a pre-stroke mRS of 0-2, which decreased to 64.29% at 90 days, a decrease of 35.71%. In contrast, 98.15% of patients who did not receive ApTOLL (placebo) had a pre-stroke mRS of 0-2, which decreased to 47.17% at 90 days, a decrease of 51.94%. Therefore, the reduction in the mRS score of 0-2 was smaller in patients who received ApTOLL compared to those who did not receive ApTOLL. Specifically, the mRS score of 0-2 decreased by 31.25% over time in patients who did not receive ApTOLL compared to those who received ApTOLL.

[0182] Thus, in one embodiment, the increase in mRS 0-2 (i.e., a decrease in mRS to 0-2) over time (e.g., from baseline to 90 days) is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% relative to a subject not treated with the ApTOLL molecule. In a specific embodiment, the increase in mRS 0-2 over time is at least about 31% relative to a subject not treated with the ApTOLL molecule. In one embodiment, the ApTOLL molecule is administered at a dose of 0.2 mg / kg, and in particular, the ApTOLL molecule is ApTOLL (SEQ ID NO: 1).

[0183] In a specific embodiment, the reduction of neural damage and the improvement of neural recovery are measured by: (i) a reduction in infarct volume; (ii) a reduction in NIHSS score; and / or (iii) a reduction in mRS score of 3-6 and / or an increase in mRS score of 0-2 (i.e., a reduction in mRS to 0-2) compared to subjects not treated with ApTOLL molecules (e.g., placebo). Specifically, the reduction of neural damage and the improvement of neural recovery are measured by: (i) a reduction in infarct volume; (ii) a reduction in NIHSS score; and (iii) an increase in mRS score of 0-2 (i.e., a reduction in mRS to 0-2).

[0184] Thus, the present invention relates to an ApTOLL molecule for use in reducing neurological damage and improving neurological recovery after acute ischemic stroke in a subject, wherein the ApTOLL molecule is administered after acute ischemic stroke, and wherein the ApTOLL molecule (i) reduces infarct volume, (ii) reduces NIHSS score, and / or (iii) increases the subject's mRS score of 0-2 (i.e., a decrease in mRS to 0-2) or reduces the mRS score of 3-6, compared to a subject not treated with the ApTOLL molecule. Specifically, the ApTOLL molecule (i) reduces infarct volume, (ii) reduces NIHSS score, and (iii) increases the subject's mRS score of 0-2 (i.e., a decrease in mRS to 0-2).

[0185] In a specific embodiment, compared to a subject not treated with the ApTOLL molecule, (i) the infarct volume at 72 hours is reduced by between about 10% and about 50%, more specifically at least about 40%; (ii) the NIHSS is reduced by between about 40% and about 60%, more specifically at least about 55%; and / or (ii) the mRS 0-2 (i.e., a decrease in mRS 0-2) is increased by between about 10% and about 40%, more specifically at least about 35%.

[0186] As described above, in the APRIL study, the reduction of neurological injury and the improvement of neurological recovery were assessed by measuring infarct volume, NIHSS score, and mRS score. However, the reduction of neurological injury and the improvement of neurological recovery can also be assessed by other scores known to those skilled in the art, for example, by measuring the Barthel Index / Score (BI), the Asian Stroke Disability Scale (ASDS), the Glasgow Outcome Score (GOS), etc.

[0187] Improve survival and reduce cerebral edema

[0188] The APRIL study showed that ApTOLL administered in combination with EVT was safe and reduced mortality at 90 days in selected stroke patients. Furthermore, the efficacy of ApTOLL as a neuroprotective agent for acute cerebral ischemia was supported by consistent positive results for most predefined primary outcome measures, including a reduction in cerebral edema (Example 1, 1.13-1.15, Table 4).

[0189] Therefore, the present invention also provides an ApTOLL molecule for use in improving survival and / or reducing brain edema in a subject after acute ischemic stroke, wherein the ApTOLL molecule is administered after acute ischemic stroke.

[0190] It will be appreciated that the uses disclosed herein may alternatively be formulated as a method of increasing survival and / or reducing cerebral edema in a subject after acute ischemic stroke, the method comprising administering to the subject an ApTOLL molecule, wherein the ApTOLL molecule is administered after acute ischemic stroke.

[0191] As described above, the inventors discovered that the reduction in intracranial hemorrhage was unexpected and resulted in improved survival and reduced brain edema.

[0192] Thus, in one embodiment, the ApTOLL molecule is used to improve survival and / or reduce cerebral edema in a subject after acute ischemic stroke, wherein the ApTOLL molecule is administered after acute ischemic stroke, and wherein the ApTOLL molecule reduces the risk of (or prevents) intracranial hemorrhage (e.g., HT and sICH); in other words, the improved survival and reduced cerebral edema are mediated by a reduced risk of intracranial hemorrhage.

[0193] Alternatively, another embodiment relates to a method of increasing survival and / or reducing cerebral edema in a subject after acute ischemic stroke, the method comprising administering an ApTOLL molecule to the subject, wherein the ApTOLL molecule is administered after acute ischemic stroke, and wherein the increase in survival and reduction in cerebral edema is mediated by reducing the risk of intracranial hemorrhage.

[0194] Table 4 shows that in the APRIL study, when administered at 0.2 mg / kg of ApTOLL, the number of deaths decreased from 18.2% to 4.85% compared to placebo, representing a 73.63% reduction in deaths. In one embodiment, administration of the ApTOLL molecule reduces mortality relative to the placebo group. Specifically, the number of deaths is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% relative to the placebo group. Specifically, the number of deaths is reduced by at least about 70%, 71%, 72%, 73%, or 74%, more specifically about 73%, relative to the placebo group. In one embodiment, the ApTOLL molecule is administered at a dose of 0.2 mg / kg, and specifically the ApTOLL molecule is ApTOLL (SEQ ID NO: 1).

[0195] In other words, the survival rate in the APRIL study was 95.2% when administered 0.2 mg / kg of ApTOLL, and 81.8% without ApTOLL treatment, a 14.08% improvement in survival. In one embodiment, administration of the ApTOLL molecule results in an increase in survival relative to the placebo group. Specifically, the increase in survival relative to the placebo group is at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, or 14%. Specifically, the increase in survival relative to the placebo group is at least about 10%, more specifically about 14%. In one embodiment, the ApTOLL molecule is administered at a dose of 0.2 mg / kg, and specifically the ApTOLL molecule is ApTOLL (SEQ ID NO: 1).

[0196] Furthermore, Table 4 shows that 2.38% of patients treated with 0.2 mg / kg ApTOLL experienced cerebral edema, compared to 7.3% of patients not treated with ApTOLL (placebo). Thus, patients treated with 0.2 mg / kg ApTOLL experienced a 67.40% reduction in cerebral edema. In one embodiment, administration of the ApTOLL molecule results in a reduction in cerebral edema relative to the placebo group. Specifically, the reduction in cerebral edema is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% relative to the placebo group. Specifically, the reduction in cerebral edema is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%, more specifically about 67%, relative to the placebo group. In one embodiment, the ApTOLL molecule is administered at a dose of 0.2 mg / kg, and particularly the ApTOLL molecule is ApTOLL (SEQ ID NO: 1).

[0197] Figure 4 This is a graphic illustration of the quality of life assessment of patients treated with ApTOLL 0.05 mg / kg, 0.02 mg / kg, or placebo. The quality of life assessment included the following criteria: mobility, self-care, daily activities, pain / discomfort, and anxiety / depression. In the APRIL study, improvements in anxiety / depression complications, daily activities, mobility, and self-care were observed with 0.2 mg / kg ApTOLL compared to placebo.

[0198] Thus, other aspects of the present invention relate to ApTOLL molecules for use in improving anxiety / depression complications, daily activities, mobility and / or self-care in a subject following acute ischemic stroke, wherein the ApTOLL molecules are administered following acute ischemic stroke.

[0199] It will be appreciated that the uses disclosed herein can alternatively be formulated as a method for improving anxiety / depression complications, daily activities, mobility and / or self-care in a subject after acute ischemic stroke, the method comprising administering an ApTOLL molecule to the subject, wherein the ApTOLL molecule is administered after acute ischemic stroke.

[0200] Administration and Dosage of ApTOLL Molecule

[0201] As described in Example 1, the APRIL study was divided into two parts, Phase Ib and Phase IIa. In Phase Ib, four increasing dose levels of SEQ ID NO: 1 (0.025, 0.05, 0.1, and 0.2 mg / kg) were administered, while in Phase IIa, two doses of SEQ ID ID: 1 (Dose A = 0.05 mg / kg and Dose B = 0.2 mg / kg) were administered. The results of Phase IIa (Example 1, 1.13-1.15) showed that Dose B at 0.2 mg / kg was effective for patients with AIS compared to patients in the placebo group and the Dose A group.

[0202] Thus, in some embodiments, the ApTOLL molecule of the invention (e.g., SEQ ID NO: 1) is administered at a dose of at least about 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.11 mg / kg, 0.12 mg / kg, 0.13 mg / kg, 0.14 mg / kg, 0.15 mg / kg, 0.16 mg / kg, 0.17 mg / kg, 0.18 mg / kg, 0.19 In some embodiments, the ApTOLL molecule is administered at a dose of at least about 0.2 mg / kg, specifically about 0.2 mg / kg. Even more specifically, SEQ ID NO: 1 is administered at a dose of at least about 0.2 mg / kg, specifically about 0.2 mg / kg.

[0203] In some embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered at a dosage ranging from about 0.05 mg / kg to about 200 mg / kg. In another embodiment, the dosage range is between about 0.06 mg / kg and about 50 mg / kg. In another embodiment, the dosage range is between about 0.06 mg / kg and about 10 mg / kg. In another embodiment, the dosage range is between about 0.1 mg / kg and about 5 mg / kg. Specifically, the dosage range is between about 0.1 mg / kg and about 1 mg / kg. Specifically, the dosage range is between about 0.15 mg / kg and about 1 mg / kg. Specifically, the dosage range is between about 0.2 mg / kg and about 1 mg / kg. Specifically, the dosage range is between about 0.2 mg / kg and about 0.5 mg / kg.

[0204] Taking into account a dosage range of about 0.1 mg / kg to about 1 mg / kg, and taking into account a standard weight of a human subject of about 70 kg, a standard single dosage amount is between about 7 mg / dose to about 70 mg / dose. Specifically, the ApTOLL molecule is administered at a dosage of at least about 14 mg / dose, specifically about 14 mg / dose. Even more specifically, SEQ ID NO: 1 is administered at a dosage of at least about 14 mg / dose, specifically about 14 mg / dose.

[0205] In some embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) can be administered by intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. In a specific embodiment, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered intravenously or intraarterially, for example, by infusion or by bolus injection.

[0206] In a more specific embodiment, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered intravenously by infusion. In a specific embodiment, the duration of the infusion is about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, or 60 minutes, and specifically about 30 minutes.

[0207] In some embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered less than 24 hours after the ischemic stroke event. In one embodiment, the ApTOLL molecule is administered less than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or less than about 24 hours after the ischemic stroke event.

