ApTOLL molecule for the treatment of ischemic stroke and intracranial hemorrhage

Abstract

The APRIL study demonstrated that infusion of ApTOLL 0.2 mg / kg (SEQ ID NO: 1) in combination with endovascular treatment in selected stroke patients was safe and reduced mortality at 90 days. ApTOLL was demonstrated to limit the occurrence of intracranial hemorrhage and reperfusion injury that can occur after recanalization, and also improve early neurological dysfunction and long-term disability. The ApTOLL molecule is provided for use in reducing the risk of intracranial hemorrhage or secondary hemorrhage, reducing neurological damage, and improving neurological recovery after acute ischemic stroke in subjects.

Description

[Technical Field]

[0001] The present invention relates to the field of oligonucleotides and therapeutic aptamers useful for human health, particularly in the treatment of intracranial hemorrhage and improving neurological recovery in patients suffering from thrombotic disorders, particularly acute ischemic stroke. [Background technology]

[0002] Reperfusion therapy has substantially improved the care of acute ischemic stroke (AIS) over the past two decades. Since 1995, tissue plasminogen activator (tPA) has been the only approved specific treatment for AIS and has positively impacted the lives of many stroke patients. However, its benefits have been significantly limited by its limited therapeutic window, set at <4.5 hours after stroke onset, and its limited efficacy. It is estimated that tPA is used in only approximately 5% of AIS patients.

[0003] In 2015, most clinical guidelines also recommended mechanical thrombectomy as the first-line treatment for ischemic stroke caused by large vessel artery occlusion (LVO).In recent years, the number of patients benefiting from endovascular thrombectomy (EVT) has rapidly increased worldwide, and its indications have been gradually extended to patients with large infarct cores and those who present with symptoms in a late time frame of up to 24 hours from onset.

[0004] However, these treatments are not without complications. The most significant concern regarding the use of recanalization therapies such as tPA or EVT is the increased risk of hemorrhage, commonly referred to as hemorrhagic transformation (HT), which refers to the transformation of a stroke into a hemorrhagic area. Other types of hemorrhage associated with ischemic stroke can also occur, such as intracranial hemorrhage (ICH), which refers to bleeding within the intracranial vault, including the brain parenchyma and surrounding meningeal spaces. Therefore, both types of hemorrhage (hereafter referred to as ICH) occur after ischemic stroke, and these are also called secondary hemorrhages, which can cause new symptoms or worsen existing symptoms. When patients suffer from secondary hemorrhage, neurological damage worsens, neurological recovery becomes more difficult, and even death may result. Several clinical trials have concluded that ICH is a presumed significant cause of neurological deterioration (NIHSS score ≥ 4 points).

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

[0006] Endovascular reperfusion therapy is now considered fully implemented, meaning that acute stroke patients now consistently undergo complete vascular evaluation (computed tomography and CT perfusion [CTA / CTP]), and patients presenting with LVO undergo EVT, which guarantees high recanalization rates of approximately 85–90%. Furthermore, several studies have demonstrated a longer treatment window for EVT (up to 24 hours after stroke onset in some specific cases) compared with 4.5 hours in tPA cases. However, despite EVT being shown to consistently achieve sufficient recanalization grades in 85–90% of cases, more than 50% of treated patients develop moderate to severe disability.

[0007] Despite these known inherent risks of reperfusion therapy, the benefit-risk ratio supports its use. In this context, it is of great interest to find new drugs with potential neuroprotective effects to improve stroke outcomes and reduce 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 that also responds to tissue damage-associated molecular patterns (DAMPs) and is directly involved in many diseases, including ischemic stroke. In particular, the inflammatory component induced during the acute phase of stroke is considered an interesting target for promoting patient recovery, and ApTOLL is expected to have a potentially high therapeutic effect in this area. WO2015197706A1 (AptaTargets SL) describes a nucleic acid aptamer, specifically ApTOLL, capable of specifically binding to and inhibiting TLR-4, and its uses, including stroke.

[0009] In fact, the efficacy of ApTOLL has been demonstrated at the preclinical level in experimental models of cerebral and myocardial ischemia. Specifically, WO2020230108A1 (AptaTargets SL) discloses methods and compositions for the treatment of ischemic stroke and demonstrates the efficacy of ApTOLL in a rodent model of stroke (Example 2). In addition, the first-in-human study described in Example 3.1 of WO2020230108A1 and Hernandez-Jimenez M et al., 2022, demonstrated the safety of ApTOLL in healthy subjects and demonstrated its pharmacokinetics. Summary of the Invention

[0010] One problem solved by the present invention is to provide new uses and methods that improve patient functional outcomes, e.g., reduction of neurological damage, while reducing hemorrhagic complications associated with ischemic stroke and reperfusion procedures.

[0011] The solution is based on administering an ApTOLL molecule (e.g., 0.2 mg / kg ApTOLL, SEQ ID NO: 1) to a subject in need thereof to reduce neurological damage and other effects, such as intracranial hemorrhage, associated with ischemic stroke and reperfusion procedures, as described below.

[0012] In this scenario, the inventors designed a Phase Ib / IIa clinical study coded APRIL (hereinafter "APRIL Study") to evaluate the safety and biological effects of ApTOLL in AIS patients eligible for EVT with or without IV thrombolysis. The protocol for this clinical study was described in Example 3.2 of WO2020230108A1 (AptaTargets SL), but the effects remain unknown.

[0013] The APRIL study was divided into two parts: (1) Phase Ib (n = 32 AIS patients): single iv administration of ApTOLL (30-minute infusion), dose escalation with 4 single dose levels (8 patients / level), randomized (1:3); and (2) Phase IIa (n = 119 patients): single dose, iv administration (30-minute infusion), parallel (3-arm, placebo:ApTOLL dose A:ApTOLL dose B), randomized (√2:1:1).

[0014] After completion of Phase Ib, a Data Safety Monitoring Board (DSMB), unblinded to the study arms, selected two doses (A, B) to be tested in Phase IIa according to the initial safety results. Patients treated with placebo, dose A, or dose B in Phase Ib were further analyzed together with patients enrolled in Phase IIa (total number of patients: 151).

[0015] The primary objective of the APRIL study was to evaluate whether different doses of intravenous ApTOLL were safe and well tolerated compared with placebo when used in combination with endovascular therapy and, when indicated, intravenous fibrinolytic therapy.The APRIL trial was not powered to draw conclusions about the effectiveness of ApTOLL in improving outcomes in patients with acute stroke.

[0016] Surprisingly, in addition to demonstrating the safety of ApTOLL, the inventors found that administration of 0.2 mg / kg of ApTOLL in combination with EVT within 6 hours of symptom onset produced significant floor effects, reducing mortality and disability rates at 90 days compared with placebo. ApTOLL exerts various effects in human patients with AIS, including increased survival, reduced intracranial hemorrhage, reduced cerebral edema, and improved neurological recovery. The Examples herein provide the results of the APRIL trial, along with detailed experimental data demonstrating the effects of ApTOLL (e.g., Example 1, Sections 1.13-1.15).

[0017] Specifically, the inventors found that ApTOLL reduced intracranial hemorrhage and significantly reduced symptomatic intracranial hemorrhage at a rate of 34.25% compared to placebo.

[0018] Furthermore, we found that ApTOLL reduced the number of deaths: all-cause deaths occurred in 10 patients assigned to placebo (18.2%) compared with 2 in the group assigned to ApTOLL 0.2 mg / kg (4.8%; absolute difference vs. placebo -13%; 95% CI: -25% to -1%), representing a 73% reduction in deaths.

[0019] Final infarct volume was 44 mL in patients assigned to placebo and 23.5 mL in patients assigned to ApTOLL 0.2 mg / kg (mean difference in log-transformed final infarct volume vs. placebo -42%; 95% CI: -66% to 1%), resulting in a 46.59% reduction in final infarct volume.

[0020] The NIHSS score (National Institutes 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 (mean difference in log-transformed 72-hour NIHSS vs. placebo: -45%; 95% CI: -67% to -10%). This represents a 57.14% reduction in NIHSS score compared with placebo.

[0021] The proportion of patients with a modified Rankin score (mRS) of 0 to 2 (no symptoms to slight disability) at 90 days was 47.1% in patients assigned to placebo and 64.3% in patients assigned to ApTOLL 0.2 mg / kg, representing a 36.29% increase (i.e., improvement) in mRS scores of 0 to 2 compared with placebo.

[0022] Furthermore, ApTOLL 0.2 mg / kg resulted in a 67.40% reduction in cerebral edema compared to placebo.

[0023] Subgroup analysis suggested similar treatment effects in patients who received ApTOLL in an early time frame (<3 hours from symptom onset) and in patients treated in a later time frame (3–6 hours), suggesting that ApTOLL may be applicable in different clinical situations.

[0024] These observed benefits of ApTOLL are remarkable given the severity of the disease studied in the clinical trial—patients suffered from a very severe, acute form of AIS—and the limited number of participants (151). These benefits are clinically meaningful and have a major impact for patients with AIS, representing an unmet medical need: improved survival and reduced functional and physical impairment. They also represent an improvement over widely used reperfusion techniques, such as EVT and thrombolysis. This is crucial because these patients currently have limited treatment options, and our findings highlight the potential importance of ApTOLL in addressing this long-standing unmet medical need.

[0025] As described above, WO2020230108A1 demonstrates the efficacy of ApTOLL in a rodent model of stroke (Example 2). ApTOLL induced a reduction in infarct size compared to vehicle, demonstrating its potential protective effect. Furthermore, mice maintained the protective effect up to 21 days after stroke. WO2020230108A1, along with Hernandez-Jimenez M et al., 2022, also describes the results of a first-in-human trial of ApTOLL. This study aimed to define the safety and pharmacokinetics of ApTOLL in healthy subjects.

[0026] Therefore, the results from the APRIL study are the first to demonstrate the efficacy of ApTOLL in human subjects with AIS. The observed effects are novel compared to the prior art, and a person skilled in the art could not have predicted the effects experimentally described herein from the prior art. In addition to the fact that human subjects suffer from AIS and their biological / clinical situation differs from that of healthy subjects, the conditions, requirements, dosage, and inclusion criteria in the APRIL trial are also different from those described in the prior art. For example, the APRIL trial included EVT and fibrinolytic techniques, which may further worsen the patient's condition and cause, for example, intracranial hemorrhage. Furthermore, most of the observed effects are associated with the 0.2 mg / kg ApTOLL dose.

[0027] Therefore, the present invention is directed to completely unforeseen new effects, specifically, the reduction of the risk of intracranial hemorrhage. Although the cause of these effects caused by ApTOLL is unknown, it suggests that ApTOLL has the ability to interact with receptors other than TLR-4, and has the ability to prevent the occurrence of intracranial hemorrhage after AIS, for example.

[0028] The improvement in neurological recovery is also a significant result that cannot be derived from the prior art, because this effect was not necessarily present in previous studies by ApTOLL, given the scenario of clinical trials.For example, the examples herein describe the reduction in infarct volume, NIHSS score and mRS score, which indicate significant neurological improvement in AIS patients.In addition, the results suggest that the improvement is not only related to mobility but also to cognition.

[0029] Therefore, the observed effects in the APRIL study, i.e., reduction in ICH and reduction in neurological damage and improved neurological recovery, may be considered as unexpected different technical effects, which may represent additional medical indications or, for example, new uses of known substances in the sense of the EPO guidelines.

[0030] Example 2 compares the effects of nerinetide and ApTOLL from the ESCAPE-NA1 and APRIL studies, respectively. ESCAPE-NA1 was a Phase III clinical trial involving 1,105 patients, with very limited results because the effect was observed only in patients not treated with tPA. Therefore, the applicability of nerinetide to future clinical practice will likely be limited to this type of patient. The results for patients not treated with tPA were a 19% increase in mRS score of 0 to 2 and a 37% reduction in mortality. In contrast, the APRIL study, conducted with only 151 patients, resulted in a 36% increase in mRS score of 0 to 2 (i.e., a reduction in mRS score of 0 to 2) and a 72% reduction in mortality for patients either treated with or not treated with tPA. Example 2 demonstrates that the effects of the APRIL study were positive and applicable to a wider range of patients and medical scenarios.

[0031] As explained above, the APRIL protocol is described in Example 3.2 of WO2020230108A1, but the outcomes associated with the defined objectives and endpoints were unexpected, particularly in that the results were beyond the reasonable experience of the experienced clinicians participating in the study and had a significant impact on AIS patients.

[0032] These surprising effects are not derived from Garcia-Culebras A et al., 2017. In this paper, TLR - / - Late tPA administration in mice upregulates TLR-4 + / + However, the study had several limitations, which are also noted in the paper: (1) The delayed administration was set at a safe time in humans; (2) the protective effect of TLR-4 absence needs to be investigated in future long-term outcome studies; and (3) The involvement of TLR-4 in thrombectomy-induced HT still requires further investigation.

[0033] Briefly, this study was conducted in a mouse model in which the TLR-4 receptor was completely knocked out, and therefore the mechanism is not the same as in the normal scenario; for example, these mice do not undergo an inflammatory response in the TLR-4 pathway when a stroke is induced. The APRIL study instead uses ApTOLL in AIS patients: first, the aptamer only partially inhibits TLR-4, and therefore does not have the same effect as complete TLR-4 deficiency in all tissues; second, patients experience inflammation in the TLR-4 pathway in the brain, which can lead to multiple complications in clinical settings, in contrast to the mouse model of Garcia-Culebras A et al., 2017.

[0034] Thus, a first aspect of the present invention relates to an ApTOLL molecule for reducing the risk of intracranial hemorrhage or secondary hemorrhage (or preventing intracranial hemorrhage or reducing the occurrence of intracranial hemorrhage) in a subject after acute ischemic stroke, wherein the ApTOLL molecule is administered after acute ischemic stroke (i.e., after the onset of the stroke).

[0035] Another embodiment relates to an ApTOLL molecule for reducing the risk of intracranial hemorrhage or secondary hemorrhage (or preventing or reducing the occurrence of intracranial hemorrhage) in a subject following a thrombotic disorder, wherein the ApTOLL molecule is administered after the thrombotic disorder.

[0036] One embodiment of the present invention relates to an ApTOLL molecule for reducing neurological damage and improving neurological recovery after an acute ischemic stroke in a subject, wherein the ApTOLL molecule is administered after an acute ischemic stroke.

[0037] In another embodiment, the invention relates to an ApTOLL molecule for increasing survival and / or reducing cerebral edema following acute ischemic stroke in a subject, wherein the ApTOLL molecule is administered following acute ischemic stroke.