[0208] In a specific embodiment, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered within about 8 hours, particularly about 6 hours, after stroke onset. In the APRIL study, the median duration of stroke onset in patients undergoing ApTOLL administration was 210 minutes (3.5 hours). Thus, in a specific embodiment, the ApTOLL molecule is administered within about 4 hours, more particularly within about 3.5 hours, after stroke onset.

[0209] In the APRIL study, ApTOLL (SEQ ID NO: 1) was administered intravenously with EVT and pharmacological thrombolysis, when indicated in the AIS target population. Generally, ApTOLL was administered before EVT and after thrombolysis, if indicated. The median time from ApTOLL infusion to recanalization (when achieved) was approximately 180 minutes (3 hours).

[0210] Thus, in some embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered prior to EVT (e.g., thrombectomy). In some embodiments, the ApTOLL molecule is administered about 6 hours prior to EVT. In other embodiments, the ApTOLL molecule is administered about 6 hours, 5.5 hours, 5 hours, 4.5 hours, 4 hours, 3.5 hours, 3 hours, 2.5 hours, 2 hours, 90 minutes, 60 minutes, 45 minutes, 30 minutes, 20 minutes, 10 minutes, or about 5 minutes prior to EVT. In one embodiment, the ApTOLL molecule is administered about 4 hours prior to EVT, particularly about 3 hours prior to EVT.

[0211] In other embodiments, the ApTOLL molecule (eg, SEQ ID NO: 1) is administered concurrently with EVT.

[0212] In other embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered after EVT. In one embodiment, the ApTOLL molecule is administered immediately after the end of EVT. In one embodiment, the ApTOLL molecule is administered approximately 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes after EVT.

[0213] In other embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered prior to and concurrently with EVT. In other embodiments, the ApTOLL molecule is administered prior to and immediately after EVT. In one embodiment, the ApTOLL molecule is administered at least about 20 minutes prior to and about 10 minutes after EVT.

[0214] In the APRIL study, patients received pharmacological thrombolysis (primarily alteplase, but some tenecteplase) when indicated. Alteplase was administered as a 1-2 minute bolus followed by a 60-minute infusion. Tenecteplase was administered as a 1-2 minute bolus. Generally, ApTOLL was administered after tPA, when indicated. Because alteplase administration lasted longer, this may coincide with the administration of ApTOLL.

[0215] In some embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered after drug thrombolysis (e.g., tPA administration). In one embodiment, the ApTOLL molecule is administered immediately after thrombolysis. In one embodiment, the ApTOLL molecule is administered about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes after thrombolysis. In another embodiment, the ApTOLL molecule is administered between about 5 minutes and about 30 minutes after thrombolysis.

[0216] In other embodiments, the ApTOLL molecule (eg, SEQ ID NO: 1) is administered concurrently with a thrombolytic drug.

[0217] In other embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered prior to and / or concurrently with drug thrombolysis. In other embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered concurrently with and / or after thrombolysis. In other embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered prior to and immediately after thrombolysis. In one embodiment, the ApTOLL molecule is administered at least about 20 minutes prior to and about 10 minutes after thrombolysis.

[0218] In some embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered after drug thrombolysis (e.g., tPA administration) and before EVT (e.g., thrombectomy). In one embodiment, the ApTOLL molecule is administered between about 5 minutes and about 30 minutes after thrombolysis and between about 4 hours and about 5 minutes before EVT.

[0219] In other embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered concurrently with and / or after drug thrombolysis and before and / or concurrently with EVT. In one embodiment, the ApTOLL molecule is administered concurrently with and immediately after thrombolysis and about 4 hours to about 5 minutes before and / or concurrently with EVT.

[0220] In some embodiments, the ApTOLL molecule (eg, SEQ ID NO: 1) is administered in multiple doses. In one embodiment, the ApTOLL molecule is administered in one, two, three, four, five, six, seven, eight, nine, or ten doses.

[0221] Arterial recanalization techniques

[0222] The uses and methods described herein further comprise additional pharmacological and / or surgical steps, for example, administration of additional ischemic stroke treatments or thrombotic disease treatments.

[0223] Thus, the ApTOLL molecules of the present invention are administered simultaneously with, before, or after another pharmacological and / or surgical procedure, particularly arterial recanalization. In particular, the ApTOLL molecules are administered in combination with arterial recanalization.

[0224] Arterial recanalization can be induced mechanically (eg, endovascular thrombectomy), pharmacologically (eg, thrombolysis), or a combination thereof. In one embodiment, arterial recanalization is mechanical, pharmacological, pharmacodynamic, or a combination thereof.

[0225] In one embodiment, arterial recanalization is mechanical. In a specific embodiment, mechanical arterial recanalization is an endovascular therapy (i.e., EVT). In a specific embodiment, EVT is selected from the group consisting of stent retriever thrombectomy, balloon embolectomy, direct aspiration embolectomy, surgical embolectomy, or a combination thereof.

[0226] In a specific embodiment, EVT is performed within 24 hours after the onset of an ischemic stroke. Specifically, EVT is performed within 8 hours after the onset of a stroke. More specifically, EVT is performed within 6 hours after the onset of a stroke.

[0227] In the APRIL study, the quality of reperfusion after EVT was assessed using the expanded Thrombolysis in Cerebral Ischemia (eTICI) scale. Each patient's recanalization status was determined as follows: (a) successful thrombectomy followed by recanalization (TICI 2b or 3); and (b) persistent LVO in patients who failed recanalization after EVT (TICI 0-2a). The recanalization rate reached 87%. In one embodiment, the final eTICI score was between 2b and 3. In one embodiment, the recanalization rate was at least about 87%.

[0228] In one embodiment, the arterial recanalization is pharmacological. In a specific embodiment, the pharmacological arterial recanalization is pharmacological thrombolysis. In a specific embodiment, the pharmacological thrombolysis is a fibrinolytic therapy. In a specific embodiment, the fibrinolytic is tissue plasminogen activator (tPA, alteplase) and modified alteplase (e.g., tenecteplase).

[0229] In a specific embodiment, the thrombolytic drug (eg, tPA) is administered within 4.5 hours of stroke onset.

[0230] In one embodiment, the arterial recanalization is pharmacomechanical. In a specific embodiment, the pharmacomechanical arterial recanalization comprises EVT and pharmacological thrombolysis (eg, tPA).

[0231] In some embodiments, the uses and methods described herein further comprise the use of imaging procedures, such as noncontrast computed tomography (NCCT), computed tomography angiography (CTA), computed tomography perfusion (CTP), and magnetic resonance imaging (MRI) images. Using published definitions and criteria, the following imaging and angiographic variables can be extracted from the imaging procedures: ASPECTS, hemorrhage, hemorrhage (Heidelberg), ischemic core (post hoc analysis DWI or CTP rCBF <30% volume (if available)), Tmax>6 seconds volume (if CTP or PWI available), baseline occlusive lesion location, CTA collateral score (based on availability - post hoc analysis), presence of proximal stenosis of the arterial occlusive lesion, arterial occlusive lesion, collateral flow grade - ASITN, eTICI on each device channel, distal embolization, new territories embolization, dissection, vessel perforation, final infarct volume after post hoc analysis DWI or CTP rCBF <30% volume (if available), etc. Imaging procedures can be performed pre-baseline (drip and ship hospital), at baseline, intra-operatively, post-operatively (24 hours), post-operatively (72 hours), or post-operatively.

[0232] Characterization of subjects

[0233] In the APRIL study, patients with moderate infarct volume (IV), cerebral artery occlusion, and disabling ischemic stroke at stroke onset (measured by NIHSS score, e.g., between 5 and 25) but functional independence before the stroke event (defined by pre-stroke mRS score, e.g., between 0 and 2) were selected to maximize the effect of ApTOLL (SEQ ID NO: 1). Therefore, different criteria were used to select subjects for ApTOLL treatment, such as age, baseline NIHSS, pre-stroke mRS score, infarct volume at stroke onset, and occlusion site, etc. However, these values are not limiting, and more patients may be eligible for treatment with the ApTOLL molecules of the present invention. All included parameters were measured at the time of stroke / symptom onset.

[0234] In some embodiments, the subject is a human subject. In one embodiment, the subject is a male. In other embodiments, the subject is a non-pregnant female.

[0235] In some embodiments, the subject is between about 18 and about 90 years old. In a specific embodiment, the subject is between about 60 and about 80 years old. In a specific embodiment, the subject is >70 years old, or about 70 years old. In a specific embodiment, the subject is about 75 years old.

[0236] The criterion for selecting subjects for treatment with the ApTOLL molecule (e.g., SEQ ID NO: 1) is the baseline NIHSS, which quantifies the severity of stroke in the acute setting. In some embodiments, the subject's baseline NIHSS is between about 5 and about 25. In some embodiments, the subject's baseline NIHSS is between about 8 and about 25. In one embodiment, the subject's baseline NIHSS is about 16. In one embodiment, the subject's baseline NIHSS is between about 11 and about 21. In one embodiment, the subject has a severe baseline NIHSS, i.e., between about 15 and about 24. In a specific embodiment, the subject's baseline NIHSS is between about 15 and about 20, specifically between about 15 and about 18.

[0237] Another criterion for selecting subjects for treatment with ApTOLL molecules (e.g., SEQ ID NO: 1) is a pre-stroke mRS score for determining neurological disability, ie, a subject's mRS score before an ischemic stroke event. In some embodiments, the subject's pre-stroke mRS score is between 0 and about 2.

[0238] In some embodiments, the criterion for selecting a subject for treatment with an ApTOLL molecule (e.g., SEQ ID NO: 1) is the time from symptom onset. Thus, in some embodiments, a subject is selected for treatment if the onset of symptoms is less than about 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or less than about 1 hour from the onset of the ischemic disease.

[0239] In some embodiments, the criterion for selecting a subject for treatment with an ApTOLL molecule (e.g., SEQ ID NO: 1) is whether the subject is a candidate for EVT, e.g., thrombectomy, with or without thrombolysis (e.g., intravenous tPA). In one embodiment, the indication for EVT is based on NCCT results (i.e., Alberta Stroke Project Early CT Score, ASPECTS of 6-10). In one embodiment, the subject's ASPECTS is between about 6 and about 10.

[0240] In some embodiments, the criterion for selecting subjects for treatment with an ApTOLL molecule (eg, SEQ ID NO: 1) is the presence of a single LVO (TICI 0 or TICI 1 flow) at the level of the M1 or M2 segments of the TICA or middle cerebral artery at the time of stroke or symptom onset.

[0241] Another criterion for selecting subjects for treatment with an ApTOLL molecule (e.g., SEQ ID NO: 1) is infarct volume. In some embodiments, the subject has a moderate infarct volume. In some embodiments, the subject has an infarct volume of between about 5 cc and about 70 cc at stroke onset or distribution. In a specific embodiment, the subject has an infarct volume (defined as cerebral blood flow (CBF)) <30% on CT perfusion (CTP), between about 5 ml and about 70 ml, as determined by automated software ( software) measurements.

[0242] In some embodiments, the criteria for selecting subjects for treatment with an ApTOLL molecule (e.g., SEQ ID NO: 1) is vascular occlusion suitable for mechanical thrombectomy, e.g., as determined or confirmed by computed tomography angiography (CTA). In one embodiment, the criteria for subjects for treatment with an ApTOLL molecule is LVO suitable for mechanical thrombectomy as determined or confirmed by neuroimaging criteria (CT or MRI), such as:

[0243] (i) Magnetic resonance imaging (MRI) criteria: Diffusion-weighted imaging (DWI) stenosis volume ≥ approximately 5 mL and ≤ approximately 70 mL, e.g., by Software determination; and / or,

[0244] (ii) Computed tomography (CT) criteria: Alberta Stroke Program Early CT Score (ASPECTS) of approximately 6 to 10 and an infarct score (CBF) of <30% and ≥ approximately 5 mL and ≤ 70 mL determined on admission, e.g. Software confirmed.

[0245] In one embodiment, the subject's DWI limiting volume is between about 5 mL and about 70 mL. In another embodiment, the subject's ASPECTS is between about 6 and about 10.

[0246] In one embodiment, the subject, at the time of stroke:

[0247] i) aged between about 18 and about 90 years old;

[0248] ii) having a baseline NIHSS between about 8 and about 25;

[0249] iii) pre-stroke mRS score between 0 and approximately 2 points;

[0250] iv) having an infarct volume of about 5 cc to about 70 cc;

[0251] v) symptom onset less than approximately 6 hours;

[0252] vi) are candidates for EVT treatment;

[0253] vii) occlusion of the TICA, M1, or M2 segment of the middle cerebral artery;

[0254] viii) mTICI score of 0 or 1;

[0255] ix) having a DWI confinement volume of about 5 mL to about 70 mL;

[0256] x) has an ASPECTS between about 6 and about 10; or

[0257] xi) Any combination of the above.

[0258] In a specific embodiment, the subject has a baseline NIHSS of between about 8 and about 25 at stroke onset or allocation, a pre-stroke mRS score of between 0 and about 2, and an infarct volume of between about 5 cc and about 70 cc.

[0259] In a specific embodiment, the subject has a single LVO at the level of the TICA or M1 or M2 segments of the middle cerebral artery, defined as an infarct core volume <30% on CTP of cerebral blood flow, between about 5 ml and about 70 ml, as determined by automated software ( iSchemaView) measurements.

[0260] In a specific embodiment, the subject is between about 18 and about 90 years old, has LVO within a 6-hour window, an ASPECTS between about 6 and about 10, and an estimated infarct core volume on CT perfusion between about 5 ml and about 70 ml.

[0261] In the APRIL study, administration of 0.2 mg / kg ApTOLL was observed to have a greater effect on final infarct volume and long-term functional improvement (mRS) in subjects over 70 years of age. Therefore, in one embodiment, the subject is >70 years of age. For patients with a baseline NIHSS greater than 15, administration of 0.2 mg / kg ApTOLL appears to have a greater effect on reduction in infarct volume and mRS score. Therefore, in one embodiment, the subject has a baseline NIHSS greater than 15.

[0262] ApTOLL molecule

[0263] As used herein, the term "AptoLL molecule" refers to an aptamer selected from SEQ ID NOs: 1-24 or SEQ ID NOs: 1-16, a variant and / or derivative thereof, or a chemically modified aptamer thereof. The APTOLL molecule has the ability to specifically bind to at least one epitope located on the extracellular domain of TLR-4 and inhibit TLR-4. Specific details and features of all of these aptamers are disclosed in WO2015197706A1, WO2020230108A1, and WO2020230109A1 (AptaTargets SL), the entire contents of which are incorporated herein by reference.

[0264] The aptamers of SEQ ID NOs: 1-16 range in length from 45 to 78 nucleotides. The aptamers of SEQ ID NOs: 17-22 are variants (mutants) of the ApTOLL sequence (SEQ ID NO: 1), and SEQ ID NOs: 23 and 24 are mutants of the 4F aptamer (SEQ ID NO: 4). SEQ ID NO: 4 is 100% identical to SEQ ID NO: 1 in its central region and has 5' and 3' extensions relative to SEQ ID NO: 1. Example 3 shows that all of these variants, which have different sequence identities and / or extension percentages at the 5' and 3' ends of ApTOLL (SEQ ID NO: 1), have comparable TLR4 antagonistic activity to ApTOLL (SEQ ID NO: 1).

[0265] In some embodiments, the ApTOLL molecule of the present invention is an aptamer selected from SEQ ID NOs: 1-24, specifically SEQ ID NOs: 1-16, more specifically SEQ ID NOs: 1-4. In a more specific embodiment, the aptamer is SEQ ID NO: 1.

[0266] In other embodiments, the ApTOLL molecule is a variant and / or derivative of an aptamer selected from SEQ ID NOs: 1-24, specifically SEQ ID NOs: 1-16, more specifically SEQ ID NOs: 1-4, and even more specifically SEQ ID NO: 1. In some embodiments, the ApTOLL molecule is a variant and / or derivative having at least 70% sequence identity with SEQ ID NOs: 1-24, wherein the variant and / or derivative is derived from SEQ ID NOs: 1-24 and retains the ability to specifically bind to and reduce and / or inhibit TLR-4 activation. Specifically, the ApTOLL molecule comprises a sequence that is at least 85% identical to SEQ ID NOs: 1-24, and more specifically, a sequence that is at least 90% or 95% identical to SEQ ID NOs: 1-24. In some embodiments, the ApTOLL molecule is a variant and / or derivative having at least 70% sequence identity to SEQ ID NOs: 1-16, wherein the variant and / or derivative is derived from SEQ ID NOs: 1-16 and retains the ability to specifically bind to and reduce and / or inhibit TLR-4 activation. Specifically, the ApTOLL molecule comprises a sequence that is at least 85% identical to SEQ ID NOs: 1-16, and more specifically, a sequence that is at least 90% or 95% identical to SEQ ID NOs: 1-16.

[0267] In other embodiments, the ApTOLL molecule is a chemically modified aptamer selected from the group consisting of SEQ ID NOs: 1-24, specifically SEQ ID NOs: 1-16, more specifically SEQ ID NOs: 1-4, and even more specifically SEQ ID NO: 1. Chemical modifications of the aptamer include base modifications (e.g., 2'-O-methyl U, 2'-O-methyl C), backbone modifications (e.g., 3'-alkylene phosphonate, PNA, inverted T), and sugar modifications (e.g., LNA), among others.

[0268] In some embodiments, the length of the ApTOLL molecule is between about 40 and about 100 nucleotides, more particularly, the length of the ApTOLL molecule is between about 45 and about 78 nucleotides, and more particularly, between about 59 and about 76 nucleotides.

[0269] In some embodiments, the ApTOLL molecule has an optional 5' extension of nucleotide sequence between 1 and 13 nucleotides in length. In other embodiments, the ApTOLL molecule has an optional 3' extension of nucleotide sequence between 1 and 4 nucleotides in length.

[0270] In a more specific embodiment:

[0271] (a) The length of the ApTOLL molecule is between 40 and 100 nucleotides and is selected from the group consisting of SEQ ID NO: 1, 2, 3 and 4, wherein

[0272] (i) the ApTOLL molecule specifically binds to an epitope on the extracellular domain of TLR-4; and,

[0273] (ii) reduced binding of the ApTOLL molecule to the epitope and / or inhibition of TLR-4 activation; or

[0274] (b) The ApTOLL molecule is a functionally equivalent variant of the ApTOLL molecule of (a) having at least 85% sequence identity to SEQ ID NO: 1, 2, 3 or 4, wherein the functionally equivalent variant is derived from SEQ ID NO: 1, 2, 3 or 4 and retains the ability to specifically bind to and reduce and / or inhibit TLR-4 activation.

[0275] In a more specific embodiment, the ApTOLL molecule is SEQ ID NO: 1 (designated ApTOLL in the APRIL study). In another specific embodiment, the ApTOLL molecule is a variant and / or derivative of SEQ ID NO: 1 having at least 70% sequence identity (more specifically 85%) to SEQ ID NO: 1, wherein the variant and / or derivative is derived from SEQ ID NO: 1 and retains the ability to specifically bind to and reduce and / or inhibit TLR-4 activation. In another embodiment, the ApTOLL molecule is a chemically modified aptamer of SEQ ID NO: 1.

[0276] The chemical formula of ApTOLL is C 575 H 723 N 223 O 351 P 58 , and its molecular weight is 18170.80Da. The tertiary structure of ApTOLL is as follows Figure 6 shown.

[0277] The ApTOLL molecule is formulated into a pharmaceutical composition suitable for administration to a subject. In the APRIL study, ApTOLL and placebo were formulated as a concentrate powder for infusion, reconstituted with 3 mL of water for injection, and diluted according to the patient's weight in a saline bag (100 mL of a 9 mg / mL [0.9%] sodium chloride injection solution). The resulting solution can be injected intravenously, for example, via an infusion pump. In one embodiment, the ApTOLL molecule is presented as a 7 mg concentrate lyophilized powder vial for intravenous administration.

[0278] In one embodiment, the ApTOLL molecule is formulated in PBS-MgCl2. In one embodiment, the formulation includes sodium chloride, potassium chloride, dehydrated sodium hydrogen phosphate, and potassium dihydrogen phosphate to produce a phosphate buffer solution at pH 7.4, which includes magnesium chloride hexahydrate. This buffer solution and conditions support the aptamer structure and its biological activity. In the placebo formulation, ApTOLL was replaced with A-trehalose dihydrate.

[0279] Specific implementation methods

[0280] It will be clear to those skilled in the art after reading this specification that each individual embodiment described and illustrated herein has its own components and features that can be combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Specific combinations of the above embodiments described in detail in different sections are described herein.

[0281] The present invention relates to ApTOLL molecules, specifically aptamers having SEQ ID NO: 1 (tested in the APRIL study) for use in reducing the risk of intracranial hemorrhage in a subject after acute ischemic stroke, wherein the aptamer is administered at a dose of about 0.5 to about 1 mg / kg after acute ischemic stroke, and wherein administration of the aptamer reduces the risk of intracranial hemorrhage by about 5% to about 40% relative to a subject not treated with the aptamer. Specifically, administration of the aptamer reduces the risk of intracranial hemorrhage by at least about 14%. In one embodiment, the intracranial hemorrhage is symptomatic intracranial hemorrhage, and administration of the aptamer reduces the risk of symptomatic intracranial hemorrhage by about 5% to about 40%, specifically by at least about 34%, relative to a subject not treated with the aptamer. Specifically, the aptamer is administered at a dose of at least about 0.2 mg / kg.

[0282] The present invention also relates to ApTOLL molecules, specifically aptamers having SEQ ID NO: 1, for use in reducing neural injury and improving neural recovery in a subject after acute ischemic stroke, wherein the reduction in neural injury and the improvement in neural recovery are mediated by reducing the risk of intracranial hemorrhage, wherein the aptamer is administered at a dose of about 0.5 to about 1 mg / kg after acute ischemic stroke, and wherein, relative to a subject not treated with the aptamer, the aptamer:

[0283] (i) reduce infarct volume,

[0284] (ii) reduction in NIHSS score, and / or

[0285] (iii) increasing the mRS score of 0-2 (ie, decreasing the mRS score to 0-2). Specifically, the aptamer is administered at a dose of at least about 0.2 mg / kg.