[0038] Another aspect of the present invention relates to an ApTOLL molecule for improving anxiety / depression complications, performance of daily activities, mobility and / or self-care following acute ischemic stroke in a subject, wherein the ApTOLL molecule is administered following acute ischemic stroke.

[0039] Throughout this description and claims, the word "comprise" and variations thereof are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of this description or may be learned by practice of the present invention. Furthermore, the present invention encompasses all possible combinations of the specific and preferred embodiments described herein. The following examples and figures are provided herein for illustrative purposes, without intending to limit the present invention. [Brief explanation of the drawings]

[0040] [Figure 1] Figure 1 shows the APRIL study flowchart (Pbo: placebo, DSMB: data safety monitoring board, AIS: acute ischemic stroke). [Figure 2] Median and interquartile range of baseline and 72-hour NIHSS scores by 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) by treatment assignment are presented. NIHSS: National Institutes of Health Stroke Scale. [Figure 3] Distribution of overall disability at 90 days by treatment assignment is shown. Stacked bar graphs represent distribution of modified Rankin score (scale of 0 to 6) at 90 days by treatment assignment. [Figure 4]1 is a graphical representation of patient quality of life assessments after treatment with ApTOLL 0.05 mg / kg, 0.02 mg / kg, or placebo. Quality of life assessments include the following criteria: mobility, self-care, daily activities, pain / discomfort, and anxiety / depression. [Figure 5] A detailed flowchart of the procedures in the APRIL study is shown. LVO: major vessel artery occlusion. TICA: distal internal carotid artery. mRS: modified Rankin score. NIHSS: National Institutes of Health Stroke Scale. C / CTA / CTP: computed tomography / computed tomography angiography / computed tomography perfusion. MRI: magnetic resonance imaging. ASPECTS: Alberta Stroke Program Early CT Score. CBF: cerebral blood flow. DWI: diffusion-weighted imaging. rt-PA: recombinant tissue plasminogen activator. EVT: endovascular thrombectomy. [Figure 6] 1 shows the primary, secondary, and tertiary structures of ApTOLL (SEQ ID NO: 1), also referred to as ApTLR#4FT. [Figure 7] Comparison of mRS and survival outcomes after administration of 0.2 mg / kg ApTOLL in the APRIL study versus 2.6 mg / kg nerinetide (NA-1) in the ESCAPE-NA1 study. mRS: modified Rankin score. tPA: tissue plasminogen activator. EVT: endovascular thrombectomy. [Figure 8] Figure 1 shows the antagonist TLR4 activity assay of ApTOLL (SEQ ID NO: 1) and its variants ApTOLL-Mut 1 to 6 (SEQ ID NOs: 17 to 22). TLR4 receptor activity is expressed as a percentage of control LPS-Ek uptake. Antagonist activity of the aptamer is determined by a decrease in the percentage of activation relative to LPS-Ek uptake. [Figure 9]1 shows a competition assay of ApTOLL (SEQ ID NO: 1) and its variants ApTOLL-Mut 1 to 6 (SEQ ID NOs: 17 to 22) for the TLR4 receptor. Mutant competition with the ApTOLL sequence for the same binding site on the TLR4 receptor is determined by a decrease in the binding percentage of the ApTOLL sequence (control) in each mixed ApTOLL / ApTOLL-Mut (1 to 6) relative to ApTOLL. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention is directed to uses and methods of ApTOLL molecules (e.g., SEQ ID NO: 1) to reduce the risk of intracranial hemorrhage in a subject after AIS; uses and methods of ApTOLL molecules to reduce the risk of intracranial hemorrhage in a subject after thrombotic injury; uses and methods of ApTOLL molecules to reduce neurological damage and improve neurological recovery in a subject after AIS (e.g., reducing infarct volume and NIHSS score, and improving mRS score); and uses and methods of ApTOLL molecules to increase survival and / or reduce cerebral edema in a subject after AIS, wherein the ApTOLL molecule is administered after AIS (or after thrombotic injury). Also provided are administration procedures and doses (e.g., 0.2 mg / kg) of ApTOLL molecules; arterial recanalization techniques (e.g., EVT or thrombolysis); subject characteristics (e.g., pre-stroke mRS score); and nucleic acid aptamers, their variants, derivatives, chemically modified aptamers, pharmaceutical compositions, and formulations.

[0042] Before describing the present invention in more detail, it should be understood that this disclosure is not limited to the particular compositions or process steps described, and as such, can, of course, vary. As will be apparent to those skilled in the art upon reading this description, each of the individual embodiments described and illustrated herein has distinct components and features that can be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the present description. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0043] The headings provided herein are not intended to limit the various aspects of the description, but such limitations can be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the description will be limited only by the appended claims.

[0044] Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0045] definition In order that this description may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0046] treatmentAs used herein, the terms "treat," "treatment," and "therapy" refer to a clinical intervention to prevent (e.g., suppress or inhibit) a disease or condition (e.g., intracranial hemorrhage); cure a disease or condition; delay the onset of a disease or condition; reduce the severity or severity of a disease or condition (e.g., reduce the extent of intracranial hemorrhage); ameliorate or eliminate one or more symptoms or sequelae associated with a disease or condition; or provide a beneficial effect to a subject having a disease or condition without necessarily curing the disease or condition.

[0047] In some embodiments, this term refers to clinical intervention to, for example, improve one or more symptoms; improve one or more sequelae; prevent (e.g., suppress, inhibit or delay) one or more symptoms; prevent (e.g., suppress, inhibit or delay) one or more sequelae; delay one or more symptoms; delay one or more sequelae; ameliorate one or more symptoms; ameliorate one or more sequelae; shorten the duration of one or more symptoms; shorten the duration of one or more sequelae; reduce the frequency of one or more symptoms; reduce the frequency of one or more sequelae; reduce the severity of one or more symptoms; reduce the severity of one or more sequelae; improve quality of life; increase survival; prevent (e.g., suppress, inhibit or delay) recurrence of a disease or condition; delay recurrence of a disease or condition; reduce the severity of the disease (e.g., reduce the extent of intracranial hemorrhage); or any combination thereof.

[0048] The term "treatment" also includes prophylaxis or prevention (e.g., suppression, inhibition, or delay) of a disease or condition or its symptoms or sequelae. Prevention refers to a therapeutic activity or course of activity used to prevent, inhibit, inhibit, reduce the risk of, reduce the occurrence of, or delay the onset of, a disease or condition, e.g., intracranial hemorrhage, or prevent, inhibit, inhibit, or delay the symptoms associated with a disease or condition.

[0049] In some embodiments, the disease or condition is a thrombotic disorder, particularly intracranial hemorrhage after ischemic stroke. In some embodiments, the ApTOLL molecule is used in the treatment of a thrombotic disorder, such as intracranial hemorrhage after ischemic stroke, or to prevent or reduce the occurrence or severity of intracranial hemorrhage.

[0050] In another embodiment, the disease or condition is neurological damage after acute ischemic stroke. In some embodiments, the ApTOLL molecule is for treating, reducing, or improving neurological recovery from neurological damage after ischemic stroke.

[0051] Ischemic strokeAs used herein, this term refers to a type of stroke (also known as cerebrovascular disease, cerebral infarction, cerebral attack, or apoplexy) characterized by neurological deficits caused by an abnormally rapid and significant reduction in cerebral blood flow. In ischemic stroke, blood perfusion is lost due to a sudden and immediate interruption of blood flow caused by occlusion of one of the arteries that irrigate the cerebral mass, resulting in the appearance of an infarcted area. Arterial occlusion is generally due to atherosclerosis or embolism (cerebral embolism) originating elsewhere, generally the heart or another artery. Ischemic stroke is a pathology characterized by increased TLR-4 expression and / or increased TLR-4 activation. Considering that the activation of TLR-4 causes a signal transduction cascade, resulting in 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, causing inflammation and cell damage, the pathology characterized by increased expression of TLR-4 and / or increased activation of TLR-4 can also be characterized as having an inflammatory component. "Acute ischemic stroke" refers to the "first stage" of ischemic stroke, where treatment (for example, with ApTOLL molecule) is administered to the patient. However, "ischemic stroke" and "acute ischemic stroke" are used interchangeably herein. Acute ischemic stroke will hereinafter be referred to as AIS.

[0052] Onset of stroke: As used herein, this term refers to the time point at which a stroke is induced. In clinical practice, stroke onset corresponds to the onset of symptoms, i.e., the first clinical symptom or sign of a particular condition, in this case, stroke, i.e., the time of onset or last known well-being (LTSW). Because the difference between stroke onset and onset is minimal, the terms "stroke onset" and "onset" are used interchangeably herein. Some stroke symptoms include sudden numbness or weakness in the face, arms, or legs (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, loss of coordination, and sudden, severe headache of unknown cause.

[0053] In the APRIL study, several parameters regarding symptom onset were defined, including EVT, pharmacologic thrombolysis, and inclusion criteria.

[0054] In the APRIL study, several time points were defined relative to the time of patient allocation to the study (similar to baseline time point) rather than the time of stroke onset: final infarct volume (measured 72 hours after allocation), NIHSS score (measured 72 hours after allocation), and mRS score (measured 90 days after allocation).

[0055] In some cases, patient allocation is proximal to symptom onset, e.g., the inclusion criterion of an infarct volume of 5cc to 70cc is measured prior to allocation, but is proximal to symptom onset. Thus, the terms "stroke onset," "onset," "allocation," and "baseline" can be used interchangeably in these cases.

[0056] Intracranial hemorrhage (ICH)As used herein, this term refers to spontaneous intracerebral hemorrhage resulting from complications of ischemic stroke, often induced by reperfusion therapy such as thrombolysis or EVT. ICH refers to any hemorrhage within the intracranial vault, including the brain parenchyma and surrounding meningeal spaces. Because ICH in this disclosure results from ischemic stroke and is not the primary cause of disease, ICH is also referred to herein as "secondary hemorrhage." ICH can be symptomatic (sICH) or asymptomatic (aICH). ICH can result in the development of new symptoms or worsening of existing symptoms, in which case it is referred to as sICH. True ICH is associated with a clear worsening of neurological status as established by the investigator in terms of a worsening of the NIHSS score by ≥ 4 points.

[0057] As used herein, 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. This occurs when the blood-brain barrier (BBB) ​​is sufficiently disrupted to allow peripheral blood to extravasate into the brain. HT, when it occurs, increases the morbidity and mortality of stroke.

[0058] In some embodiments, the intracranial hemorrhage is symptomatic. In some embodiments, the intracranial hemorrhage is a hemorrhagic transformation.

[0059] Intracranial hemorrhage can be measured by imaging procedures such as non-contrast CT, CTA, CPT, and MRI. Intracranial hemorrhage can be classified, for example, according to the Heidelberg Classification of Hemorrhage.

[0060] time frameAs used herein, this term, also referred to as "therapeutic window," refers to the time period between injury and treatment during which treatment is effective. Herein, treatment can refer to drug therapy with fibrinolytic / thrombolytic agents (e.g., alteplase or tenecteplase), also known as intravenous thrombolysis (IVT), with a time frame in accordance with the European Stroke Organization (ESO) guidelines. Berge E et al., 2021, provides recommendations for the time frame for using different thrombolytic agents, which is typically <4.5 hours from the onset of stroke. Herein, treatment can also refer to EVT (thrombectomy), which is recommended within 24 hours after the onset of stroke, specifically within 8 hours (Jovin TG et al., 2015). Note that the APRIL study protocol set the EVT time frame at 6 hours, aiming to adhere to the 8-hour period.

[0061] Neurological damage : As used herein, this term refers to neurological dysfunction or deterioration after suffering from ischemic stroke. Neurological dysfunction includes, but is not limited to, neuromuscular dysfunction that causes physical disabilities such as mobility problems, apraxia, pain syndrome, limb spasticity, and incontinence; cognitive dysfunction that negatively affects the patient's cognitive ability, ranging from memory loss to impairments in reasoning, speech, learning ability, language processing, and problem-solving skills; and psychiatric disorders (emotional problems), such as mood disorders, for example, depression, anxiety, emotional lability, crisis reaction, and post-stroke fatigue. This can lead to general physical disability and dependency in daily activities for people who have suffered from stroke. In the APRIL study and herein, the damage is quantified by infarct volume, NIHSS score, and mRS score, as defined herein below. "Neurological damage" and "neurological dysfunction" are used interchangeably herein. In some embodiments, the ApTOLL molecule is for reducing neurological damage / impairment and also for improving neurological recovery after AIS in a subject.

[0062] In one embodiment, the neurological damage comprises a functional impairment and / or a physical disability, hi another embodiment, the neurological damage comprises a neuromuscular dysfunction, a cognitive impairment, and / or a psychiatric disorder.

[0063] In some embodiments, the ApTOLL molecule is for treating, reducing, or improving neurological recovery after ischemic stroke. In this context, the term "neurological recovery" refers to the improvement or amelioration of neurological damage.

[0064] NIHSS score The National Institutes of Health Stroke Scale (NIHSS) is a tool used by healthcare providers to objectively quantify the functional impairment caused by stroke. The NIHSS consists of 11 items, each of which scores a specific ability from 0 to 4. For each item, a score of 0 typically indicates normal function in that specific ability, while higher scores indicate some level of functional impairment. Individual scores from each item are summed to calculate a patient's total NIHSS score. The maximum possible score is 42, and the minimum is 0. Stroke severity scores are: 0 = no stroke symptoms; 1–4 = mild stroke; 5–15 = moderate stroke; 16–20 = moderate-to-severe stroke; and 21–42 = severe stroke (Lyden P et al., 1994). The NIHSS can help physicians quantify stroke severity in acute settings. In one embodiment, the reduction in neurological damage and improvement in neurological recovery is measured by the NIHSS score.

[0065] mRS scoreThe modified Rankin Scale (mRS) is a commonly used scale for measuring the degree of disability and dependency on daily activities in people who have suffered a stroke or other causes of neurological disability. The scale ranges from 0 to 6, ranging from complete health with no symptoms to death: 0 - no symptoms; 1 - no significant disability. Able to perform all daily activities despite some symptoms; 2 - slight disability. Cannot perform all previous activities but able to care for oneself without assistance; 3 - moderate disability. Requires some help but able to walk without assistance; 4 - moderate to severe disability. Unable to meet one's physical needs without assistance and unable to walk without assistance; 5 - severe disability. Bedridden, incontinent, requiring regular care and attention; 6 - death (Van Swieten JC et al., 1988). In one embodiment, reduction in neurological damage and improvement in neurological recovery are measured by the mRS score.