[0286] Specifically, the present invention also relates to ApTOLL molecules, specifically an aptamer having SEQ ID NO: 1, for use in improving neurological recovery in a subject after acute ischemic stroke, wherein the improvement in neurological recovery is mediated by reducing the risk of intracranial hemorrhage, wherein the aptamer is administered at a dose of about 0.5 to about 1 mg / kg after acute ischemic stroke, and wherein, relative to a subject not treated with the aptamer:

[0287] (i) reduce NIHSS score, and / or

[0288] (ii) reducing mRS to 0-2. Specifically, the aptamer is administered at a dose of at least about 0.2 mg / kg. In another embodiment, the aptamer further reduces infarct volume.

[0289] In one embodiment, the reduction in infarct volume at 72 hours is between about 10% and about 50%, specifically at least about 46%, relative to a subject not treated with the aptamer. In another embodiment, the reduction in infarct volume over time is between about 10% and about 50%, specifically at least about 40%, relative to a subject not treated with the aptamer.

[0290] In one embodiment, the reduction in NIHSS score at 72 hours is between about 40% and about 60%, specifically at least about 57%, relative to a subject not treated with the aptamer.

[0291] In one embodiment, the increase in mRS score 0-2 (i.e., a decrease in mRS to 0-2) at 90 days is between about 10% and about 40%, specifically at least about 36%, relative to subjects not treated with the aptamer. In another embodiment, the increase in mRS score 0-2 (i.e., a decrease in mRS to 0-2) over time is between about 10% and about 50%, specifically at least about 31%, relative to subjects not treated with the aptamer.

[0292] In one embodiment, the aptamer reduces brain edema by about 40% to about 80%, specifically at least about 67%, relative to a subject not treated with the aptamer.

[0293] In one embodiment, the aptamer is administered within about 8 hours after stroke onset, specifically within about 4 hours after stroke onset.

[0294] In one embodiment, the aptamer is administered in conjunction with arterial recanalization.

[0295] In one embodiment, the aptamer is administered in conjunction with an intravascular treatment. In a specific embodiment, the aptamer is administered prior to or concurrently with the intravascular treatment. Specifically, the aptamer is administered less than about 4 hours prior to the intravascular treatment.

[0296] In one embodiment, the aptamer is administered in combination with a thrombolytic drug, specifically a tissue plasminogen activator. Specifically, the aptamer is administered simultaneously with the thrombolytic drug or after the thrombolytic drug.

[0297] In one embodiment, the aptamer is administered intravenously by infusion over about 30 minutes.

[0298] In one embodiment, the subject, at the time of stroke:

[0299] i) aged between about 18 and about 90 years old;

[0300] ii) having a baseline NIHSS between about 8 and about 25;

[0301] iii) pre-stroke mRS score between 0 and approximately 2 points;

[0302] iv) having an infarct volume of about 5 cc to about 70 cc;

[0303] v) occlusion of the TICA, M1, or M2 segments of the middle cerebral artery;

[0304] vi) mTICI score of 0 or 1;

[0305] vii) having a diffusion-weighted imaging limiting volume between about 5 mL and about 70 mL;

[0306] viii) having an ASPECTS between about 6 and about 10; or

[0307] ix) any combination of the above,

[0308] The contents of all citations (including literature references, patents, patent applications, and websites) that may be cited in this application are hereby expressly incorporated by reference in their entirety for any purpose, as if they were references cited herein.

[0309] The following examples are offered by way of illustration only and not limitation.

[0310] Example

[0311] Example 1: APRIL: Double-blind, placebo-controlled, randomized, Phase Ib / IIa clinical trial of ApTOLL for the treatment of AIS

[0312] Trial Identifier: EudraCT:2020-002059-38 and ClinicalTrials.gov Identifier: NCT04734548 Methods and Analysis

[0313] Overall study design

[0314] APRIL was a multicenter, double-blind, randomized, placebo-controlled, parallel-group, Phase Ib / IIa clinical trial designed to evaluate the safety and tolerability, pharmacokinetics, and biologic effects of ApTOLL in patients with AIS who had established LVO and were candidates for endovascular therapy with or without intravenous tPA. The study population consisted of eligible men and women in whom ApTOLL could be administered within 6 hours of symptom onset. Phase IIa allocation was stratified into 3 strata: initial infarct core size (<35 vs. ≥35 cc), patient age (<70 vs. ≥70 years), and NIHSS score (<15 vs. ≥15). For patients with awake stroke, onset was considered the time of first symptom onset.

[0315] The APRIL clinical trial was divided into two parts:

[0316] (1) Phase Ib (n=32) single intravenous administration (30 min infusion), dose escalation across 4 single dose levels (8 patients / level), randomized (1:3); and

[0317] (2) Phase IIa (n=119) Single dose, intravenous administration (30 min infusion), parallel (3 groups, placebo: ApTOLL dose A: ApTOLT dose B), randomized (√2:1:1). Therefore, after the completion of Phase Ib, the Data Safety Monitoring Board (DSMB) conducted an unblinded review of the study groups based on preliminary safety results and selected two doses (A and B) for testing in Phase IIa.

[0318] Prior to enrollment, all patients underwent a complete neuroimaging study, including noncontrast computed tomography (NCCT), CT angiography, and CT perfusion (CTP). Only patients with an indication for EVT based on NCCT results (i.e., Alberta Stroke Program Early CT Score, ASPECTS>5) were assessed as candidates for the APRIL study. Further neuroimaging inclusion criteria were the presence of a single LVO at the level of the TICA or middle cerebral artery M1 or M2 segments and the identification of a favorable CTP spectrum to maximize the chance of identifying the biological effect of the study drug. The predicted infarct core volume on CTP, defined as CBF <30% and between 5 and 70 ml, had to be determined using automated software ( software) to be eligible for the APRIL study. Full inclusion and exclusion criteria are detailed in Section 1.3. After obtaining informed consent, Figure 5 As shown, patients were randomly assigned to receive EVT plus ApTOLL versus EVT plus placebo. Study medication was administered after imaging acquisition and before the start of EVT (groin puncture). All patients were then treated according to institutional protocols and national and European Stroke Organization (ESO) guidelines. Figure 1 The protocol of the APRIL study is summarized.

[0319] 1.2. Goals and End Points

[0320] The aim of the APRIL study was to evaluate whether ApTOLL is safe and to show any biologic effect in AIS patients with LVO.

[0321] Primary objective: To assess whether ApTOLL administered intravenously at escalating doses is safe and well tolerated compared with placebo when administered EVT in the target population of AIS.

[0322] Secondary objectives: Although this is a unique study, the secondary objectives of the two study parts are different:

[0323] Phase Ib :

[0324] To evaluate the pharmacokinetic characteristics of ApTOLL in patients with AIS by measuring ApTOLL levels in plasma and urine.

[0325] • Two doses administered in Phase IIa were selected based on their safety profile.

[0326] To provide preliminary estimates of the biological effects of ApTOLL on final infarct volume (measured by MRI-FLAIR [magnetic resonance image-fluid attenuated inversion recovery] at 72 ± 24 hours) and on pro-inflammatory biomarkers associated with AIS at baseline (before dosing) and at the end of infusion (within [1 hour], 6 hours, 24 hours, 48 hours, and 72 hours after dosing).

[0327] Stage IIa :

[0328] The biological effects of ApTOLL on final infarct volume (measured by MRI-FLAIR at 72 ± 24 h) and on pro-inflammatory biomarkers associated with AIS (before administration and at 6, 24, 48, and 72 h after administration) were evaluated.

[0329] To determine the biological effects of ApTOLL, as measured by functional impairment 72 hours after stroke (NIHSS [National Institute of Health Stroke Scale]) or hospital discharge (whichever occurs first) and disability 90 days after stroke (modified Ranking Scale [mRS]).

[0330] To achieve these objectives, the following endpoints were assessed:

[0331] Primary endpoint: Safety of ApTOLL combined with EVT, as determined by mortality, adverse events occurring during the study period, physical examinations, laboratory tests, recurrent stroke rate, and symptomatic intracranial hemorrhage (sICH) rate.

[0332] Secondary endpoints:

[0333] The mean final infarct volume measured by MRI was 1.72±24h.

[0334] 2. Pro-inflammatory markers in the blood.

[0335] 3. Early clinical course (NIHSS at 72 hours).

[0336] 4. Long-term results (mRS at 90 days).

[0337] 5. Neuroimaging proinflammatory biomarkers in MRI at 72 hours and 90 days after stroke (substudy conducted only at some APRIL Spanish sites).

[0338] Inclusion and exclusion criteria

[0339] Participants were recruited at comprehensive stroke centers in Spain and France. Mechanical thrombectomy involved the use of any commercially available stent retriever or aspiration catheter, or a combination of these, as recommended by the ESO guidelines.

[0340] 1.3.1. Inclusion criteria

[0341] 1. Aged ≥ 18 and ≤ 90 years.

[0342] 2. Obtain informed consent from the subject or an acceptable surrogate (i.e., next of kin or legal representative).

[0343] 3. A new focal disabling neurologic deficit consistent with acute cerebral ischemia.

[0344] 4. Baseline NIHSS score obtained before randomization ≥8 points and ≤25 points.

[0345] 5. The mRS score before stroke was 0-2.

[0346] 6. Treatment is available as soon as possible and at least within 6 hours of symptom onset, defined as the time the subject last appeared normal (baseline). For awakening stroke, onset is considered the time of first symptom onset. (Treatment initiation is defined as administration of study drug).

[0347] 7. Candidates receiving EVT with or without intravenous tPA. For such patients who are to receive intravenous tPA, tPA should be initiated according to the European Stroke Organization recommendations for the early management of patients with AIS, meaning as soon as possible and within 4.5 hours after stroke onset (onset time defined as the last time the patient was confirmed to be well at baseline), with the investigator verifying that the subject has received / is receiving the correct intravenous tPA dose for estimated weight. For any reason, intravenous tPA should be stopped prematurely, and the reason and total administered dose should be recorded. In addition, once a patient is enrolled in the study, if recanalization is observed and documented before thrombectomy, the patient will continue in the trial and no protocol deviation will be recorded.

[0348] Specific neuroimaging inclusion criteria

[0349] 8. Patients with TICA, middle cerebral artery M1, or M2 segment occlusion (TICI 0 or TICI 1 flow) confirmed by CTA are suitable for mechanical thrombectomy. This may include tandem intracranial and extracranial lesions.

[0350] 9. Admission neuroimaging examinations should also meet the following imaging criteria:

[0351] a)MRI standards: Software-determined DWI (diffusion-weighted imaging) limit volume ≥5 mL and ≤70 mL, or

[0352] b) CT criteria: Alberta Stroke Program Early CT Score (ASPECTS) 6 to 10 on baseline CT and CBF <30% of the infarct core determined by CT perfusion on admission: ≥5 mL and ≤70 mL, determined by Software confirmed.

[0353] Note: ASPECTS are determined by investigator criteria.

[0354] 10. The subject is eligible and plans to receive endovascular treatment for stroke according to ESO guidelines.