[0066] Infarct volume Infarct volume is a direct measurement of damaged brain tissue, one of the final pathological steps resulting in clinical deficits caused by ischemic stroke. Therefore, the final infarct volume derived from magnetic resonance imaging (MRI) represents an objective and validated measure of the impact of stroke. 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 discrepant determinations were read to determine the ischemic core at baseline. The final infarct volume (FIV) after the procedure (72 hours) was determined by MRI-FLAIR (MRI-point recovery imaging) or by CT scan if MRI was not available.

[0067] Hydrocephalus or cerebral edemaAs used herein, this term refers to a severe complication of AIS, which is the cause of death in 5% of all patients with cerebral infarction. Cerebral edema is caused by capillary endothelial dysfunction, leading to the breakdown of the blood-brain barrier (BBB). Edema causes tissue changes and increased intracranial pressure, which can lead to death. In clinical practice, edema is determined using imaging techniques, i.e., computed tomography or magnetic resonance imaging.

[0068] ASPECTS: The Alberta Stroke Program Early CT Score (ASPECTS) is a 10-point quantitative topographic CT scan score developed to provide the reliability and utility of standard CT examinations with a reproducible grading system for assessing early ischemic changes (<3 hours from onset) in pretreatment CT studies of patients with anterior circulation AIS. This CT score is simple, reliable, and identifies stroke patients who are unlikely to recover independently despite thrombolytic treatment. The score divides the middle cerebral artery (MCA) territory into 10 regions of interest. ASPECTS is therefore a topographic scoring system that applies a quantitative approach, avoiding the need for physicians to estimate volume from two-dimensional images (Pexman JH et al., 2011).

[0069] subject The terms "subject," "patient," and "individual," and variations thereof, are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired, including, but not limited to, humans, domestic animals (e.g., dogs, cats, etc.), livestock (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.). The uses and methods described herein are applicable to both human therapy and veterinary applications. In certain embodiments, the subject is a human, particularly a human having the characteristics described in the "Subject Characteristics" section of this description.

[0070] identity: As used herein, this term refers to overall monomer conservation between polymer molecules, for example, between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules). The term "identical" without any additional qualifier, for example, nucleic acid A is identical to nucleic acid B, means 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." Sequence comparison and percent identity determination between two sequences can be achieved 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 MAFFT, Clustal (ClustalW, Clustal X, or Clustal Omega), MUSCLE, etc.

[0071] about The term "about" is used herein to mean approximately, roughly, around, or within the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" can modify a numerical value above or below the stated value by, for example, a 10 percent up or down (higher or lower) variance. As used herein, the terms "about" or "at least about," when applied to a series of values ​​or ranges, apply equally to all members of the list. Thus, "at least about 1, 2, 3, 4..." is interchangeable with "at least about 1, at least about 2, at least about 3, at least about 4...."

[0072] Clinical outcomes Reducing the risk of intracranial hemorrhage after AIS As discussed above, bleeding complications are common after thrombolytic therapy such as tPA or mechanical thrombectomy such as EVT, which can lead to poor functional outcomes and even death in stroke patients.

[0073] The APRIL study demonstrated that administration of ApTOLL (SEQ ID NO: 1) in combination with EVT in selected stroke patients limited the incidence of intracranial hemorrhage and reperfusion injury that can occur after recanalization (Example 1). Only 4.8% of stroke patients treated with a combination of ApTOLL and EVT developed symptomatic intracranial hemorrhage, compared with 7.3% of stroke patients treated with EVT alone (Table 4). Thus, the risk of developing symptomatic intracranial hemorrhage after suffering an ischemic stroke was reduced by up to 34.25%, an unexpected reduction that may be a major solution for AIS patients and may result in increased survival. Table 4 also shows that 40.47% of patients treated with ApTOLL 0.2 mg / kg suffered from intracranial hemorrhage, compared with 47.27% of patients with intracranial hemorrhage who were not treated with ApTOLL (placebo). Thus, there is a 14.39% reduction in the risk of suffering from intracranial hemorrhage for patients treated with ApTOLL 0.2 mg / kg.

[0074] Thus, the present invention provides an ApTOLL molecule for reducing the risk of intracranial hemorrhage or secondary hemorrhage (or preventing or reducing the occurrence of intracranial hemorrhage) in a subject after acute ischemic stroke, wherein the ApTOLL molecule is administered after acute ischemic stroke (i.e., after the onset or onset of the stroke).

[0075] It is understood that the uses disclosed herein may alternatively be formulated as methods for reducing the risk of (suffering from) intracranial hemorrhage (or preventing or reducing the occurrence of intracranial hemorrhage) in a subject following an acute ischemic stroke, comprising administering an ApTOLL molecule to the subject, wherein the ApTOLL molecule is administered after the acute ischemic stroke.

[0076] In one embodiment, the intracranial hemorrhage is a hemorrhagic transformation. In another embodiment, the intracranial hemorrhage is symptomatic. In another embodiment, the intracranial hemorrhage is asymptomatic.

[0077] In some embodiments, the reduction in risk of intracranial hemorrhage is 5% to 90% compared to a control condition, e.g., compared to the risk in a subject not treated with an ApTOLL molecule. In certain embodiments, the reduction in risk of intracranial hemorrhage is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% compared to a subject not treated with an ApTOLL molecule. In certain embodiments, the reduction in risk of intracranial hemorrhage is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% compared to a subject not treated with an ApTOLL molecule. In certain embodiments, the reduction in risk of intracranial hemorrhage is at least about 10%, 11%, 12%, 13%, or 14%, 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, specifically, the ApTOLL molecule is ApTOLL (SEQ ID NO: 1).

[0078] In some embodiments, the reduction in risk of symptomatic intracranial hemorrhage is a 5% to 90% reduction compared to a control condition, e.g., compared to the risk in a subject not treated with an ApTOLL molecule. In certain embodiments, the reduction in risk of symptomatic intracranial hemorrhage is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% reduction relative to a subject not treated with an ApTOLL molecule. In certain embodiments, the reduction in risk of symptomatic intracranial hemorrhage is at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% reduction relative to a subject not treated with the ApTOLL molecule, specifically, the reduction is at least about a 30%, 31%, 32%, 33%, or 34%, more specifically, a reduction of about 34%. In one embodiment, the ApTOLL molecule is administered at a dose of 0.2 mg / kg, specifically, the ApTOLL molecule is ApTOLL (SEQ ID NO: 1).

[0079] As described, the primary criterion for selecting candidates for reperfusion therapy is the time from the onset of stroke. Reperfusion therapy must be administered within a narrow time window: up to 4.5 hours after the onset of stroke for tPA and up to 24 hours for EVT. The restriction on tPA treatment beyond 4.5 hours would disqualify the majority (around 85%) of stroke patients admitted beyond this time frame, thereby significantly limiting the eligible population. The timing of treatment is important because administering a strong anticoagulant such as tPA during a stroke can cause bleeding within the brain.

[0080] In the APRIL study, administration of ApTOLL together with EVT was shown to reduce intracranial hemorrhage, such as ICH and HT, i.e., treatment with tPA and EVT did not result in significant intracranial hemorrhage within the brain due to the effects of ApTOLL. Therefore, ApTOLL may be able to effectively reach at-risk tissues, exerting a direct protective effect in the ischemic penumbra region, potentially extending the therapeutic window of reperfusion therapy.

[0081] Thus, in certain embodiments, administration of an ApTOLL molecule of the present invention (eg, SEQ ID NO: 1) to a subject following an ischemic stroke extends the therapeutic window of reperfusion therapy.

[0082] In certain embodiments, administration of an ApTOLL molecule to a subject after an ischemic stroke extends the therapeutic window of pharmacological thrombolysis (e.g., tPA). In certain embodiments, the therapeutic window of pharmacological thrombolysis (e.g., tPA) is extended to at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours from the onset of the stroke.

[0083] In certain embodiments, administration of an ApTOLL molecule to a subject after an ischemic stroke extends the therapeutic window of EVT (e.g., thrombectomy). In certain embodiments, the therapeutic window of 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 from the onset of the stroke.

[0084] In addition to reducing secondary hemorrhage, the APRIL study also demonstrated that administration of ApTOLL 0.2 mg / kg resulted in reduced mortality, reduced final infarct volume, improved early neurological dysfunction and long-term disability (as measured by NIHSS and mRS scores), and reduced cerebral edema.

[0085] Thus, in some embodiments, administration of an ApTOLL molecule to a subject after an ischemic stroke comprises: (i) Reduction of infarct volume; (ii) reduction in NIHSS score; (iii) an increase in mRS score of 0–2 (i.e., a reduction in mRS score to 0–2); (iv) increased survival rate; (v) reduction of cerebral edema; and (vi) any combination thereof results.

[0086] The uses disclosed herein alternatively relate to an ApTOLL molecule (e.g., SEQ ID NO: 1) for treatment (i.e., therapy or method) for reducing the risk of intracranial hemorrhage or secondary hemorrhage following acute ischemic stroke in a subject, the treatment comprising: (a) Selecting patients with acute ischemic stroke within approximately 6 hours of onset; (b) administering a dose of the aptamer to the patient of at least 0.2 mg / kg; and (c) measuring intracranial hemorrhage or secondary hemorrhage, specifically by imaging procedures; Including, administration of the ApTOLL molecule reduces the risk of intracranial hemorrhage or secondary hemorrhage relative to a reference value from a subject not treated with the ApTOLL molecule; The ApTOLL molecule may be formulated as a present.

[0087] Reduced risk of intracranial hemorrhage after thrombotic disorders As mentioned above, the reduction of 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 suffering from ischemic stroke. Furthermore, the evidence provided in the APRIL study makes it plausible that ApTOLL may also be useful for reducing intracranial hemorrhage in similar conditions, specifically in thrombotic disorders. For example, fibrinolysis is also administered after myocardial infarction, which may cause systemic (remote) hemorrhage transformation, such as digestive bleeding or local hemorrhage. Therefore, the effect of ApTOLL in reducing intracranial hemorrhage is also valid for thrombotic disorders other than ischemic stroke.

[0088] Thus, the present invention also relates to ApTOLL molecules for reducing the risk of intracranial hemorrhage or secondary hemorrhage (or preventing intracranial hemorrhage or reducing the occurrence of intracranial hemorrhage) in a subject following a thrombotic disorder, wherein the ApTOLL molecule is administered after the thrombotic disorder.

[0089] Alternatively, the present invention also relates to a method for reducing the risk of (suffering from) intracranial hemorrhage (or preventing or reducing the occurrence of intracranial hemorrhage) in a subject following a thrombotic disorder, comprising administering an ApTOLL molecule to the subject, wherein the ApTOLL molecule is administered after the thrombotic disorder.

[0090] In some embodiments, thrombotic disorder is associated with thrombotic event, i.e., thrombosis. In some embodiments, thrombotic disorder is a disease or disorder selected from the group consisting of arterial thrombosis, for example, atherothrombosis, venous thrombosis, ischemic event, acute coronary syndrome, thrombotic occlusion of coronary artery, coronary thrombosis, cerebrovascular accident due to atherosclerosis (particularly thrombosis), myocardial infarction (heart attack), acute cerebrovascular ischemia (ischemic stroke), percutaneous coronary intervention, stent thrombosis, restenosis, disease of the aorta and its branches (for example, aortic aneurysm, thrombosis), peripheral arterial disease, venous thrombosis, acute phlebitis and pulmonary embolism, cancer-related thrombosis (Trousseau syndrome), inflammatory thrombosis and thrombosis associated with inflammation, and deep vein thrombosis, among other embolism.

[0091] In certain embodiments, the thrombotic disorder is selected from the group consisting of cerebral artery disease, cerebrovascular disease, or coronary artery disease.

[0092] In certain embodiments, the thrombotic disorder is a cerebrovascular disease (e.g., stroke or ischemia). In certain embodiments, the cerebrovascular disease is a stroke.

[0093] In certain embodiments, the thrombotic disorder is coronary artery disease, including, but not limited to, cardiovascular diseases such as 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, and cardiac ischemia, including, but not limited to, complications associated with coronary procedures, e.g., percutaneous coronary angioplasty (balloon angioplasty) procedures.

[0094] In certain embodiments, the thrombotic disorder is venous thromboembolic disease, e.g., diseases including leg swelling, pain, and ulcers, pulmonary embolism, abdominal vein thrombosis, hi another embodiment, the thrombotic disorder is thrombotic microangiopathy, vascular purpura, etc.

[0095] Medical conditions associated with thrombolytic agents include arterial thrombosis, deep vein thrombosis, acute myocardial infarction, acute ischemic stroke, IV catheter clots, pulmonary embolism, and thrombotic / thromboembolic disorders.

[0096] 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).

[0097] In some embodiments, the uses and methods described herein for reducing the risk of intracranial hemorrhage following a thrombotic disorder in a subject further include the use of a thrombolytic drug. Thrombolytic drugs or thrombolytic agents include, for example, streptokinase, alteplase, reteplase, tenecteplase, urokinase, prourokinase, anistreplase (APSAC), and the like.

[0098] Reducing neurological damage and improving neurological recovery The APRIL study showed consistent positive results in most predefined secondary endpoints, including final infarct volume, early neurological dysfunction, and long-term disability (Example 1). Of note, these three measures improved simultaneously in the APRIL study.

[0099] Thus, the present invention also provides an ApTOLL molecule for reducing neurological damage and improving neurological recovery after an acute ischemic stroke in a subject, wherein the ApTOLL molecule is administered after an acute ischemic stroke.

[0100] The uses disclosed herein may alternatively be formulated as methods for reducing neurological damage and improving neurological recovery after an acute ischemic stroke in a subject, comprising administering an ApTOLL molecule to the subject, wherein the ApTOLL molecule is administered after an acute ischemic stroke.

[0101] As noted above, the inventors have found that the reduction in intracranial hemorrhage was unexpected and consistently resulted in reduced neurological damage and improved neurological recovery.

[0102] Thus, in one embodiment, the ApTOLL molecule is used to reduce neurological damage and improve neurological recovery after acute ischemic stroke in a subject, wherein the ApTOLL molecule is administered after acute ischemic stroke and the ApTOLL molecule reduces the risk of (or prevents) intracranial hemorrhage (e.g., HT and sICH); in other words, the reduction in neurological damage and improvement in neurological recovery is mediated by the reduction in the risk of (or prevention of) intracranial hemorrhage.

[0103] Alternatively, another embodiment relates to a method of reducing neurological damage and improving neurological recovery after acute ischemic stroke in a subject, comprising administering an ApTOLL molecule to the subject, wherein the ApTOLL molecule is administered after acute ischemic stroke, and the reduction in neurological damage and improvement in neurological recovery is mediated by a reduced risk of intracranial hemorrhage.

[0104] In one embodiment, the ApTOLL molecule is used to reduce neurological damage after acute ischemic stroke in a subject, wherein the ApTOLL molecule is administered after acute ischemic stroke, and the ApTOLL molecule reduces the risk of (or prevents) intracranial hemorrhage (e.g., HT and sICH), or 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 physical disability. In another embodiment, the neurological damage includes neuromuscular dysfunction, cognitive impairment, and / or psychiatric disorder.

[0105] 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 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.

[0106] In the APRIL study, reduction in neurological damage and improvement in neurological recovery were assessed by measuring several parameters at baseline, 72 hours, and / or 90 days after subject allocation: infarct volume, NIHSS score, and mRS score.

[0107] Thus, in some embodiments, the reduction in neurological damage and improved neurological recovery is achieved by: (i) infarct volume; (ii) NIHSS score; (iii) mRS score; or (iv) any combination thereof It is measured by

[0108] In certain embodiments, neurological damage is measured by infarct volume, NIHSS score and mRS score, particularly infarct volume.In another specific embodiment, the improvement of neurological recovery is measured by infarct volume, NIHSS score and mRS score, particularly NIHSS score and mRS score, more particularly mRS score.

[0109] In certain embodiments, functional disability is measured by the NIHSS score. In another embodiment, physical disability is measured by the mRS score.

[0110] The uses disclosed herein alternatively include an ApTOLL molecule (e.g., SEQ ID NO: 1) for use in a treatment (i.e., a therapy or method) for reducing neurological damage and improving neurological recovery after acute ischemic stroke in a subject, wherein the treatment comprises: (a) Selecting patients with acute ischemic stroke within approximately 6 hours of onset; (b) administering a dose of the aptamer to the patient of at least 0.2 mg / kg; and (c) measuring infarct volume, NIHSS score, mRS score, or a combination thereof; Including, A reduction in neurological damage and an improvement in neurological recovery relative to a reference value from a subject not treated with the ApTOLL molecule. (i) reduction of infarct volume; (ii) reduction in NIHSS score, and / or (iii) reduction of mRS score to 0–2 Consists of: The ApTOLL molecule may be formulated as a present.

[0111] Infarct volume 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 within 72 ± 24 hours, CT was measured 24 hours later. The baseline infarct volume cannot be considered FIV in any case. In cases where post-treatment (meaning after ApTOLL administration) imaging was not available, the patient was considered "missing." In patients who developed AIS, a reduction in infarct volume directly correlates with anatomical protection, functional, and neurological performance.

[0112] Thus, in certain embodiments, reduced neurological damage and improved neurological recovery is measured by infarct volume.

[0113] The biological effect of ApTOLL on infarct volume was assessed by the change in infarct volume from baseline to final infarct volume at 72 hours and also by comparison of final infarct volume at 72 hours between study groups (placebo vs. ApTOLL).

[0114] Therefore, 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 the ApTOLL molecule reduces the infarct volume of the subject compared to a subject not treated with the ApTOLL molecule.

[0115] The reduction in neurological damage and improvement in neurological recovery is measured by the reduction in final infarct volume at a specific time point (e.g., 72 hours from assignment) compared to subjects not treated with the ApTOLL molecule (e.g., placebo). Table 4 and Figure 2 show that patients treated with ApTOLL 0.2 mg / kg had a median final infarct volume of 23.5 ml at 72 hours, compared to patients not treated with ApTOLL (placebo), who had a final infarct volume of 44 ml. Thus, patients treated with ApTOLL had a 46.6% reduction in final infarct volume relative to subjects not treated with ApTOLL.

[0116] Thus, in certain embodiments, the reduction in final infarct volume at short term, e.g., 72 hours, is at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% reduction relative to subjects not treated with the ApTOLL molecule. In certain embodiments, the reduction in final infarct volume at 72 hours is at least about a 46% reduction relative to subjects not treated with the ApTOLL molecule.

[0117] In another embodiment, the final infarct volume in the short term, for example, 72 hours, is about 10 ml to about 45 ml, more specifically, 20 ml to 25 ml.

[0118] 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).

[0119] Reduction in neurological damage and improvement in neurological recovery can also be measured by reduction in infarct volume over time (e.g., from baseline to 72 hours) compared to subjects not treated with the ApTOLL molecule (e.g., placebo). Table 5 shows that patients treated with ApTOLL 0.2 mg / kg had a baseline infarct volume of 14 ml and a final infarct volume at 72 hours of 23.5 ml, thereby resulting in a 67.86% increase in infarct volume caused by the evolution of the stroke. Instead, patients not treated with ApTOLL (placebo) had a baseline infarct volume of 20.50 ml and a final infarct volume at 72 hours of 44 ml, thereby resulting in a 114.63% increase in infarct volume. Therefore, patients treated with ApTOLL experienced a smaller increase in infarct volume compared to patients not treated with ApTOLL. In particular, patients not treated with ApTOLL have a 40.80% increase in infarct volume over time relative to patients treated with ApTOLL.

[0120] Thus, in certain embodiments, the reduction in infarct volume over time, for example, from baseline to 72 hours, is at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% reduction relative to a subject not treated with the ApTOLL molecule. In particular embodiments, the reduction in infarct volume over time is at least about a 40% reduction relative to a subject not treated with the ApTOLL molecule. In certain embodiments, the ApTOLL molecule is administered at a dose of 0.2 mg / kg, and specifically, the ApTOLL molecule is ApTOLL (SEQ ID NO: 1).

[0121] To assess the early clinical course and long-term neurological outcomes, the changes from baseline in the NIHSS score and mRS scale, respectively, were calculated in the APRIL study and comparisons were made between groups at the end (72 hours for NIHSS and 90 days for mRS).

[0122] NIHSS score 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 vs. placebo: -45%; 95% CI: -67% to -10%) (Table 4, Figure 2). This represents a 57.14% reduction compared to placebo.

[0123] Thus, in certain embodiments, reduction in neurological damage and improvement in neurological recovery are measured by NIHSS score. In certain embodiments, neurological damage is functional impairment.

[0124] Therefore, the present invention relates to an ApTOLL molecule for 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 the ApTOLL molecule reduces the subject's NIHSS score compared to a subject not treated with the ApTOLL molecule.

[0125] In certain embodiments, the reduction in neurological damage and improvement in neurological recovery are measured by a reduction in NIHSS score over the short term (e.g., 72 hours) compared to subjects not treated with the ApTOLL molecule (e.g., placebo). In certain embodiments, the reduction in NIHSS score is 5% to 90% relative to subjects not treated with the ApTOLL molecule. In certain embodiments, 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 reduction in NIHSS score is at least about a 40%, 45%, 50%, 55%, or 60% reduction relative to subjects not treated with the ApTOLL molecule, specifically, the reduction is at least about a 50%, 51%, 52%, 53%, 54%, 55%, 56%, or 57%, more specifically, a reduction of about 57%. In one embodiment, the ApTOLL molecule is administered at a dose of 0.2 mg / kg, specifically, the ApTOLL molecule is ApTOLL (SEQ ID NO: 1).

[0126] mRS score In the APRIL study, the mRS scale was measured at baseline (pre-stroke mRS) and at 72 hours and 90 days after baseline to assess long-term neurological outcomes. If data were missing at 72 hours or 90 days, the last measurement after treatment (meaning after ApTOLL administration) was considered.

[0127] Thus, in certain embodiments, reduction in neurological damage and improvement in neurological recovery are measured by mRS score. In certain embodiments, the neurological damage is a disability.

[0128] Thus, the present invention relates to an ApTOLL molecule for 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 an mRS score of 3 to 6, and / or increases an mRS score of 0 to 2 (i.e., reduces the mRS score to 0 to 2), compared to a subject not treated with the ApTOLL molecule. An improvement in the mRS score is reflected by a decrease in the mRS score, toward lower values ​​between 0 and 2.

[0129] Reduction in neurological damage and improvement in neurological recovery are measured at a specific time point (e.g., 90 days) by a reduction in an mRS score of 3 to 6 or an increase in an mRS score of 0 to 2 (i.e., a reduction in an mRS score to 0 to 2) compared to subjects not treated with the ApTOLL molecule (e.g., placebo). Table 4 and Figure 3 show that patients treated with ApTOLL 0.2 mg / kg had a 32.40% reduction in an mRS score of 3 to 6 (moderate to severe disability to death) relative to subjects not treated with ApTOLL, i.e., there were 32.40% fewer patients with an mRS score of 3 to 6 assigned to the ApTOLL 0.2 mg / kg group. Furthermore, Table 4 and Figure 3 also show that patients treated with ApTOLL 0.2 mg / kg had a 36.29% increase in mRS score from 0 to 2 (0 being no symptoms to 2 being slight disability) relative to subjects not treated with ApTOLL, i.e., there were 36.29% more patients with an mRS score of 0 to 2 assigned to the ApTOLL 0.2 mg / kg group (meaning their mRS score was reduced to 0 to 2 from a higher mRS value); specifically, patients treated with ApTOLL 0.2 mg / kg had a 71.23% increase in mRS score from 0 to 1 (0 being no symptoms to 1 being minor disability) relative to subjects not treated with ApTOLL (meaning their mRS score was reduced to 0 to 2 from a higher mRS value). The results suggest that patients treated with ApTOLL within 6 hours of stroke onset had milder disability in the long term (90 days), with more patients with mRS scores of 0-1 and 0-2, and fewer patients with mRS scores of 3-6, compared with the placebo group.

[0130] Thus, in certain embodiments, the long-term, e.g., 90-day, reduction in an mRS score of 3-6 is at least about a 10%, 15%, 20%, 25%, 30%, or 35% reduction relative to subjects not treated with an ApTOLL molecule. In certain embodiments, the 90-day reduction in an mRS score of 3-6 is at least about a 32% reduction relative to subjects not treated with an ApTOLL molecule.

[0131] In another embodiment, the long-term, e.g., 90-day, increase in mRS score from 0 to 2 (i.e., reduction in mRS score to 0 to 2) is at least about a 10%, 15%, 20%, 25%, 30%, 35%, or 40% increase relative to a subject not treated with the ApTOLL molecule. In a specific embodiment, the increase in mRS score from 0 to 2 at 90 days is at least about a 36% increase relative to a subject not treated with the ApTOLL molecule.

[0132] In another embodiment, the long-term, e.g., 90-day, increase in mRS score from 0 to 1 (i.e., reduction in mRS score to 0 to 1) is at least about a 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% increase relative to a subject not treated with the ApTOLL molecule. In a specific embodiment, the increase in mRS score from 0 to 1 at 90 days is at least about a 71% increase relative to a subject not treated with the ApTOLL molecule.

[0133] In certain embodiments, the increase in mRS score of 0 at 90 days (i.e., reduction in mRS score to 0) is at least about a 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, or 120% increase relative to subjects not treated with the ApTOLL molecule. In specific embodiments, the increase in mRS score of 0 at 90 days is at least about a 120% increase relative to subjects not treated with the ApTOLL molecule.

[0134] In another embodiment, the increase in mRS score of 1 at 90 days (i.e., reduction in mRS score to 1) is at least about a 30%, 35%, 40%, 45%, or 50% increase relative to a subject not treated with an ApTOLL molecule. In a specific embodiment, the increase in mRS score of 1 at 90 days is at least about a 50% increase relative to a subject not treated with an ApTOLL molecule.

[0135] In another embodiment, the reduction in an mRS score of 4 at 90 days is at least about a 10%, 15%, 20%, or 25% reduction relative to subjects not treated with the ApTOLL molecule. In a specific embodiment, the reduction in an mRS score of 4 at 90 days is at least about a 25% reduction relative to subjects not treated with the ApTOLL molecule.

[0136] In another embodiment, the reduction in mRS score of 5-6 at 90 days is at least about a 40%, 45%, 50%, 60%, 65%, 70%, 75%, or 80% reduction relative to a subject not treated with the ApTOLL molecule. In a specific embodiment, the reduction in mRS score of 5-6 at 90 days is at least about a 75% reduction relative to a subject not treated with the ApTOLL molecule.

[0137] 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).

[0138] Reduction in neurological damage and improvement in neurological recovery can also be measured by an increase in mRS score of 0-2 over time (e.g., baseline - pre-stroke mRS to 90 days) compared to subjects not treated with the ApTOLL molecule (e.g., placebo). Table 5 shows that 100% of patients treated with ApTOLL 0.2 mg / kg had a pre-stroke mRS of 0-2, which decreased to 64.29% at 90 days, representing a 35.71% reduction. In contrast, 98.15% of patients not treated with ApTOLL (placebo) had a pre-stroke mRS of 0-2, which decreased to 47.17% at 90 days, representing a 51.94% reduction. Therefore, patients treated with ApTOLL have a smaller reduction in mRS score of 0-2 compared to patients not treated with ApTOLL. In particular, patients not treated with ApTOLL had a 31.25% reduction in mRS score of 0 to 2 over time compared to patients treated with ApTOLL.

[0139] Thus, in some embodiments, the increase in mRS from 0 to 2 over time, e.g., from baseline to 90 days (i.e., reduction in mRS to 0 to 2), is at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% increase relative to subjects not treated with the ApTOLL molecule. In particular embodiments, the increase in mRS from 0 to 2 over time is at least about a 31% increase relative to subjects not treated with the ApTOLL molecule. In some embodiments, the ApTOLL molecule is administered at a dose of 0.2 mg / kg, and specifically, the ApTOLL molecule is ApTOLL (SEQ ID NO: 1).

[0140] In certain embodiments, the reduction in neurological damage and the improvement in neurological 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 to 6 and / or an increase in mRS score of 0 to 2 (i.e., a reduction in mRS to 0 to 2) compared to subjects not treated with the ApTOLL molecule (e.g., a placebo). Specifically, the reduction in neurological damage and the improvement in neurological 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 to 2 (i.e., a reduction in mRS to 0 to 2).