[0355] 1.3.2. Exclusion criteria

[0356] 1. The subject has had a stroke in the past year.

[0357] 2. Occlusion of the basilar artery, vertebral artery, posterior cerebral artery, or anterior cerebral artery (TICI 0 or TICI 1 blood flow).

[0358] 3. Clinical symptoms suggestive of bilateral or multiple strokes.

[0359] 4. Patients receiving oral anticoagulant therapy with known bleeding diathesis, coagulation factor deficiency, or INR>3.0.

[0360] 5. Baseline platelet count <50,000 / μL.

[0361] 6. Baseline blood glucose <50 mg / dL or >400 mg / dL.

[0362] 7. Severe, persistent hypertension (systolic blood pressure > 185 mmHg or diastolic blood pressure > 110 mmHg).

[0363] NOTE: Patients may be enrolled if their blood pressure can be successfully lowered and maintained at an acceptable level using medications recommended by the European Stroke Organization (ESO) guidelines, including intravenous antihypertensive drops.

[0364] 8. Severe, advanced or terminal illness with life expectancy of less than 1 year.

[0365] 9. Subjects with identifiable intracerebral tumors (meningiomas are considered extracerebral tumors and are therefore not included in this exclusion criterion).

[0366] 10. History of life-threatening allergic reaction to contrast media (more than rash).

[0367] 11. Known renal insufficiency, creatinine ≥3 mg / dL or glomerular filtration rate (GFR) <30 mL / min.

[0368] 12. Cerebral vasculitis.

[0369] 13. Evidence of active systemic infection.

[0370] 14. Known cocaine use at the time of treatment.

[0371] 15. Patients participating in studies involving investigational drugs or devices that may affect this study.

[0372] 16. Patients for whom 90-day follow-up is unlikely (e.g., no fixed home address, visitors from overseas).

[0373] 17. Women who are pregnant or breastfeeding or have a positive pregnancy test at the time of admission.

[0374] Specific neuroimaging exclusion criteria

[0375] 18. CT or MRI evidence of bleeding (microbleeding is allowed).

[0376] 19. Obvious mass effect and midline shift.

[0377] 20. Suspected aortic dissection with possible septic embolus or suspected bacterial endocarditis.

[0378] 1.4. Enrollment and Randomization

[0379] All patients who met the eligibility criteria were eligible to participate in the APRIL clinical trial. This included patients who were admitted directly to the study site and those who were transferred (drip and ship) from a primary stroke center.

[0380] 1. Treatment blinding

[0381] The study was conducted in a double-blind fashion, meaning that neither the patients nor the treating physicians knew which treatment was being administered. The ApTOLL or matching placebo solutions were clear, transparent, and colorless.

[0382] 2. Enrollment and Randomization Phase Ib

[0383] In Phase Ib, four ascending dose levels (0.025, 0.05, 0.1, and 0.2 mg / kg) were completed. Within each dose level cohort, eight patients were randomized to receive ApTOLL plus EVT versus placebo plus EVT in a 3:1 ratio.

[0384] A staggered dosing schedule was used to maximize patient safety. After each dose level (with a 72-hour follow-up of the last patient included in the corresponding dose level), the DSMB evaluated the safety results and approved the next dose level if appropriate. Safety parameters to be evaluated included: (1) any SUSAR (suspected serious adverse reaction), SAE (serious adverse event), or AE that may be related to drug administration, and (2) blood biochemical parameters.

[0385] 3. Transition from Phase Ib to Phase IIa

[0386] Prior to initiating Phase IIa, once safety data from the last Phase Ib patient were available, the DSMB evaluated the overall safety results to select the two optimal doses (Dose A, Dose B) for the Phase IIa study:

[0387] 1. Safety parameters related to stroke:

[0388] -die.

[0389] - Intracranial hemorrhage (ICH) and hemorrhagic transformation (HT) leading to new symptoms or worsening of existing symptoms.

[0390] - Cerebral edema leading to brain herniation and neurologic deterioration or death. Other potential AEs related to stroke include seizures; cardiac conduction disorders, arrhythmias; effects on coagulation and fibrinolysis; hypotension / hypertension; hyperglycemia; hyperpyrexia; serious infections; deep vein thrombosis, pulmonary embolism, and venous thromboembolism; vomiting; and anxiety, hallucinations, and agitation.

[0391] 2. Drug-related safety parameters:

[0392] - Complement activation: CH50 (50% hemolytic complement) and C3 / C4 (complement factors 3 and 4) levels.

[0393] -Biochemistry: CK (creatine kinase) and CRP (C-reactive protein)

[0394] - Coagulation parameters: aPTT (activated partial thromboplastin time), prothrombin activity (PT) and INR (International Normalized Ratio).

[0395] 4. Phase IIa Enrollment and Randomization

[0396] In Phase IIa, three arms were studied (placebo and ApTOLL at doses A or B). Eligible patients were randomized in a √2:1:1 ratio to placebo plus EVT, ApTOLL (dose A) plus EVT, and ApTOLL (dose B) plus EVT, resulting in randomization probabilities of 0.41, 0.29, and 0.29, respectively.

[0397] In Phase IIa, the DSMB analyzed AEs and SAEs when each treatment group included approximately 20 patients and all patients had reached 72-hour follow-up. In Phase Ib and Phase IIa, the CAC (Clinical Review Committee) analyzed all AEs and SAEs to determine which were drug-related and communicated this determination to the DSMB. Based on this information, the DSMB reviewed the reported AEs and SAEs.

[0398] Study Treatment

[0399] Both treatments (ApTOLL and placebo) were administered intravenously over 30 minutes using an infusion pump.

[0400] 1. Study Treatment

[0401] ApTOLL is presented as a 7 mg concentrate powder vial for intravenous administration as an infusion solution. ApTOLL and placebo are formulated as a concentrate powder for infusion, reconstituted with 3 mL of water for injection, and diluted according to patient weight in a saline bag (100 mL of 9 mg / mL [0.9%] sodium chloride injection solution). The placebo treatment matched the appearance of ApTOLL, both as a concentrate powder for infusion and upon reconstitution. The formulation containing ApTOLL consists of a lyophilisate containing sodium chloride, potassium chloride, sodium hydrogen phosphate dehydrate, and potassium dihydrogen phosphate to produce a phosphate buffer solution at pH 7.4, which includes magnesium chloride hexahydrate. In the placebo formulation, the API (i.e., ApTOLL) was replaced with A-trehalose dihydrate.

[0402] For example, for a 70 kg Grade 3 patient, 7 mg should be administered, so 3 mL should be taken from the stock solution and subsequently diluted with 100 mL of 9 mg / mL (0.9%) Sodium Chloride Injection.

[0403] Administration of ApTOLL / placebo should begin before EVT (before groin puncture). In each case, the infusion rate should not exceed 2.67 mg / min (equivalent to 3.33 mL / min upon reconstitution and dilution). ApTOLL and placebo should be stored at -20°C prior to reconstitution and dilution. Reconstitution should be completed just prior to administration (maximum 10 minutes before injection into the saline bag).

[0404] The dose regimen for Phase Ib is as follows (Table 1):

[0405] Table 1. Phase Ib dose escalation

[0406]

[0407] 2. Drugs / treatments allowed and not allowed before and during clinical studies

[0408] In addition to the study drug, patients were treated according to ESO guidelines.

[0409] 1.6. Study Assessment and Surgery

[0410] Treatment process such as Figure 5 Each subject underwent the following surgeries (Table 2). The study duration for each subject was a maximum of 90 days.

[0411] Table 2. Timeline of events

[0412]

[0413]

[0414] X 1 - NIHSS performed on day 5 or at discharge (whichever occurs first).

[0415] X 2 - In Phase Ib, pro-inflammatory biomarker samples were collected before dosing, at the end of infusion (up to 1 hour), and 6, 24, 48, and 72 hours after dosing. In Phase IIa, pro-inflammatory biomarkers were analyzed before dosing and 6, 24, 48, and 72 hours after dosing.

[0416] X 3 -Pharmacokinetic samples collected before dosing and at the end of the infusion (up to 1 hour), and 6 hours, 24 hours, 48 hours, and 72 hours after the end of the infusion.

[0417] X 4- If possible, collect a urine sample up to 24 hours after study drug administration.

[0418] X 5 -First ECG examination performed 6 hours after drug administration.

[0419] X 6 - Only hospitals included according to MRI criteria (ie, France).

[0420] X 7 - Applicable only to sites participating in the IMAGE substudy.

[0421] EVT Details 8 - Inguinal puncture performed before 8 hours after symptom onset. For wake-up stroke, onset time is considered to be the time of first symptom awareness.

[0422] X 9 -Please fill in the cause of stroke if any.

[0423] Endovascular Thrombectomy (EVT details) EVT (inguinal puncture) was initiated after randomization and study drug administration. EVT was initiated within 8 hours of symptom onset (ApTOLL was administered within 6 hours of stroke onset) (for wake-up stroke, onset was considered the time of first symptom onset). Individual investigators used any approved device or any combination of devices to clear thrombi in the TICA, MCAM1 segment, or, if indicated, the M2 segment of the intracranial circulation.

[0424] Imaging procedures : Plain CT, CTA, CTP and MRI.

[0425] CT images were read by appropriately trained local clinicians. ASPECTS for patient selection was independently determined by local clinicians. CTP images with mismatch determination were read by iSchemaView automated RAPID software. All brain imaging from stroke onset to discharge, including MRI, CTP / MRP, and CT, as well as angiographic images obtained for the diagnostic and therapeutic portions of the procedure, were evaluated independently of each other at the APRIL Central Imaging Core Laboratory (ICL) and were blinded to treatment allocation. The following imaging and angiographic variables were extracted using published definitions and criteria based on previous direct experience with these measures and scales:

[0426] Baseline:

[0427] ASPECTS

[0428] Ischemic core – where available, post hoc DWI or CTP rCBF <30% volume

[0429] Tmax > 6 seconds capacity (if CTP or PWI is available)

[0430] Bleeding

[0431] Baseline occlusive lesion location

[0432] CTA parallel scoring – based on usability (after the fact)

[0433] Operation:

[0434] Stenosis proximal to the arterial occlusive lesion

[0435] Arterial occlusive disease

[0436] Parallel mobility level-ASITN

[0437] Each eTICI passing through the device

[0438] Distal embolization

[0439] Embolization to new areas

[0440] ·Anatomy

[0441] Vascular perforation

[0442] Post-operatively (24 hours):

[0443] Infarct volume

[0444] Hemorrhage (Heidelberg)

[0445] Post-operatively (72 hours):

[0446] Final infarct volume on post-hoc DWI (or on CT if MRI is not available)

[0447] Hemorrhage (Heidelberg)

[0448] Postoperative neurological deterioration or course of disease:

[0449] Relevant imaging findings and measurements based on availability (infarct volume on all available follow-up images)

[0450] In the Phase IIa substudy, a second MRI was performed on day 90 after surgery within the context of the imaging substudy, and the total and relative infarct volumes (gray matter / white matter) at 90 days were determined. In addition, cerebral perfusion parameters using IVIM (intravoxel randomized motion) sequences at 72 ± 24 hours, microstructural indices using DTI (diffusion tensor imaging) at 72 ± 24 hours and 90 days, and brain tissue iron burden were measured at 72 ± 24 hours and 90 days.