[0141] Thus, the present invention relates to an ApTOLL molecule for 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 the ApTOLL molecule (i) reduces infarct volume, (ii) reduces the NIHSS score, and / or (iii) increases the subject's mRS score from 0 to 2 (i.e., a reduction in the mRS to 0 to 2) or reduces the mRS score from 3 to 6, compared to a subject not treated with the ApTOLL molecule. Specifically, the ApTOLL molecule (i) reduces infarct volume, (ii) reduces the NIHSS score, and (iii) increases the subject's mRS score from 0 to 2 (i.e., a reduction in the mRS to 0 to 2).

[0142] In certain embodiments, compared to subjects not treated with the ApTOLL molecule, (i) the reduction in infarct volume at 72 hours is about 10% to about 50%, more specifically at least about 40%; (ii) the reduction in NIHSS is about 40% to about 60%, more specifically at least about 55%; and / or (ii) the increase in mRS from 0 to 2 (i.e., a reduction in mRS from 0 to 2) is about 10% to about 40%, more specifically at least about 35%.

[0143] As described above, in the APRIL study, the reduction of neurological damage and the improvement of neurological recovery are evaluated by measuring infarct volume, NIHSS score and mRS score.However, the reduction of neurological damage and the improvement of neurological recovery can also be evaluated by measuring other scores known to those skilled in the art, such as Barthel Index / Score (BI), Asian Stroke Disability Scale (ASDS), Glasgow Outcome Score (GOS), etc.

[0144] Increased survival and reduced cerebral edema The APRIL study demonstrated that administration of ApTOLL in combination with EVT in selected stroke patients was safe and reduced mortality at 90 days. Furthermore, the efficacy of ApTOLL as a neuroprotective agent in acute cerebral ischemia is supported by consistent positive results for most of the prescribed primary endpoints, including reduction of cerebral edema (Examples 1, 1.13-1.15, Table 4).

[0145] Thus, the present invention also provides an ApTOLL molecule for increasing survival and / or reducing cerebral edema following acute ischemic stroke in a subject, wherein the ApTOLL molecule is administered following acute ischemic stroke.

[0146] It is understood that the uses disclosed herein may alternatively be formulated as methods for increasing survival and / or reducing cerebral edema following acute ischemic stroke in a subject, comprising administering an ApTOLL molecule to the subject, wherein the ApTOLL molecule is administered following acute ischemic stroke.

[0147] As noted above, the inventors have found that the reduction in intracranial hemorrhage was unexpected and resulted in increased survival and reduced cerebral edema.

[0148] Thus, in one embodiment, the ApTOLL molecule is used to increase survival and / or reduce cerebral edema after acute ischemic stroke in a subject, wherein the ApTOLL molecule is administered after acute ischemic stroke and the ApTOLL molecule reduces the risk of intracranial hemorrhage (e.g., HT and sICH) (or prevents intracranial hemorrhage), in other words, the increased survival and reduced cerebral edema are mediated by a reduced risk of intracranial hemorrhage.

[0149] Alternatively, another embodiment relates to a method of increasing survival rate and / or reducing cerebral edema following acute ischemic stroke in a subject, comprising administering an ApTOLL molecule to the subject, wherein the ApTOLL molecule is administered following acute ischemic stroke, and wherein the increased survival rate and reduced cerebral edema are mediated by a reduced risk of intracranial hemorrhage.

[0150] Table 4 shows that in the APRIL study, mortality was reduced from 18.2% to 4.85% when 0.2 mg / kg of ApTOLL was administered compared to placebo, representing a 73.63% reduction in mortality. In certain embodiments, administration of an ApTOLL molecule causes a reduction in mortality relative to the placebo group. Specifically, the reduction in mortality is 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 reduction in mortality is 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).

[0151] In other words, the APRIL study achieved a 95.2% survival rate throughout the study period when 0.2 mg / kg of ApTOLL was administered, and an 81.8% survival rate without ApTOLL treatment, representing a 14.08% increase in survival. In one embodiment, administration of an ApTOLL molecule results in an increase in survival relative to the placebo group. Specifically, the increase in survival is at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, or 14% relative to the placebo group. Specifically, the increase in survival is at least about 10%, more specifically about 14%, 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).

[0152] Table 4 also shows that 2.38% of patients treated with ApTOLL 0.2 mg / kg suffered from cerebral edema, compared to 7.3% of patients with cerebral edema who were not treated with ApTOLL (placebo). Thus, there is a 67.40% reduction in cerebral edema for patients treated with ApTOLL 0.2 mg / kg. In certain embodiments, administration of an ApTOLL molecule causes 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 a 67% reduction relative to the placebo group. In one embodiment, the ApTOLL molecule is administered at a dose of 0.2 mg / kg, specifically, the ApTOLL molecule is ApTOLL (SEQ ID NO: 1).

[0153] Figure 4 is a graphical representation of patient quality of life assessments after treatment with ApTOLL 0.05 mg / kg, 0.02 mg / kg, or placebo. Quality of life assessments include the following criteria: mobility, self-care, daily activities, pain / discomfort, and anxiety / depression. In the APRIL study, improvements in anxiety / depression complications, performance of daily activities, mobility, and self-care were observed with administration of 0.2 mg / kg ApTOLL compared to placebo.

[0154] Thus, another aspect of the present invention relates to an ApTOLL molecule for improving anxiety / depression complications, performance of daily activities, mobility and / or self-care following acute ischemic stroke in a subject, wherein the ApTOLL molecule is administered following acute ischemic stroke.

[0155] It is understood that the uses disclosed herein may alternatively be formulated as methods for improving anxiety / depression complications, performance of daily activities, mobility and / or self-care in a subject following acute ischemic stroke, comprising administering an ApTOLL molecule to the subject, wherein the ApTOLL molecule is administered following acute ischemic stroke.

[0156] Administration and dosage of ApTOLL molecules As detailed in Example 1, the APRIL study was conducted in two parts, Phase Ib and Phase IIa. During Phase Ib, four escalating dose levels of SEQ ID NO: 1 (0.025, 0.05, 0.1, and 0.2 mg / kg) were administered, while two doses of SEQ ID NO: 1 (Dose A = 0.05 mg / kg and Dose B = 0.2 mg / kg) were administered during Phase IIa. The results of Phase IIa (Example 1, 1.13-1.15) show that Dose B at 0.2 mg / kg was effective in patients suffering from AIS compared to patients in the placebo and Dose A groups.

[0157] Thus, in some embodiments, the ApTOLL molecule of the present invention (e.g., SEQ ID NO: 1) has a cytotoxicity 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 mg / kg, 0.20 mg / kg, 0.21 mg / kg, 0.22 mg / kg, 0.23 mg / kg, 0.24 mg / kg, 0.25 mg / kg, 0.26 mg / kg, 0.27 mg / kg, 0.28 mg / kg, 0.29 mg / kg, 0.30 mg / kg, 0.31 mg / kg, 0.32 mg / kg, 0.33 mg / kg, 0.34 mg / kg, 0.35 mg / kg, 0.36 mg / kg, 0.37 mg / kg, 0.38 mg / kg, 0.39 mg / kg, 0.40 mg / kg, 0.41 mg / kg, 0.42 mg / kg, 0.43 mg / kg, 0.44 mg / kg, 0.45 mg / kg, 0.46 mg / kg, 0.47 mg / kg, 0.48 mg / kg, 0.49 mg / kg, 0.50 mg / kg, 0.51 mg / kg, 0.52 mg / kg, 0.53 mg / kg, 0.54 mg / kg, 0.55 mg / kg, 0.56 mg / kg, 0. In certain 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.

[0158] In some embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered at a dose range of about 0.05 mg / kg to about 200 mg / kg. In another embodiment, the dose range is about 0.06 mg / kg to about 50 mg / kg. In another embodiment, the dose range is about 0.06 mg / kg to about 10 mg / kg. In another embodiment, the dose range is about 0.1 mg / kg to about 5 mg / kg. Specifically, the dose range is about 0.1 mg / kg to about 1 mg / kg. Specifically, the dose range is about 0.15 mg / kg to about 1 mg / kg. Specifically, the dose range is about 0.2 mg / kg to about 1 mg / kg. Specifically, the dose range is about 0.2 mg / kg to about 0.5 mg / kg.

[0159] A typical single dose amount is about 7 mg / dose to about 70 mg / dose, taking into account a dose range of about 0.1 mg / kg to about 1 mg / kg, and taking into account a typical body weight of a human subject of about 70 kg. 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.

[0160] 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, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. In certain embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered intravenously or intraarterially, for example, by infusion or by bolus.

[0161] In more specific embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered intravenously by infusion. In specific embodiments, the infusion has a duration of about 5, 10, 15, 20, 25, 30, 35, 40, or 60 minutes, specifically about 30 minutes.

[0162] In some embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered less than 24 hours after the ischemic stroke event. In certain embodiments, 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.

[0163] In certain embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered within about 8 hours, specifically within about 6 hours, of the onset of stroke. In the APRIL study, patients had a median time of 210 minutes (3.5 hours) from the onset of stroke to ApTOLL administration in the study. Thus, in certain embodiments, the ApTOLL molecule is administered within about 4 hours, more specifically within about 3.5 hours, of the onset of stroke.

[0164] In the APRIL study, intravenous administration of ApTOLL (SEQ ID NO: 1) was administered in conjunction with EVT and, when indicated, pharmacologic thrombolysis in the AIS target population. In general, ApTOLL was administered before EVT and after thrombolysis, when indicated. The median time from ApTOLL infusion to recanalization (when achieved) was approximately 180 minutes (3 hours).

[0165] 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 certain embodiments, the ApTOLL molecule is administered about 4 hours prior to EVT, specifically about 3 hours prior to EVT.

[0166] In other embodiments, the ApTOLL molecule (eg, SEQ ID NO: 1) is administered simultaneously with EVT.

[0167] In other embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered after EVT. In certain embodiments, the ApTOLL molecule is administered immediately after EVT. In certain embodiments, the ApTOLL molecule is administered about 5, 10, 15, 20, 25, or 30 minutes after EVT.

[0168] In other embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered prior to and simultaneously with EVT. In other embodiments, the ApTOLL molecule is administered prior to and immediately after EVT. In certain embodiments, the ApTOLL molecule is administered at least about 20 minutes prior to EVT and about 10 minutes after EVT.

[0169] In the APRIL study, patients received concomitant pharmacologic thrombolysis (primarily alteplase, but occasionally tenecteplase) when indicated. Alteplase was administered as a 1- to 2-minute bolus and a 60-minute infusion. Tenecteplase was administered as a 1- to 2-minute bolus. Generally, ApTOLL was administered after tPA administration when indicated. Because alteplase administration is longer-lasting, this occurred simultaneously with ApTOLL administration.

[0170] In some embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered after pharmacological thrombolysis (e.g., tPA administration). In certain embodiments, the ApTOLL molecule is administered immediately after thrombolysis. In certain embodiments, 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 about 5 minutes to about 30 minutes after thrombolysis.

[0171] In other embodiments, the ApTOLL molecule (eg, SEQ ID NO: 1) is administered concomitantly with pharmacological thrombolysis.

[0172] In other embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered prior to and / or concurrently with pharmacological 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 certain embodiments, the ApTOLL molecule is administered at least about 20 minutes prior to thrombolysis and about 10 minutes after thrombolysis.

[0173] In some embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered after pharmacological thrombolysis (e.g., tPA administration) and before EVT (e.g., thrombectomy). In certain embodiments, the ApTOLL molecule is administered about 5 to about 30 minutes after thrombolysis and about 4 hours to about 5 minutes before EVT.

[0174] In other embodiments, the ApTOLL molecule (e.g., SEQ ID NO: 1) is administered simultaneously with and / or after pharmacological thrombolysis, and before and / or simultaneously with EVT. In certain embodiments, the ApTOLL molecule is administered simultaneously with and immediately after thrombolysis, and about 4 hours and about 5 minutes before and / or simultaneously with EVT.

[0175] In some embodiments, the ApTOLL molecule (eg, SEQ ID NO: 1) is administered in multiple doses. In certain embodiments, the ApTOLL molecule is administered in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

[0176] Arterial recanalization method The uses and methods described herein further include administering additional pharmacological and / or surgical steps, for example, additional ischemic stroke or thrombotic disorder treatments.

[0177] Thus, the ApTOLL molecules of the present invention are administered simultaneously with, before, or after additional pharmacological and / or surgical steps, particularly arterial recanalization. Specifically, the ApTOLL molecules are administered in combination with arterial recanalization.

[0178] Arterial recanalization can be induced mechanically (e.g., endovascular thrombectomy), pharmacologically (e.g., thrombolysis), or a combination thereof. In certain embodiments, arterial recanalization is mechanical, pharmacological, pharmaco-mechanical, or a combination thereof.

[0179] In some embodiments, the arterial recanalization is mechanical. In certain embodiments, the mechanical arterial recanalization is endovascular therapy (i.e., EVT). In certain embodiments, the EVT is selected from the group consisting of stent retrieval thrombectomy, balloon embolectomy, direct aspiration thrombectomy, surgical embolectomy, or a combination thereof.

[0180] In certain embodiments, EVT is performed within 24 hours of the onset of ischemic stroke. Specifically, EVT is performed within 8 hours of the onset of stroke. More specifically, EVT is performed within 6 hours of the onset of stroke.

[0181] In the APRIL study, the quality of reperfusion after EVT was assessed using the Extended Thrombolysis in Cerebral Ischemia (eTICI) scale. Recanalization status was assessed for all patients as follows: (a) vascular recanalization after successful thrombectomy (TICI 2b or 3); (b) persistent LVO in patients who failed to recanalize after EVT (TICI 0-2a). A recanalization rate of 87% was achieved. In one embodiment, the final eTICI score is 2b-3. In one embodiment, the recanalization rate is at least about 87%.

[0182] In some embodiments, the arterial recanalization is pharmacological. In certain embodiments, the pharmacological arterial recanalization is pharmacological thrombolysis. In certain embodiments, the pharmacological thrombolysis is fibrinolytic therapy. In certain embodiments, the fibrinolytic is tissue plasminogen activator (tPA, alteplase) and modified alteplase (e.g., tenecteplase).

[0183] In certain embodiments, pharmacological thrombolysis (eg, tPA) is administered within 4.5 hours of stroke onset.

[0184] In certain embodiments, the arterial recanalization is pharmaco-mechanical. In certain embodiments, the pharmaco-mechanical arterial recanalization comprises EVT and pharmaco-thrombolysis (e.g., tPA).