[0451] Fibrinolytic therapyPatients received standard ESO guideline-directed medical therapy, which may include intravenous tPA for patients presenting within the first 4.5 hours after their last normal examination and meeting other ESO labeling criteria. Post-tPA patients were treated according to the standard study site protocol for these patients. The timing of tPA infusion and the administered dose were collected.

[0452] result

[0453] 1.7. Study Design and Participants

[0454] APRIL is a double-blind, randomized, multicenter, placebo-controlled Phase Ib / IIa clinical study designed to evaluate whether ApTOLL administration is safe, well-tolerated, and shows any biological effects in stroke patients with confirmed LVO undergoing EVT (with or without intravenous thrombolysis). The trial was conducted at 15 comprehensive stroke centers in Spain (12 sites) and France (3 sites). The study was approved by the ethics committees and national regulatory authorities at each site. Informed consent was obtained from patients or their legally authorized representatives.

[0455] Eligible patients were men and nonpregnant women aged 18 to 90 years who had a disabling ischemic stroke at randomization (baseline National Institutes of Health Stroke Scale [NIHSS] 5 to 25; range 0-42, with higher scores indicating greater stroke severity); functional independence before stroke, defined as a modified Rankin Scale (mRS) score between 0 and 2, with scores ranging from 0 (no symptoms) to 6 (death); and ApTOLL administered within 6 hours of symptom onset. For patients with stroke who woke up, the onset time was considered the time of first symptom detection, as safety is not expected to be questioned if the study drug is administered within 6 hours of actual stroke onset.

[0456] Patients with a single LVO at the level of the M1 or M2 segments of the middle cerebral artery who were considered candidates for EVT on non-invasive vascular imaging based on noncontrast computed tomography (NNCT) findings (Alberta Stroke Program Early CT Score [ASPECTS] 6-10; range 0-10, minus 1 point for any evidence of early ischemic changes in each defined area on the CT scan) were evaluated as candidates for the APRIL study. To maximize the chance of determining the biological effect of the study drug, a specific CT perfusion (CTP) profile as previously defined (Olive-Gadea M et al., 2021) was also required for inclusion. The predicted infarct core volume on CTP was defined as a relative cerebral blood flow <30% between 5 and 70 ml and had to be determined using previously validated automated software ( iSchemaView) to identify eligible for the APRIL study.

[0457] Eligible imaging was planned to be performed at an endovascular center; in a few cases (n=2), eligible imaging was performed at the primary stroke center before transfer to a thrombectomy center. No screening log was maintained. A complete list of eligibility criteria is provided in Section 1.3 of Example 1.

[0458] 1.8. Enrollment, Randomization, and Concealment

[0459] The APRIL clinical trial is divided into two parts. In Phase Ib, 32 patients were divided into four ascending dose groups (0.025, 0.05, 0.1 and 0.2 mg / kg, intravenous injection over 30 minutes) (6 APRIL per level: 2 placebos). A staggered dosing regimen was used to maximize patient safety. After the Data Safety Monitoring Board (DSMB) evaluated the results of the 72-hour follow-up, the next dose level was approved if deemed appropriate. The safety results evaluated were any suspected serious adverse reactions, serious adverse events (SAEs) or adverse events (AEs) that may be related to drug administration and blood biochemical parameters.

[0460] In Phase Ib and Phase IIa, a masked clinical adjudication committee analyzed all AEs and SAEs to determine their potential relationship to the study drug. Based on this information, a masked DSMB reviewed all reported AEs and SAEs. After the completion of Phase Ib, the DSMB was instructed to select two doses for testing in the subsequent Phase IIa based on preliminary safety results. Patients who received placebo or one of the two selected doses in Phase Ib were further analyzed together with patients enrolled in Phase IIa. In Phase IIa, three groups were studied, with patients randomized in a 1:1:√2 ratio to one of the two selected doses of ApTOLL or placebo, resulting in assignment probabilities of 0.293, 0.293, and 0.414, respectively. ApTOLL and placebo were prepared as colorless solutions in numbered, refrigerated (-20°C) vials. Aside from the unique vial number, the vials were visually identical, so all trial personnel and patients were completely blinded to treatment assignment.

[0461] During both study phases, patients were randomly assigned using a real-time, internet-based system. This process was automated from the outset of the study and allowed complete concealment of the allocation sequence. The randomization system was originally planned to stratify allocation according to three levels: predicted infarct core size on admission CTP (<35 vs. ≥35 cc), patient age (<70 vs. ≥70 years), and admission NIHSS (<15 vs. ≥15). However, due to an error in the system that was not noticed until study termination, no stratification was applied. To compensate for the potential bias introduced by this error, post hoc analyses of the primary outcomes after adjustment for the preplanned stratification factors are reported.

[0462] In phase IIa, after 100 patients completed 72 hours of follow-up, the DSMB performed a preplanned interim analysis and decided not to make any changes to the regimen for the remaining patients. Figure 1 The protocol of the APRIL study is summarized.

[0463] 1.9. Surgery

[0464] After qualifying imaging, all patients underwent EVT and, if indicated, intravenous thrombolysis (before or during EVT, at the primary hospital or endovascular center before transfer) and were treated according to local institutional protocols and national and European Stroke Organization guidelines (Turc G et al., 2019). Patients had to meet the inclusion and exclusion criteria of a thrombectomy-capable center. Trial medication was administered as soon as possible after randomization, and investigators were instructed to ensure that drug infusion began after imaging acquisition and before EVT (arterial puncture).

[0465] Sites were expected to adhere to national guidelines for stroke unit, stroke rehabilitation, and stroke prevention care. All patients underwent standard assessments of demographic characteristics, past medical history, laboratory values, and stroke severity (NIHSS score). The quality of reperfusion after EVT was assessed using the expanded Thrombolysis in Cerebral Ischemia (eTICI) scale. Follow-up brain imaging was performed with CT at 24 hours and MRI at 72 hours, if possible. Clinical follow-up was performed 90 days after randomization to assess the degree of disability determined by the mRS, if possible in person. If in-person follow-up was not possible, videoconference or telephone follow-up was performed. In all patients participating in Phase Ib, up to six serial blood samples were collected before and after dosing (baseline, 1, 6, 24, 48, and 72 hours after randomization) for pharmacokinetic analysis of ApTOLL concentrations and plasma concentrations of different proinflammatory biomarkers associated with acute stroke response. In Phase IIa, blood biomarkers were measured at 5 time points (baseline and 6, 24, 48, and 72 hours after randomization). Imaging interpretation was blinded and performed at a central core laboratory (University of California, Los Angeles, CA). Clinical data were validated by an independent monitoring organization (Anagram ESIC, Barcelona, Spain).

[0466] 1.10. Results

[0467] The primary objective of the study was to evaluate whether different doses of ApTOLL administered intravenously were safe and well-tolerated compared with placebo when administered with EVT and intravenous fibrinolytics (if indicated). The safety of ApTOLL was determined by monitoring adverse events occurring during the study period, which were detected by physical examination, laboratory tests, or neuroimaging. The primary endpoint was defined as the presence of any of the following events: death from any cause, intracranial hemorrhage resulting in new symptoms or worsening of existing symptoms (sICH, symptomatic intracranial hemorrhage), cerebral edema leading to brain herniation, neurologic deterioration or death, and recurrent stroke.

[0468] Although APRIL is a unique study, the two study parts have different secondary objectives:

[0469] Phase Ib Objective: (1) To evaluate the pharmacokinetic characteristics of ApTOLL in patients with AIS by measuring ApTOLL levels in plasma and urine. (2) To select two doses for Phase IIa administration based on their respective safety profiles. (3) To preliminarily estimate the therapeutic effect of ApTOLL on final infarct volume (measured by MRI fluid-attenuated inversion recovery [MRI-FLAIR] at 72 ± 24 hours) and proinflammatory biomarkers (at baseline [before dosing] and at the end of infusion [within 1 hour], and at 6 hours, 24 hours, 48 hours, and 72 hours after dosing).

[0470] Stage IIa : To assess the therapeutic and clinical effects of ApTOLL on: (1) final infarct volume (measured by MRI-FLAIR at 72 ± 24 h); (2) proinflammatory plasma biomarkers (before and 6, 24, 48, and 72 h after dosing, (3) early clinical course (NIHSS at 72 h or discharge [whichever occurs first]), and (4) long-term functional outcome (mRS at 90 days after stroke).

[0471] In the event of missing one or more NIHSS measurements, the last measurement after study drug administration was considered. Deceased patients were included in the "designated population" and had an mRS score of 6. For patients known to be alive 3 months after randomization but unable to undergo follow-up assessment, the discharge mRS was carried forward. If a 72-hour MRI was unavailable, the final infarct volume was determined on the last CT scan after ApTOLL administration. If no follow-up images were available, the final infarct volume was considered missing.

[0472] Statistical analysis

[0473] Before the study database was locked, a statistical analysis plan (SAP) was published, which included a list of all tables, lists, and figures.

[0474] Statistical analyses were performed by an independent external statistical consulting group. All analyses were performed on the designated population, i.e., all patients who were randomly assigned to the trial, regardless of treatment. The total sample size was defined as 151 patients: Phase Ib = 32 patients, Phase IIa = 119 patients. Due to the exploratory nature of the study, no statistical power was estimated. The analysis assumed an asymptotic relationship (i.e., any dose above an unsafe dose is considered unsafe). Phase Ib patients who received placebo or one of the two selected doses of ApTOLL were combined with Phase IIa patients. In order to successfully combine patients from the two study phases, the design, follow-up, and data collection were strictly identical in the Phase Ib and Phase IIa portions of the trial, with the only exception being serial blood and urine sampling for pharmacokinetic analysis purposes, which was performed only in Phase Ib.

[0475] Categorical variables were summarized using counts and percentages. Continuous variables were summarized using mean, standard deviation (SD), median, and corresponding interquartile range. These summaries were reported by treatment and follow-up, where available. Infarct volumes, which exhibited skewed distributions, are reported as medians (interquartile ranges).

[0476] The primary study outcomes were compared between the two groups based on the absolute difference in proportions and 95% asymptotic Wald confidence intervals. When both compared proportions were zero, generalized linear models were used. For confidence intervals of odds ratios, 95% asymptotic Wald confidence limits based on the logarithmic transformation of the odds ratios were used. Differences in continuous secondary outcomes (final infarct volume at 72 hours and NIHSS score) were tested between the groups in a one-way analysis of variance after adjustment with the Scheffe method; mean differences and 95% confidence intervals between placebo and ApTOLL doses are reported. To assess for shifts in mRS scores at 90 days, ordinal logistic regression models of proportion odds were constructed with placebo as the reference category to estimate unadjusted common odds ratios and 95% confidence intervals for a better outcome at 90 days.

[0477] To compensate for potential imbalances in baseline patient characteristics due to the lack of stratified randomization, post hoc binomial logistic regression models adjusted for predicted infarct core volume at admission, age, and baseline NIHSS score were performed to estimate odds ratios and 95% confidence intervals. A detailed statistical analysis plan was reported before study completion. The trial is registered with EudraCT: 2020-002059-38 and ClinicalTrials.gov: NCT04734548.