[0185] In some embodiments, the uses and methods described herein further include the use of imaging procedures, such as non-contrast computed tomography (NCCT), computed tomography angiography (CTA), computed tomography perfusion (CTP), and magnetic resonance imaging (MRI). The following imaging and angiographic variations can be extracted from the imaging procedures using published definitions and standards: ASPECTS, hemorrhage, hemorrhage (Heidelberg), ischemic core (post-hoc DWI or CTP rCBF<30% volume if available), Tmax>6 sec volume (if CTP or PWI is available), baseline occlusive lesion location, CTA collateral score (based on availability - post-hoc), presence of stenosis proximal to arterial occlusive lesion, arterial occlusive lesion, collateral flow grade - ASITN, eTICI at each device pass, distal embolization, embolization to new territory, dissection, vessel perforation, post-hoc DWI of final infarct volume or CTP rCBF<30% volume if available, etc. Imaging procedures can be performed pre-baseline (IV infusion and transport hospital), baseline, intra-procedure, post-procedure (24 hours), post-procedure (72 hours), or during the post-procedure course.

[0186] Subject characteristics In the APRIL study, patients who were functionally independent before the stroke event (defined by a pre-stroke mRS score, e.g., 0-2) but had an ischemic stroke (measured by an NIHSS score, e.g., 5-25) resulting in moderate infarct volume (IV), occlusion of a large cerebral artery, and disability at the onset of the stroke were selected to maximize the effect of ApTOLL (SEQ ID NO: 1). Therefore, different criteria, such as age, baseline NIHSS, pre-stroke mRS score, infarct volume at the onset of the stroke, and occlusion site, were used to select subjects for treatment with ApTOLL. Nevertheless, these values ​​are not restrictive, and more patients may be eligible for treatment with the ApTOLL molecule of the present invention. All inclusion parameters were measured at the time of stroke / onset.

[0187] In some embodiments, the subject is a human subject. In certain embodiments, the subject is a male. In other embodiments, the subject is a non-pregnant female.

[0188] In some embodiments, the subject is about 18 to about 90 years old. In certain embodiments, the subject is about 60 to about 80 years old. In certain embodiments, the subject is >70 years old or about 70 years old. In certain embodiments, the subject is about 75 years old.

[0189] The criterion used for selecting subjects for treatment with an ApTOLL molecule (e.g., SEQ ID NO: 1) is a baseline NIHSS score, which is used to quantify the severity of stroke in acute settings. In some embodiments, the subject has a baseline NIHSS score of about 5 to about 25 points. In some embodiments, the subject has a baseline NIHSS score of about 8 to about 25 points. In certain embodiments, the subject has a baseline NIHSS score of about 16 points. In certain embodiments, the subject has a baseline NIHSS score of about 11 to about 21 points. In certain embodiments, the subject has a severe baseline NIHSS score, i.e., about 15 to about 24 points. In certain embodiments, the subject has a baseline NIHSS score of about 15 to about 20 points, specifically about 15 to about 18 points.

[0190] Another criterion used to select subjects for treatment with an ApTOLL molecule (e.g., SEQ ID NO: 1) is the pre-stroke mRS score, which is used to determine neurological disability, i.e., the subject's mRS score before the ischemic stroke event. In some embodiments, the subject has a pre-stroke mRS score of 0 to about 2 points.

[0191] In some embodiments, the criterion used to select subjects for treatment with an ApTOLL molecule (e.g., SEQ ID NO: 1) is time from onset. Thus, in some embodiments, subjects are selected for treatment if onset 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 condition.

[0192] In some embodiments, the criterion used to select 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., ivtPA). In some embodiments, the indication for EVT is based on NCCT findings (i.e., Alberta Stroke Program Initial CT Score, ASPECTS, of 6 to 10). In some embodiments, the subject has an ASPECTS of about 6 to about 10.

[0193] In some embodiments, the criterion used to select subjects for treatment with an ApTOLL molecule (e.g., SEQ ID NO: 1) is the presence of a stroke or a single LVO (TICI 0 or TICI 1 flow) at the level of the TICA or M1 or M2 segment of the middle cerebral artery at the time of onset.

[0194] Another criterion used to select 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 about 5 cc to about 70 cc at the time of stroke onset or assignment. In certain embodiments, the subject has an infarct volume on CT perfusion (CTP), defined as a cerebral blood flow (CBF) < 30%, of about 5 ml to about 70 ml, as measured by automated software (RAPID® software).

[0195] In some embodiments, the criteria used for selecting subjects for treatment with an ApTOLL molecule (e.g., SEQ ID NO: 1) are vascular occlusions suitable for mechanical thrombectomy, for example, as determined or confirmed by computed tomography angiography (CTA). In certain embodiments, the criteria used for selecting subjects for treatment with an ApTOLL molecule are neuroimaging criteria (CT or MRI), for example, (i) magnetic resonance imaging (MRI) criteria: a volume of a restricted region on diffusion-weighted imaging (DWI) of ≥ about 5 mL and ≤ about 70 mL, as determined, for example, by RAPID® software; and / or (ii) Computed tomography (CT) criteria: Alberta Stroke Program Initial CT Score (ASPECTS) of about 6 to about 10, and an infarction score determined, for example, by RAPID® software, at admission cerebral blood flow (CBF) < 30% of ≥ about 5 mL and ≤ about 70 mL. is an LVO suitable for mechanical thrombectomy as determined or confirmed by

[0196] In one embodiment, the subject has a DWI restricted diffusion volume of about 5 mL to about 70 mL, hi another embodiment, the subject has ASPECTS of about 6 to about 10.

[0197] In one embodiment, the subject, at the onset of the stroke, i) Approximately 18 to approximately 90 years old; ii) have a baseline NIHSS of approximately 8 points to approximately 25 points; iii) have a pre-stroke mRS score of 0 points to approximately 2 points; iv) have an infarct volume of about 5 cc to about 70 cc; v) have an onset of less than about 6 hours; vi) being a candidate for EVT treatment; vii) TICA, with occlusion in the M1 or M2 segment of the middle cerebral artery; viii) have an mTICI score of 0 or 1; ix) having a DWI restricted diffusion volume of approximately 5 mL to approximately 70 mL; x) having about 6 to about 10 ASPECTS; or xi) any combination thereof is.

[0198] In certain embodiments, the subject has a baseline NIHSS score of about 8 to about 25 points, a pre-stroke mRS score of 0 to about 2 points, and an infarct volume of about 5 cc to about 70 cc at the time of stroke onset or assignment.

[0199] In certain embodiments, the subject has a single LVO at the level of the TICA or M1 or M2 segment of the middle cerebral artery, and a measurement of infarct core volume on CTP defined as cerebral blood flow <30% of about 5 ml to about 70 ml by automated software (RAPID®, iSchemaView).

[0200] In certain embodiments, the subject is about 18 to about 90 years old, has an LVO within a 6 hour window, ASPECTS of about 6 to about 10, and an estimated infarct core volume on CT perfusion of about 5 ml to about 70 ml.

[0201] In the APRIL study, higher effects were observed in final infarct volume and long-term functional improvement (mRS) in populations over 70 years old with 0.2 mg / kg ApTOLL administration. Thus, in one embodiment, the subject is >70 years old. Administration of 0.2 mg / kg ApTOLL also appears to have a higher effect on reducing infarct volume and mRS score in patients with a baseline NIHSS score of more than 15 points. Thus, in one embodiment, the subject has a baseline NIHSS score of >15 points.

[0202] ApTOLL molecule As used herein, the term "ApTOLL molecule" refers to an aptamer selected from SEQ ID NOs: 1-24 or SEQ ID NOs: 1-16, variants and / or derivatives of said aptamers, or chemically modified aptamers thereof. APTOLL molecules specifically bind to at least one epitope located on the extracellular domain of TLR-4 and have the ability to inhibit TLR-4. The specificities and features of all these aptamers are disclosed in WO2015197706A1, WO2020230108A1, and WO2020230109A1 (AptaTargets SL), which are incorporated herein by reference in their entireties.

[0203] The aptamers of SEQ ID NOs: 1-16 have lengths between 45 and 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 variants 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 these variants with different percentages of sequence identity and / or 5' and 3' extensions of ApTOLL (SEQ ID NO: 1) have TLR4 antagonist activity equivalent to that of ApTOLL (SEQ ID NO: 1).

[0204] In some embodiments, the ApTOLL molecule of the present invention is an aptamer selected from SEQ ID NOs: 1 to 24, specifically selected from SEQ ID NOs: 1 to 16, and more specifically selected from SEQ ID NOs: 1 to 4. In a more particular embodiment, the aptamer is SEQ ID NO: 1.

[0205] In other embodiments, the ApTOLL molecule is a variant and / or derivative of an aptamer selected from SEQ ID NOS: 1-24, specifically selected from SEQ ID NOS: 1-16, more specifically SEQ ID NOS: 1-4, and even more specifically SEQ ID NOS: 1. In some embodiments, the ApTOLL molecule is a variant and / or derivative having at least 70% sequence identity to SEQ ID NOS: 1-24, wherein the variant and / or derivative is derived from SEQ ID NOS: 1-24 and maintains the ability to specifically bind and reduce and / or inhibit TLR-4 activation. Specifically, the ApTOLL molecule comprises a sequence at least 85% identical to SEQ ID NOS: 1-24, more specifically, a sequence 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 maintains the ability to specifically bind 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, more specifically, a sequence that is at least 90% or 95% identical to SEQ ID NOs: 1-16.

[0206] In other embodiments, the ApTOLL molecule is a chemically modified aptamer selected from SEQ ID NOs: 1-24, specifically selected from SEQ ID NOs: 1-16, more specifically SEQ ID NOs: 1-4, and even more specifically SEQ ID NO: 1. Chemical modifications of aptamers 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 other modifications.

[0207] In some embodiments, the ApTOLL molecule has a length of about 40 to about 100 nucleotides. Specifically, the ApTOLL molecule has a length of about 45 to about 78 nucleotides, more specifically, about 59 to about 76 nucleotides.

[0208] In some embodiments, the ApTOLL molecule has an optional 5' extension of a nucleotide sequence 1 to 13 nucleotides in length, hi other embodiments, the ApTOLL molecule has an optional 3' extension of a nucleotide sequence 1 to 4 nucleotides in length.

[0209] In a more particular embodiment, (a) the ApTOLL molecule has a length of 40 to 100 nucleotides and is selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4, wherein: (i) the ApTOLL molecule specifically binds to an epitope on the extracellular domain of TLR-4; and (ii) binding of the ApTOLL molecule to the epitope reduces and / or inhibits TLR-4 activity; or (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 maintains the ability to specifically bind to and reduce and / or inhibit TLR-4 activation.

[0210] In a more particular embodiment, the ApTOLL molecule is SEQ ID NO: 1 (named ApTOLL in the APRIL study). In another particular embodiment, the ApTOLL molecule is a variant and / or derivative of SEQ ID NO: 1 having at least 70% sequence identity (more particularly, 85%) to SEQ ID NO: 1, where the variant and / or derivative is derived from SEQ ID NO: 1 and maintains 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.

[0211] The chemical formula of ApTOLL is C 575 H 723 N 223 O 351 P 58 The molecular weight of ApTOLL is 18,170.80 Da. The tertiary structure of ApTOLL is shown in Figure 6.

[0212] The ApTOLL molecule is formulated into a pharmaceutical composition suitable for administration to a subject. In the APRIL study, ApTOLL and placebo are formulated as powders for a concentrate for infusion solution, which are reconstituted with 3 mL of water for injection, and diluted in a saline solution bag according to the patient's weight (100 mL of a 9 mg / mL [0.9%] solution of sodium chloride for injection). The resulting solution can be administered intravenously, for example, by an infusion pump. In one embodiment, the ApTOLL molecule is presented as one vial of 7 mg of lyophilized powder for a concentrate for infusion solution for intravenous administration.

[0213] In one embodiment, the ApTOLL molecule is formulated in PBS-MgCl2. In one embodiment, the formulation contains sodium chloride, potassium chloride, anhydrous disodium hydrogen phosphate, and potassium dihydrogen phosphate to create a phosphate buffer solution of pH 7.4 containing magnesium chloride hexahydrate. This buffer solution and conditions support the aptamer structure and its biological activity. ApTOLL is substituted for A-trehalose dihydrate in the placebo formulation.

[0214] Specific Embodiments As will be apparent to one of ordinary skill in the art upon reading this description, each of the individual embodiments described and illustrated herein has distinct components and features that can be combined with features of any of the other several embodiments without departing from the scope or spirit of the invention. Specific combinations of the above embodiments are described herein, as detailed in different sections.

[0215] The present invention relates to an ApTOLL molecule, particularly an aptamer having SEQ ID NO: 1 (tested in the APRIL study), for reducing the risk of intracranial hemorrhage following acute ischemic stroke in a subject, wherein the aptamer is administered at a dose of about 0.5 to about 1 mg / kg following acute ischemic stroke, and wherein administration of the aptamer reduces the risk of intracranial hemorrhage by about 5% to about 40% relative to subjects 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, 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 subjects not treated with the aptamer. Specifically, the aptamer is administered at a dose of at least about 0.2 mg / kg.

[0216] The present invention provides an ApTOLL molecule, particularly an aptamer having SEQ ID NO: 1, for reducing neurological damage and improving neurological recovery after acute ischemic stroke in a subject, wherein the reduction in neurological damage and improvement in neurological recovery is mediated by a reduced 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 the aptamer is administered to a subject who has not been treated with the aptamer. (i) reducing infarct volume; (ii) reduce NIHSS score, and / or (iii) increasing an mRS score of 0–2 (i.e., reducing the mRS to 0–2); Also pertaining to the ApTOLL molecule is the aptamer, specifically administered at a dose of at least about 0.2 mg / kg.

[0217] Specifically, the present invention provides an ApTOLL molecule, particularly an aptamer having SEQ ID NO: 1, for improving neurological recovery after acute ischemic stroke in a subject, wherein the improved neurological recovery is mediated by a reduced 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 the aptamer is administered at a dose of about 0.5 to about 1 mg / kg after acute ischemic stroke, relative to a subject not treated with the aptamer. (i) reduce NIHSS score, and / or (ii) reducing mRS to 0–2; The present invention also relates to an ApTOLL molecule. 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.

[0218] In one embodiment, the reduction in infarct volume at 72 hours is about 10% to 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 about 10% to about 50%, specifically at least about 40%, relative to a subject not treated with the aptamer.

[0219] In certain embodiments, the reduction in NIHSS score at 72 hours is about 40% to about 60%, specifically at least about 57%, relative to subjects not treated with the aptamer.