[0478] 1.12. Results. Study population

[0479] Between November 2020 and June 2021, 32 patients were enrolled in Phase Ib and randomized to receive ApTOLL or placebo ( Figure 1 ) to complete four dose-escalation cohorts. ApTOLL plasma concentrations were obtained from 22 patients in Phase Ib. The DSMB did not identify any safety concerns and selected doses of 0.05 mg / kg and 0.2 mg / kg for Phase IIa. The 0.05 mg / kg dose was chosen because it should be sufficient to produce some benefit, and all patients achieved adequate ApTOLL concentrations in the pharmacokinetic analysis. The 0.2 mg / kg dose is expected to achieve the greatest therapeutic effect because this dose achieved the greatest bioavailability in the first-in-human study in 2022 by Hernández Jiménez M et al. Furthermore, there do not appear to be any toxicity risks that would limit this option.

[0480] From July 2021 to April 2022, 119 patients were assigned to receive ApTOLL 0.05 mg / kg (n=36), ApTOLL 0.2 mg / kg (n=36), or placebo (n=47). The combined Ib / IIa population consisted of 42 patients assigned to ApTOLL 0.05 mg / kg, 42 patients assigned to ApTOLL 0.2 mg / kg, and 55 patients assigned to placebo ( Figure 1 Baseline characteristics were similar between the groups (Table 3). Patient compliance with eligibility criteria and treatment management, major protocol noncompliance, patient withdrawals and reasons for withdrawal (e.g., AEs, protocol noncompliance, loss to follow-up, consent withdrawal, and other reasons), and assignment to each analysis population are reported. Major protocol deviations occurred in 10 patients.

[0481] Of the 151 patients in the combined Ib / IIa population, all received the assigned intervention (active dose or placebo), with no crossover. All patients received the assigned intervention, although the amount or duration was incorrect in 3 patients: ApTOLL 0.05 mg / kg, 2 patients (4.8%); placebo, 1 patient (1.82%). All but one patient in the ApTOLL 0.2 mg / kg group (99.3%) underwent a trial of EVT; intravenous thrombolysis was administered in 29 patients (69.1%) in the ApTOLL 0.05 mg / kg group, 27 patients (64.3%) in the ApTOLL 0.2 mg / kg group, and 29 patients in the placebo group. Overall workflow (from onset to randomization, from onset to study drug administration, and from study drug to reperfusion) and reperfusion quality (on the expanded eTICI scale) were similar across all groups (Table 3).

[0482] 1.13. Result. Ending

[0483] At 90 days, data for the primary safety outcome were missing for 2 patients (1.3%; 2 patients withdrew consent) ( Figure 1 Death from any cause occurred in 10 patients assigned to the placebo group (18.2%), 11 patients assigned to the ApTOLL 0.05 mg / kg group (26.2%; absolute difference compared to the placebo group, 8%; 95% CI: -9% to 25%), and 2 patients assigned to the ApTOLL 0.2 mg / kg group: 3 (4.8%; absolute difference compared to the placebo group, -13%; 95% CI: -25% to -1%). Table 4 shows the distribution of primary and secondary endpoints by study group.

[0484] Final infarct volume was 44 mL (IQR 26-89) in patients assigned to placebo, 46 mL (IQR 18-100) in patients assigned to ApTOLL 0.05 mg / kg (mean difference in log-transformed final infarct volume from placebo, -12%; 95% CI, -49% to 35%), and 23.5 mL (IQR 13-42) in patients assigned to ApTOLL 0.2 mg / kg (mean difference in log-transformed final infarct volume from placebo, -42%; 95% CI, -66% to 1%). The NIHSS assessed at 72 hours was 7 (IQR 3-17) for patients assigned to the placebo group, 8 (IQR 3-17) for patients assigned to the ApTOLL 0.05 mg / kg group (mean difference in log-transformed 72-hour NIHSS from placebo was -1%; 95% CI: -41% to 40%), and 3 (0-11) for patients assigned to the ApTOLL 0.2 mg / kg group (mean difference in log-transformed 72-hour NIHSS from placebo was -45%; 95% CI: -67% to -10%). The proportion of patients with an mRs score of 0-2 at day 90 was 47.1% in the placebo group, 37.5% in the ApTOLL 0.05 mg / kg group (common odds ratio for a better outcome compared with placebo, 0.76; 95% CI, 0.37-1.56), and 64.3% in the ApTOLL 0.2 mg / kg group (common odds ratio for a better outcome compared with placebo, 2.44; 95% CI, 1.76-5.00). Analyses of multiple blood biomarkers did not differ significantly between the study groups. Figure 2-4 Graphical representations of the secondary efficacy outcomes are shown.

[0485] Discussion

[0486] The APRIL study showed that ApTOLL 0.2 mg / kg (SEQ ID NO: 1) infused with EVT was safe and reduced 90-day mortality in selected stroke patients. Furthermore, the efficacy of ApTOLL as a neuroprotective agent in acute cerebral ischemia was supported by consistent positive results for most predefined secondary outcome measures, including final infarct volume, early neurological damage, and long-term disability.

[0487] ApTOLL has been shown to: (1) effectively reach tissues at risk and exert protective effects directly in the ischemic penumbra region, potentially extending the therapeutic window for reperfusion therapy; and (2) limit hemorrhagic transformation and reperfusion injury that may occur after recanalization.

[0488] A key advantage of this trial is that the design largely matches the preclinical ischemia / reperfusion model of transient middle cerebral artery occlusion. The median duration of stroke onset before study drug administration was 210 minutes, and reperfusion rates after EVT were high (final eTICI 2b-3 score 87%). The observed pharmacokinetic profile suggests that ApTOLL can exert its neuroprotective effects within 12 hours of administration, including the initial phase of ischemia and several hours after reperfusion. The median time from drug infusion to recanalization (when achieved) was approximately 180 minutes, corresponding to the potential duration of neuroprotection during ischemia.

[0489] Subgroup analysis showed that treatment effects were similar between patients who received the drug in the early time window (<3 hours after symptom onset) and those who received treatment in the later window (3-6 hours).

[0490] This study did not yield results regarding the efficacy of ApTOLL in improving outcomes in patients with acute stroke, but rather determined a safe dose for these patients. However, promising results have been observed at this stage. Table 5 and Figure 4 Showing more results.

[0491] Conclusion

[0492] In AIS, ApTOLL 0.2 mg / kg administered within 6 h after onset combined with EVT was safe and associated with a meaningful clinical effect of reducing 90-day mortality and disability compared with placebo.

[0493] Table 3. Baseline characteristics expressed as mean (standard deviation) or median [interquartile range] (mRS: modified Rankin Scale, NIHSS: National Institutes of Health Stroke Scale, LTSW: last seen normal, ASPECTS: Alberta Stroke Project Early CT Score, ICA: internal carotid artery, MCA: middle cerebral artery, EVT: endovascular therapy, eTICI: extended thrombolysis of ischemic encephalopathy).

[0494]

[0495]

[0496] Table 4. Primary and secondary outcome measures. IQR: interquartile range. In the “Effect comparison of placebo with ApTOLL 0.2 mg / kg” column, positive % values indicate a decrease, and negative % values indicate an increase.

[0497]

[0498]

[0499] Table 5. Secondary outcome measures over time

[0500]

[0501]

[0502] Example 2: Comparison of ApTOLL (APRIL study) and Nerinetide (ESCAPE-NA1 study)

[0503] Nerinetide (NA-1) is a neuroprotective agent that focuses on inhibiting postsynaptic density protein 95, which is involved in excitotoxic processes after stroke. The ESCAPE-NA1 clinical trial, with a total of 1105 patients, had negative global results, as nerinetide did not increase the global proportion of patients with a good clinical outcome after EVT compared with patients who received placebo. This negative effect can be explained by the fact that the populations treated with EVT and EVT+tPA were mixed in the final analysis, but in subsequent sub-analyses, researchers observed evidence of a modified effect of nerinetide, resulting in a suppressed effect of nerinetide in patients who received alteplase (Hill M et al., 2020). Figure 7 Summarizing the results considering only patients who did not receive tPA, an mRS of 0-2 increased by 19% and reduced mortality by 37%.

[0504] It is important to note that ESCAPE-NA1, a Phase III clinical trial involving 1,105 patients, yielded very limited results, as efficacy was observed only in patients who had not received tPA. Therefore, the applicability of nerinetide in future clinical practice will be limited to patients who are ineligible for tPA treatment. In contrast, the APRIL study, conducted in only 151 patients, demonstrated a 36% increase in mRS of 0-2 and a 72% reduction in mortality compared to patients who received or did not receive tPA.

[0505] Example 3: ApTOLL mutation analysis

[0506] The experiment tested the effect of the percentage of sequence identity and extension at the 5' and 3' ends of the ApTOLL sequence (SEQ ID NO: 1).

[0507] First, six variants of the ApTOLL sequence shown in Table 6 were obtained and tested. The variants had sequence identities ranging from 89.83% to 81.53% with SEQ ID NO: 1 and included mutations related to the aptamer sequence of SEQ ID NO: 1, such as additions to the 5' end of the sequence, additions to both the 5' and 3' ends of the sequence, internal deletions, conservative substitutions, and non-conservative substitutions.

[0508] Table 6. Mutants (Mut) of the ApTOLL sequence. Mutations in the ApTOLL sequence are indicated in bold.

[0509]

[0510]

[0511] Antagonistic activity against TLR4 was assessed using the secreted embryonic alkaline phosphatase (SEAP) assay in HEK-Blue hTLR4 cells (InvivoGen, catalog code Hkb-htlr4). LPS-EK up (InvivoGen catalog code tlrl-peklps) is a TLR4 agonist control. HEK-Blue hTLR4 cells were generated by co-transfecting human TLR4, MD-2, and CD14 co-receptor genes and an inducible SEAP reporter gene into HEK293 cells. The SEAP reporter gene was placed under the control of the IL-12p40 minimal promoter fused to five NF-κB and AP-1 binding sites. TLR4 ligand stimulation activates NF-κB and AP-1, which induces SEAP production. The TLR4 receptor of HEK-Blue hTLR4 cells seeded in P96 plates was activated by adding LPS-EK up. One hour later, aptamers were added at two final concentrations (20 nM, 200 nM), and the expression of the reporter protein SEAP was quantified 16-20 hours later.

[0512] Figure 8 The activity of the SEAP reporter protein produced by TLR4 receptor activation is shown. The natural agonist ligand of TLR4 is LPS (bacterial lipopolysaccharide); LPS-EK up was used in this assay. The agonist control bar on the left indicates that the TLR4 receptor is 100% activated by the above agonist. When the aptamers (control ApTOLL and ApTOLL-Mut 1-6) are added to the activated cells, TLR4 activity decreases and the aptamer bar becomes shorter than the agonist control bar. Therefore, the aptamers have concentration-dependent TLR4 antagonist activity, with the exception of ApTOLL-Mut 4, which is not concentration-dependent. The mutants tested have similar behavior, therefore, Figure 8 It was shown that all tested ApTOLL variants (ApTOLL-Mut, SEQ ID NOs: 17-22) had TLR4 antagonist activity comparable to that of the ApTOLL aptamer (SEQ ID NO: 1).