[0220] In one embodiment, the increase in mRS score from 0 to 2 (i.e., reduction in mRS to 0 to 2) at 90 days is about 10% to about 40%, specifically at least about 36%, relative to subjects not treated with the aptamer. In another embodiment, the increase in mRS score from 0 to 2 (i.e., reduction in mRS to 0 to 2) over time is about 10% to about 50%, specifically at least about 31%, relative to subjects not treated with the aptamer.

[0221] In certain embodiments, the aptamer reduces cerebral edema by about 40% to about 80%, particularly by at least about 67%, relative to a subject not treated with the aptamer.

[0222] In certain embodiments, the aptamer is administered within about 8 hours of the onset of stroke, particularly within about 4 hours of the onset of stroke.

[0223] In certain embodiments, the aptamer is administered in combination with arterial recanalization.

[0224] In some embodiments, the aptamer is administered in combination with intravascular treatment. In certain embodiments, the aptamer is administered before or simultaneously with intravascular treatment. Specifically, the aptamer is administered less than about 4 hours before intravascular treatment.

[0225] In certain embodiments, the aptamer is administered in combination with pharmacological thrombolysis, particularly administration of tissue plasminogen activator, particularly administered simultaneously with or subsequent to pharmacological thrombolysis.

[0226] In one embodiment, the aptamer is administered intravenously by infusion over about 30 minutes.

[0227] In one embodiment, the subject, at the onset of the stroke, i) Approximately 18 to approximately 90 years old; ii) have a baseline NIHSS of approximately 8 points to approximately 25 points; iii) have a pre-stroke mRS score of 0 points to approximately 2 points; iv) have an infarct volume of about 5 cc to about 70 cc; v) TICA, with occlusion in the M1 or M2 segment of the middle cerebral artery; vi) have an mTICI score of 0 or 1; vii) having a volume of the restricted area of ​​diffusion-weighted imaging of approximately 5 mL to approximately 70 mL; viii) having about 6 to about 10 ASPECTS; or ix) any combination thereof.

[0228] The contents of all cited references (including literature references, patents, patent applications, and websites) that may be cited throughout this application are expressly incorporated by reference in their entirety for any purpose, as are the references cited therein.

[0229] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0230] Example 1: APRIL: A double-blind, placebo-controlled, randomized, Phase Ib / IIa clinical trial of ApTOLL for the treatment of AIS Trial identification: EudraCT: 2020-002059-38 and ClinicalTrials.gov identifier: NCT04734548

[0231] Methods and analysis 1.1. Overall study design APRIL is a multicenter, double-blind, randomized, placebo-controlled, parallel-group, Phase Ib / IIa clinical trial designed to evaluate the safety and tolerability, pharmacokinetics, and biological effects of ApTOLL in patients with AIS and confirmed LVO who were candidates for endovascular treatment with or without ivtPA. The study population consisted of eligible men and women who were able to receive ApTOLL within 6 hours of symptom onset. Allocation in Phase IIa consisted of three strata: initial infarct core size (<35 vs. ≥35 cc), patient age (<70 vs. ≥70 years), and NIHSS score (<15 vs. ≥15). For patients with morning stroke, time of onset was considered the time when symptoms were first detected.

[0232] The APRIL clinical trial was divided into two parts: (1) Phase Ib (n=32) single iv administration (30-minute infusion), dose escalation with four single dose levels (8 patients / level), randomized (1:3); and (2) Phase IIa (n=119) single-dose, iv administration (30-minute infusion), parallel (3-arm, placebo:ApTOLL dose A:ApTOLL dose B), randomized (√2:1:1). Therefore, after the completion of Phase Ib, the Data Safety Monitoring Board (DSMB) unblinded the study arms, selecting the two doses (A, B) to be tested in Phase IIa according to the initial safety results.

[0233] Prior to enrollment, all patients underwent a complete neuroimaging examination, including non-contrast computed tomography (NCCT), CT angiography, and CT perfusion (CTP). Only patients with an indication for EVT based on NCCT findings (i.e., Alberta Stroke Program Initial CT Score, ASPECTS > 5) were evaluated as candidates for the APRIL study. Inclusion criteria for further neuroimaging were the presence of a single LVO at the level of the TICA or the M1 or M2 segment of the middle cerebral artery, and identification of a favorable CTP to maximize the opportunity to identify the biological effect of the study drug. A predicted infarct core volume of 5–70 ml at CTP, defined as CBF < 30%, must be confirmed using automated software (RAPID® software) to be eligible for the APRIL study. Complete inclusion and exclusion criteria are detailed in section 1.3. After obtaining informed consent, patients were randomized to receive EVT + ApTOLL versus EVT + placebo, as shown in Figure 5. Study medication must be administered after imaging acquisition and before EVT initiation (groin puncture). All patients were then treated according to institutional protocols, as well as national and European Stroke Organization (ESO) guidelines. Figure 1 summarizes the scheme of the APRIL study.

[0234] 1.2. Purpose and Endpoints The objective of the APRIL study was to evaluate whether ApTOLL is safe and shows any biological effects in AIS patients with LVO. Primary objective: To assess whether escalating doses of intravenous ApTOLL are safe and well tolerated compared with placebo when administered with EVT in the AIS target population. Secondary Objectives: Although unique, the secondary objectives differed between the two study parts: Phase Ib : To determine the pharmacokinetic profile of ApTOLL in patients with AIS, as assessed by determining ApTOLL levels in plasma and urine. · To select the two doses to be administered in Phase IIa according to their safety profile. To provide an initial estimate of the biological effect of ApTOLL on final infarct volume (measured by MRI-FLAIR [magnetic resonance imaging-point recovery imaging] at 72 ± 24 hours) and pro-inflammatory biomarkers associated with AIS (baseline (pre-dose), and at the end of infusion [up to 1 hour], 6 hours, 24 hours, 48 ​​hours, and 72 hours after dosing). Phase IIa : To evaluate the biological effects of ApTOLL on final infarct volume (measured by MRI-FLAIR at 72±24 hours) and pro-inflammatory biomarkers associated with AIS (before dosing and at 6, 24, 48, and 72 hours after dosing). To determine the biological effect of ApTOLL as measured by functional disability (NIHSS [National Institutes of Health Stroke Scale]) at 72 hours or at discharge (whichever occurs first) and physical disability (modified Rankin Score [mRS]) 90 days after stroke.

[0235] To achieve these objectives, the following endpoints were assessed: Primary endpoint: Safety of ApTOLL in combination with EVT as determined by mortality, adverse events occurring during the study, physical examination, laboratory tests, recurrent stroke rate, and symptomatic intracranial hemorrhage (sICH) rate. Secondary endpoints: Mean final infarct volume (MRI) measured at 1.72 ± 24 hours. 2. Pro-inflammatory markers in the blood. 3. Initial clinical course (NIHSS at 72 hours). 4. Long-term outcome (mRS at 90 days). Neuroimaging pro-inflammatory biomarkers on MRI at 5.72 hours and 90 days after stroke (sub-study conducted only in some APRIL Spanish centers).

[0236] 1.3. Registration inclusion and exclusion criteria Participants were recruited at participating comprehensive stroke centers in Spain and France.Mechanical thrombectomy included the use of any commercially available stent retrieval or aspiration catheter recommended by the ESO guidelines, or a combination of the above.

[0237] 1.3.1. Inclusion criteria 1. Age ≥ 18 and ≤ 90 years. 2. Informed consent obtained from the subject or the subject's acceptable representative (i.e., next of kin or legal guardian). 3. There is a neurological deficit resulting in a new focal deficit consistent with acute cerebral ischemia. 4. Baseline NIHSS score ≥ 8 points and ≤ 25 points obtained before randomization. 5. Pre-stroke mRS score of 0-2. 6. Treatable as soon as possible from onset and within at least 6 hours of onset, defined as the point when the subject last appeared healthy (baseline). For morning stroke, time of onset was considered the time when symptoms were first detected (treatment initiation defined as study drug administration). 7. Candidates for EVT treatment with or without ivtPA. For such patient candidates for ivtPA therapy, tPA must be initiated as recommended by the European Stroke Organization for the initial management of patients with AIS. This means that the investigator must verify that the subject received / had received the correct ivtPA dose for their estimated body weight, and that tPA should be initiated as soon as possible and within 4.5 hours of stroke onset (time of onset defined as the last time the patient reported being healthy at baseline). If ivtPA was discontinued early for any reason, the cause and total administered dose were recorded. Additionally, once a patient was enrolled in the study, if recanalization was observed or confirmed before thrombectomy, the patient continued in the trial and was enrolled without protocol deviations.

[0238] Inclusion criteria for specific neuroimaging studies 8. Occlusion of the TICA, M1, or M2 segments of the middle cerebral artery (TICI 0 or TICI 1 flow) suitable for mechanical embolectomy and confirmed on CTA. Multiple extracranial and intracranial lesions may be included. 9. The following imaging criteria must also be met on admission neuroimaging: a) MRI criteria: a volume of a restricted region on DWI (diffusion weighted imaging) of ≥ 5 mL and ≤ 70 mL as determined by RAPID® software, or b) CT criteria: Alberta Stroke Program Initial CT Score (ASPECTS) of 6-10 at baseline CT, and infarct core determined at admission CT perfusion with CBF <30%: ≥5 mL and ≤70 mL as determined by RAPID® software. NOTE: ASPECTS were established according to investigator criteria. 10. Subjects were eligible and planned to undergo endovascular treatment of stroke according to ESO guidelines.

[0239] 1.3.2. Exclusion criteria 1. The subject has had a stroke within the past year. 2. Occlusion of the base of the skull or spine or posterior or anterior cerebral artery (TICI 0 or TICI 1 blood flow). 3. Clinical symptoms suggestive of bilateral stroke or stroke in multiple territories. 4. Known bleeding diathesis, coagulation factor deficiency, or receiving oral anticoagulant therapy due to INR > 3.0. 5. Baseline platelet count <50,000 / μL. 6. Baseline blood glucose level <50 mg / dL or >400 mg / dL. 7. Severe persistent hypertension (systolic blood pressure >185mmHg or diastolic blood pressure >110mmHg). NOTE: Patients can be enrolled if blood pressure is successfully reduced and maintained at an acceptable level using medical therapy (including iv antihypertensive infusions) recommended by the European Stroke Organization (ESO) guidelines. 8. Serious, progressive, or terminal illness with a predicted life expectancy of less than 1 year. 9. Patients with an identifiable intracerebral tumor (meningiomas are considered extracerebral tumors and are not included in this exclusion criterion). 10. History of life-threatening allergy (beyond a rash) to contrast media. 11. History of renal insufficiency with creatinine ≥ 3 mg / dL or glomerular filtration rate (GFR) < 30 mL / min. 12. If you have cerebral vasculitis. 13. History of active systemic infection. 14. Known to be using cocaine at the time of treatment. 15. Patients participating in a study involving an investigational drug or device that would affect this study. 16. Patients who may not be able to participate in the 90-day follow-up survey (e.g., no fixed home address, visitors from overseas). 17. Women who were pregnant or breastfeeding or had a positive pregnancy test at the time of admission.

[0240] Exclusion criteria for specific neuroimaging tests 18. CT or MRI findings of bleeding (microbleeds are allowed). 19. Significant mass effect with midline shift. 20. Suspected aortic dissection with suspected septic emboli or suspected bacterial endocarditis.

[0241] 1.4. Enrollment and Randomization All patients who met the eligibility criteria were eligible to enroll in the APRIL clinical trial, including both patients directly admitted to the study site and patients transferred (infusion and transport) from primary stroke centers.

[0242] 1. Treatment blinding The study was double-blind and masked, i.e., neither the patients nor the treating physicians knew the treatment being administered. The ApTOLL or matching placebo solution was clear, transparent, and colorless.

[0243] 2. Enrollment and Randomization Phase Ib During Phase Ib, four escalating dose levels (0.025, 0.05, 0.1, and 0.2 mg / kg) were completed. Within each dose level group, eight patients were randomized in a 3:1 ratio to receive ApTOLL plus EVT versus placebo plus EVT.

[0244] A staged dosing scheme was used to maximize patient safety. After each dose level (including the last patient follow-up 72-hour visit for the corresponding dose level), the DSMB evaluated safety outcomes and approved the next dose level, if appropriate. Safety parameters evaluated were (1) any SUSARs (unanticipated serious adverse events), SAEs (serious adverse events), or AEs potentially related to drug administration, and (2) blood chemistry parameters.

[0245] 3. Transition from Phase Ib to Phase IIa Prior to initiating Phase IIa and once safety data from the last patient in Phase Ib were available, the DSMB evaluated the overall safety results and selected the two optimal doses (Dose A, Dose B) to be studied in Phase IIa:

[0246] 1. Safety parameters related to stroke: -death. - Intracranial hemorrhage (ICH) and hemorrhagic transformation (HT) resulting in new symptoms or worsening of existing symptoms. -Cerebral edema resulting in herniation and neurological deterioration or death.Additional potential stroke-related AEs: seizures; cardiac conduction disturbances, arrhythmias; effects on coagulation and fibrinolysis; hypotension / hypertension; hyperglycemia; hyperpyrexia; severe infections; deep vein thrombosis, pulmonary embolism, and venous thromboembolism; vomiting; and anxiety, hallucinations, and agitation.

[0247] 2. Drug-related safety parameters: - Complement activation: CH50 (complement 50%) and C3 / C4 (complement factors 3 and 4) levels. -Biochemistry: CK (creatine kinase) and CRP (C-reactive protein) - Coagulation parameters: aPTT (activated partial thromboplastin time), prothrombin activity (PT) and INR (international normalized ratio).

[0248] 4. Enrollment and Randomization in Phase IIa 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 and EVT versus ApTOLL (dose A) and EVT versus ApTOLL (dose B) and EVT, yielding probabilities of random assignment of 0.41, 0.29, and 0.29, respectively.

[0249] In Phase IIa, the DSMB analyzed AEs and SAEs when approximately 20 patients were included in each treatment group and all of them reached the 72-hour follow-up visit. In both Phase Ib and Phase IIa, the CAC (Clinical Adjudication Committee) analyzed all AEs and SAEs to determine which were related to medication and communicated this determination to the DSMB. With this information, the DSMB reviewed the reported AEs and SAEs.

[0250] 1.5. Research treatment Both treatments (ApTOLL and placebo) were administered intravenously using an infusion pump over a period of 30 minutes.