[0513] In addition, these aptamers were evaluated in a TLR4 receptor competition assay. The ELO-NA cell assay was performed using an ApTOLL aptamer (SEQ ID NO: 1) modified with a digoxigenin tag (ApTOLL-Dig) and HEK-Blue hTLR4 cells. Equimolecular mixtures containing ApTOLL-Dig (100 nM) and each of ApTOLL-Mut 1-6 (100 nM) were used to determine the potential reduction of digoxigenin-associated signal relative to a positive ApTOLL-Dig control (100 nM). The positive ApTOLL-Dig control and each ApTOLL-Dig / ApTOLL-Mut mixture were added to the wells and incubated in a CO2 incubator at 37°C for 15 minutes. The wells containing the cells were then washed with PBS, and anti-dig antibodies were added. Finally, ApTOLL-Dig was detected using the ABTS protocol (Roche Reference No. 11684302001).

[0514] Figure 9The results of the competition assay are depicted, which show that when the ApTOLL control aptamer (SEQ ID NO: 1) was added to the cells, its binding percentage to the TLR4 receptor was 100%. In contrast, when the ApTOLL-Mut aptamers (1-6) in an equimolecular mixture were added to the cells separately, the binding percentage bar of the ApTOLL aptamer decreased, which means that the ApTOLL-Mut aptamers (1-6) competed with the ApTOLL control aptamer for the same TLR4 receptor binding site. ApTOLL-Mut6 was the aptamer with the highest binding affinity to the TLR4 receptor (35.9%), and ApTOLL-Mut2 had the lowest. ApTOLL-Mut 1 and ApTOLL-Mut 4 obtained similar binding percentages (20% and 21%, respectively). Therefore, Figure 9 The experimental data presented in show that ApTOLL-Mut aptamers (SEQ ID NOs: 17-22) compete with ApTOLL (SEQ ID NO: 1), indicating that they are structurally similar and therefore bind to the same binding site on TLR4 as the original ApTOLL aptamer.

[0515] In addition, the effects of extending the 5' and 3' ends of ApTOLL (SEQ ID NO: 1) and simultaneously introducing mutations were tested. Three variants of SEQ ID NO: 1 were tested (see Table 7 below):

[0516] Aptamer 4F (SEQ ID NO: 4): Compared to the ApTOLL sequence (SEQ ID NO: 1), it has 13 additional nucleotides at the 5' end and 4 additional nucleotides at the 3' end. If only the central region is considered, the sequence identity to SEQ ID NO: 1 would be 100%. Considering the entire sequence, the sequence identity percentage is 77.6%.

[0517] Aptamers 4F-Mut2 and 4F-Mut3 (SEQ ID NOs: 23 and 24): These two aptamers correspond to the most divergent sequences relative to the ApTOLL sequence (SEQ ID NO: 1).

[0518] To test whether any 5' or 3' extensions were effective, the added 5' and 3' regions of 4F-Mut2 and 4F-Mut3 were highly mismatched with the extensions in aptamer 4F. The 5' extensions in 4F-Mut2 and 4F-Mut3 had 0% sequence identity with the 5' extension of aptamer 4F. Similarly, the 3' extensions in 4F-Mut2 and 4F-Mut3 had 0% sequence identity with the 3' extension of aptamer 4F.

[0519]

[0520] Table 7. Regions of homology to ApTOLL (SEQ ID NO: 1) in 4F, 4F-Mut2, and 4F-Mut3 are shaded grey, while mutations in 4F-Mut2 and 4F-Mut3 relative to ApTOLL (SEQ ID NO: 1) are in bold.

[0521]

[0522]

[0523] The ability of the aptamer to inhibit TLR4 and the ability of the aptamer to bind to TLR4 (antagonist activity against TLR4) were measured by the above-mentioned method. The results are shown in Table 8.

[0524] Table 8. TLR4 antagonistic activity of 4F, 4F-Mut2 and 4F-Mut3 aptamers

[0525]

[0526] Therefore, as experimentally confirmed in Table 8, the addition of nucleotides to the 5' and 3' ends of SEQ ID NO: 1 does not affect the formation of an effective binding structure and its activity. The results in Table 8 confirm that the 4F-Mut2 and 4F-Mut3 aptamers, which have 90% sequence identity with SEQ ID NO: 1 and have 13 nucleotides added to the 5' end and 4 nucleotides added to the 3' end of the aptamer of SEQ ID NO: 1, both retain the ability to inhibit TLR4 and the ability to bind to TLR4 (antagonistic activity against TLR4), similar to their original aptamers.

[0527] Thus, variant aptamers having at least 90% sequence identity (or at least 69% sequence identity taking into account the entire sequence) to the original nucleic acid sequence (SEQ ID NO: 1) and optionally extended by 1-13 or 1-4 nucleotide sequences at the 5' and 3' ends, respectively, retain the function of the original nucleic acid sequence (i.e., the ability to specifically bind to and inhibit TLR4).

[0528] References

[0529] WO2015197706A1(AptaTargets SL)

[0530] WO2020230108A1(AptaTargetsSL) WO2020230109A1(AptaTargets SL)

[0531] Hernández-Jiménez M,et al.First-in-human phase I clinical trial of aTLR4-binding DNAaptamer,ApTOLL:Safety and pharmacokinetics in healthyvolunteers.Mol Ther Nucleic Acids.2022;28:124-135.doi:10.1016 / j.omtn.2022.03.005

[0532] García-Culebras A,et al.Toll-Like Receptor 4 Mediates HemorrhagicTransformation AfterDelayed Tissue Plasminogen Activator Administration in InSitu Thromboembolic Stroke.Stroke.2017 Jun;48(6):1695-1699.doi:10.1161 / STROKEAHA.116.015956Berge E,et al.European Stroke Organisation(ESO)guidelineson intravenous thrombolysis foracute ischaemic stroke.European StrokeJournal.2021;6(1):I-LXII.doi:10.1177 / 2396987321989865

[0533] Jovin TG,et al.Thrombectomy within 8 Hours after Symptom Onset inIschemic Stroke.NEngl J Med 2015;372:2296-2306.doi:10.1056 / NEJMoa1503780

[0534] Lyden P,et al.Improved reliability of the NIH Stroke Scale usingvideo training.NINDS TPAStroke Study Group.Stroke.1994;25(11):2220-2226.doi:10.1161 / 01.str.25.11.2220

[0535] Van Swieten JC,et al.Interobserver agreement for the assessment ofhandicap in stroke patients.Stroke.1988;19(5):604-607.doi:10.1161 / 01.str.19.5.604

[0536] Chen S,et al.Cerebral edema formation after stroke:emphasis on blood-brain barrier and thelym-phatic drainage system of thebrain.Front.Cell.Neurosci.15:716825.doi:10.3389 / fncel.2021.716825

[0537] Pexman JH,et al.Use of the Alberta Stroke Program Early CT Score(ASPECTS)for assessingCT scans in patients with acute stroke.AJNR Am JNeuroradiol.2001;22(8):1534-1542Olivé-Gadea M,et al.Defining a targetpopulation to effectively test a neuroprotective drug.Stroke.2021;52(2):505-510.doi:10.1161 / STROKEAHA.120.032025

[0538] Turc G,et al.European Stroke Organisation(ESO)-European Society forMinimally InvasiveNeu-rological Therapy(ESMINT)guidelines on mechanicalthrombectomy in acute ischemicstroke.J Neurointerv Surg.2019;11(6):535-538.doi:10.1136 / neurintsurg-2018-014568Hill M et al.Efficacy and safety ofnerinetide for the treatment of acute ischaemic stroke(ESCAPE-NA1):amulticentre,double-blind,randomised controlled trial.2020.The Lancet.395(10227):878-887.doi:10.1016 / S0140-6736(20)30258-0。

Claims

1. An aptamer having SEQ ID NO: 1, for use in reducing the risk of intracranial hemorrhage in a subject after acute ischemic stroke, wherein the aptamer is administered at a dose of at least about 0.2 mg / kg after acute ischemic stroke, and wherein administration of the aptamer reduces the risk of intracranial hemorrhage by at least about 14% relative to a subject not treated with the aptamer.

2. The aptamer for use according to claim 1, wherein the intracranial hemorrhage is symptomatic intracranial hemorrhage, and wherein administration of the aptamer reduces the risk of symptomatic intracranial hemorrhage by at least about 34% relative to a subject not treated with the aptamer.

3. An aptamer having SEQ ID NO: 1 for use in improving neurological recovery in a subject after acute ischemic stroke, wherein the improvement in neurological recovery is mediated by reducing the risk of intracranial hemorrhage, wherein the aptamer is administered at a dose of at least about 0.2 mg / kg after acute ischemic stroke, and wherein, relative to a subject not treated with the aptamer: (i) reduce NIHSS scores, and (ii) Reduce the mRS score to 0-2. 4 . The aptamer for use according to claim 3 , wherein the reduction in NIHSS score at 72 hours is at least about 57% relative to a subject not treated with the aptamer.

5. The aptamer for use according to any one of claims 3-4, wherein the reduction in mRS score to 0-2 at 90 days is at least about 36% relative to a subject not treated with the aptamer.

6. The aptamer for use according to any one of claims 1 to 5, wherein the aptamer reduces brain edema by at least about 67% relative to a subject not treated with the aptamer. 7 . The aptamer for use according to any one of claims 1 to 6 , wherein the aptamer is administered within about 8 hours of stroke onset, specifically within about 4 hours of stroke onset.

8. The aptamer for use according to any one of claims 1 to 7, wherein the aptamer is administered in combination with arterial recanalization.

9. The aptamer for use according to claim 8, wherein the aptamer is administered in combination with intravascular therapy. 10 . The aptamer for use according to claim 9 , wherein the aptamer is administered before or simultaneously with the intravascular treatment.

11. The aptamer for use according to claim 10, wherein the aptamer is administered less than about 4 hours before the intravascular treatment. 12 . The aptamer for use according to claim 8 , wherein the aptamer is administered in combination with a thrombolytic drug, particularly in combination with a tissue plasminogen activator. 13 . The aptamer for use according to claim 12 , wherein the aptamer is administered simultaneously with or after administration of a thrombolytic drug.

14. The aptamer for use according to any one of claims 1 to 13, wherein the aptamer is administered intravenously by infusion over about 30 minutes.

15. The aptamer for use according to any one of claims 1 to 14, wherein at the onset of a stroke, the subject: i) aged between about 18 and about 90 years old; ii) having a baseline NIHSS between about 8 and about 25; iii) pre-stroke mRS score between 0 and approximately 2 points; iv) having an infarct volume of about 5 cc to about 70 cc; v) Occlusion of the terminal internal carotid artery or the M1 or M2 segment of the middle cerebral artery; vi) mTICI score of 0 or 1; vii) having a diffusion-weighted imaging limiting volume between about 5 mL and about 70 mL; viii) having an ASPECTS between about 6 and about 10; or ix) Any combination of the above.

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