[0251] 1. Research treatment ApTOLL was presented as a single vial of 7 mg powder for the concentrate for infusion solution for intravenous administration. ApTOLL and placebo were formulated as powder for the concentrate for infusion solution to be reconstituted with 3 mL of water for injection and diluted according to patient weight in a saline bag (100 mL of a 9 mg / mL [0.9%] solution of sodium chloride for injection). The placebo treatment matched the appearance of ApTOLL as powder for the concentrate for infusion solution and when reconstituted. The formulation with ApTOLL consisted of lyophilized sodium chloride, potassium chloride, anhydrous disodium hydrogen phosphate, and potassium dihydrogen phosphate to create a pH 7.4 phosphate buffer solution containing magnesium chloride hexahydrate. The API (i.e., ApTOLL) was substituted for α-trehalose dihydrate in the placebo formulation.

[0252] In the example for a 70 kg patient at level 3, 7 mg had to be administered, so 3 mL was taken from the stock solution and then diluted in 100 mL of a 9 mg / mL (0.9%) solution of sodium chloride for injection.

[0253] Administration of ApTOLL / placebo must begin before EVT is initiated (before groin puncture). The rate of infusion must not exceed 2.67 mg / min (equivalent to 3.33 mL / min when reconstituted and diluted) in all cases. Prior to reconstitution and dilution, ApTOLL and placebo were stored at -20°C. Reconstitution occurred immediately prior to administration (maximum 10 minutes prior to injection in saline bags).

[0254] The dosing scheme for Phase Ib was as follows (Table 1):

[0255] [Table 1]

[0256] 2. Acceptable and unacceptable medications / treatments prior to and during the clinical study In addition to the study medication, patients were treated according to ESO guidelines.

[0257] 1.6. Study Assessment and Procedures The treatment flow is shown in Figure 5. Each subject underwent the following procedure (Table 2). The maximum study period for each subject was 90 days.

[0258] [Table 2] TIFF2026506313000003.tif54170

[0259] X 1 NIHSS was performed on day 5 or at discharge (whichever occurred first). X 2 In Phase Ib, pro-inflammatory biomarker samples were collected pre-dose, and at the end of the infusion (up to 1 hour), 6 hours, 24 hours, 48 ​​hours, and 72 hours post-dose. In Phase IIa, pro-inflammatory biomarkers were analyzed pre-dose and 6 hours, 24 hours, 48 ​​hours, and 72 hours post-dose. X 3 -Pharmacokinetic samples were collected pre-dose, and at the end of the infusion (up to 1 hour), 6 hours, 24 hours, 48 ​​hours, and 72 hours after the end of the infusion. X 4 - If possible, collect a urine specimen up to 24 hours after administration of study drug. X 5 - The first ECG was performed 6 hours after administration. X 6 Only hospitals where inclusion was based on MRI criteria were included (i.e., France). X 7 -Only for centers participating in the imaging substudy. More about EVT 8 Inguinal puncture was performed 8 hours before the onset of symptoms. For morning stroke, the time of onset was considered to be the time when symptoms were first detected. X 9 - Etiology of stroke was provided, if available.

[0260] Endovascular thrombectomy (EVT) EVT was initiated (groin puncture) after randomization and administration of study medication. EVT was initiated within 8 hours of symptom onset (ApTOLL was administered within 6 hours of stroke onset) (for morning stroke, time of onset was considered the time when symptoms were first detected). Individual investigators used any approved device or any combination of devices to remove thrombus from the TICA, MCA M1 segment, or, if necessary, the M2 segment of the intracranial circulation.

[0261] Imaging Procedures : Non-contrast CT, CTA, CTP and MRI. CT images were read by appropriately trained local clinicians. Aspects for patient selection were determined independently by local clinicians. CTP images with discordant determinations were read by iSchemaView automated RAPID software. All brain imaging studies, including MRI, CTP / MRP, and CT, from stroke onset to discharge, as well as angiographic images obtained for the diagnostic and therapeutic portions of the procedure, were evaluated independently of each other and blinded to treatment assignment at the APRIL Central Imaging Core Lab (ICL). The following imaging and angiographic variables were extracted based on direct prior experience with these measurements and scales using published definitions and standards:

[0262] Baseline: ASPECTS Ischemic core - postmortem DWI, or if available, CTP rCBF <30% volume Tmax > 6 sec volume (if CTP or PWI is available) ·bleeding Baseline occlusive lesion location CTA collateral score – based on availability (post-hoc) procedure: Presence of stenosis proximal to the arterial occlusion Arterial occlusion Collateral flow grade - ASITN eTICI for each device passing through Distal embolization Embolization to new territories ·Incision ·Vessel perforation Post-procedure (24 hours): Infarct volume Hemorrhage (Heidelberg) Post-procedure (72 hours): Post-mortem DWI of final infarct volume (or CT if MRI is not available) Hemorrhage (Heidelberg) Neurological deterioration or post-procedure course: Relevant imaging findings and measurements based on availability (infarct volume for all available follow-up images)

[0263] In Phase IIa, a second MRI was performed 90 days after the procedure in the context of an imaging substudy to determine the total and relative size (gray / white) of the infarct volume at 90 days. In addition, cerebral perfusion parameters from IVIM (Intravoxel Incoherent Motion) sequences at 72 ± 24 hours, microstructural indices from DTI (Diffusion Tensor Imaging) at 72 ± 24 hours and 90 days, and iron content in brain tissue at 72 ± 24 hours and 90 days were measured.

[0264] Fibrinolytic therapy Patients received standard ESO guideline-directed medical therapy, including iv tPA infusion in patients who presented within the first 4.5 hours of their last apparent normal event and met other ESO label criteria. Post-tPA patients were treated according to standard institutional protocols for these patients. The time of tPA infusion and the dose administered were collected.

[0265] result 1.7. Study design and participants APRIL is a double-blind, randomized, multicenter, placebo-controlled, Phase Ib / IIa clinical study designed to evaluate whether administration of ApTOLL, with or without intravenous thrombolysis, in stroke patients with confirmed LVO who are candidates for EVT is safe, well-tolerated, and demonstrates any biological effects. The trial was conducted at 15 comprehensive stroke centers in Spain (12 centers) and France (3 centers). The study was approved by the ethics committees of each center and by the national regulatory agency. Signed informed consent was obtained from patients or their legally authorized representatives.

[0266] Eligible patients were men and non-pregnant women aged 18–90 years who had a disabling ischemic stroke at the time of randomization (baseline National Institutes of Health Stroke Scale [NIHSS] of 5–25; range 0–42, with higher scores indicating greater stroke severity); were functionally independent before the stroke, defined as a modified Rankin Scale (mRS) score of 0–2, with scores ranging from 0 (no symptoms) to 6 (death); and were able to administer ApTOLL within 6 hours of stroke onset. For patients with morning-onset stroke, the time of onset was considered the time when symptoms were first detected, as safety concerns were not anticipated if the study drug was administered more than 6 hours after the actual stroke onset.

[0267] Patients with a single LVO at the level of the TICA or M1 or M2 segment of the middle cerebral artery on noninvasive vascular imaging who were considered to undergo EVT based on non-contrast computed tomography (NNCT) findings (Alberta Stroke Program Early CT Score [ASPECTS] of 6–10; range 0–10, 1 point is subtracted for any evidence of early ischemic changes in each defined region on the CT scan) were evaluated as candidates for the APRIL study. To maximize the opportunity for identifying the biological effect of the study drug, identification of a previously defined (Olive-Gadea M et al., 2021) specific CT perfusion (CTP) profile was also required for enrollment. A predicted infarct core volume on CTP, defined as a relative cerebral blood flow <30% of 5–70 ml, had to be identified using previously validated automated software (RAPID®, iSchemaView) to be eligible for the APRIL study.

[0268] Quantitative imaging was scheduled to be obtained at the endovascular center; in a few cases (n=2), quantitative imaging was performed at the primary stroke center prior to transfer to the thrombectomy center. Screening logs were not kept. A complete list of eligibility criteria is available in Example 1, section 1.3.

[0269] 1.8. Enrollment, Randomization and Masking The APRIL clinical trial was divided into two parts. In Phase Ib, 32 patients were assigned to four escalating dose levels (0.025, 0.05, 0.1, and 0.2 mg / kg administered intravenously over 30 minutes) (six ApTOLL patients at each level; two placebo patients). A staged dosing regimen was used to maximize patient safety. After the results of the 72-hour follow-up visit were evaluated by the Data Safety Monitoring Board (DSMB), the next dose level was approved if deemed appropriate. The safety outcomes evaluated were any unexpected serious side effects, serious adverse events (SAEs), or adverse events (AEs) that could be related to drug administration and blood biochemistry parameters.

[0270] In both Phase Ib and Phase IIa, a masked clinical adjudication committee analyzed all AEs and SAEs to determine their possible relationship to study medication. With this information, a masked DSMB reviewed all reported AEs and SAEs. After Phase Ib was completed, the DSMB was instructed to select two doses to be tested in the subsequent Phase IIa study according to the initial safety results. Patients treated with either placebo or one of the two selected doses in Phase Ib were further analyzed alongside patients enrolled in Phase IIa. In Phase IIa, three arms were studied, and patients were randomized to one of the two selected doses of ApTOLL or placebo in a 1:1:√2 ratio, resulting in allocation probabilities of 0.293, 0.293, and 0.414, respectively. ApTOLL and placebo were prepared as colorless solutions in numbered, refrigerated (-20°C) vials. These vials were visually identical except for the unique vial number so that all study personnel and patients were completely masked to treatment assignment.

[0271] In both study phases, patients were randomly assigned using a real-time internet-based system. This process was automated from study launch, allowing complete concealment of the allocation sequence. The randomization system was originally designed to stratify allocation according to three strata: predicted infarct core size on admission CTP (<35 vs. ≥35 cc), patient age (<70 vs. ≥70 years), and admission NIHSS score (<15 vs. ≥15). However, due to a system error that went unnoticed until the end of the study, stratification was not applied. To account for potential bias introduced by this error, a post-hoc analysis of the primary outcome adjusting for the preplanned stratification factors was reported.

[0272] During Phase IIa, after 100 patients completed 72 hours of follow-up, the DSMB conducted a preplanned interim analysis and decided not to adopt any changes to the protocol for the remaining patients. Figure 1 summarizes the scheme of the APRIL study.

[0273] Procedure After imaging quantification, all patients underwent EVT and, if indicated, intravenous thrombolysis (either before or during EVT, at the primary hospital before transfer to the endovascular center, or at the endovascular center) in accordance with local institutional protocols and national and European stroke organization guidelines (Turc G et al., 2019). Patients must meet the inclusion and exclusion criteria at centers capable of thrombectomy. The study drug was administered as soon as possible after randomization, and investigators were required to ensure that the drug infusion began after imaging acquisition and before the start of EVT (arterial puncture).

[0274] Facilities were expected to adhere to national guidelines for stroke units, stroke rehabilitation, and stroke prevention care. All patients had demographic characteristics, past medical history, laboratory values, and standard assessment of stroke severity (NIHSS score). The quality of reperfusion after EVT was assessed using the Extended Thrombolysis in Cerebral Ischemia (eTICI) scale. Follow-up brain imaging, if available, was performed at 24 hours by CT and at 72 hours by MRI. Clinical follow-up was obtained in person, if possible, and at 90 days from randomization to assess the degree of disability as determined by the mRS. If in-person follow-up was not possible, follow-up was obtained via videoconference or telephone. In all patients recruited to Phase 1b, up to six consecutive blood samples (baseline, 1, 6, 24, 48, and 72 hours after randomization) were obtained before and after dosing for pharmacokinetic analysis of ApTOLL concentrations and determination of plasma concentrations of different pro-inflammatory biomarkers related to the acute stroke response. In Phase IIa, blood biomarkers were determined at five time points (baseline and 6, 24, 48, and 72 hours after randomization). Interpretation of imaging studies was blinded and performed in a central core laboratory (UCLA, Los Angeles, CA). Clinical data were verified by an independent monitor (Anagram-ESIC, Barcelona, ​​Spain).

[0275] Outcome The primary objective of the study was to evaluate whether intravenous ApTOLL administration at different doses, combined with EVT and, when indicated, IV fibrinolysis, was safe and well-tolerated compared with placebo. The safety of ApTOLL was determined by monitoring adverse events occurring during the study, identified during medical examinations, by clinical examination or neuroimaging. The primary endpoint was defined as the presence of any of the following events: death from any cause, intracranial hemorrhage (sICH, symptomatic intracranial hemorrhage) resulting in new symptoms or worsening of existing symptoms, herniation, cerebral edema resulting in neurological deterioration or death, and recurrent stroke.

[0276] Even though APRIL was the only study, the secondary objectives differed in the two study parts.

[0277] Phase Ib To: (1) determine the pharmacokinetic profile of ApTOLL in patients with AIS, assessed by determining ApTOLL levels in plasma and urine; (2) select the two doses to be administered in Phase IIa according to their individual safety profiles; and (3) provide an initial estimate of the therapeutic effect of ApTOLL on final infarct volume (measured by MRI-FLAIR at 72 ± 24 hours) and pro-inflammatory biomarkers (baseline [pre-dose], and at the end of infusion [up to 1 hour], 6 hours, 24 hours, 48 ​​hours, and 72 hours after dosing).

[0278] Phase IIa To evaluate the therapeutic and clinical effects of ApTOLL on: (1) final infarct volume (measured by MRI-FLAIR at 72±24 hours); (2) pro-inflammatory plasma biomarkers (before dosing, and at 6, 24, 48, and 72 hours after dosing); (3) early clinical course (NIHSS at 72 hours or discharge [whichever occurs first]); and (4) long-term functional outcome (mRS at 90 days after stroke).

[0279] In cases where one or more NIHSS measurements were missing, the last measurement after study drug administration was considered. Affected patients were included in the "assigned population" with an mRS score of 6. For patients known to be alive 3 months after randomization whose follow-up assessment was not possible, the discharge mRS was performed first. In cases where an MRI could not be obtained at 72 hours, the final infarct volume was determined on the last available CT scan after ApTOLL administration. If follow-up images were not available, the final infarct volume was considered missing.

[0280] 1.11.Statistical analysis A statistical analysis plan (SAP) including all tables, listings, and graphs was published prior to locking of the study database.

[0281] Statistical analyses were performed by an independent external statistical consulting group. All analyses were performed in the assigned population, defined as all patients randomly assigned to the trial, regardless of the treatment they received. The total sample size was 151 patients: 32 patients in Phase Ib and 119 patients in Phase IIa. Due to the exploratory nature of the study, no statistical power estimates were made. The analysis assumed a progressive relationship (i.e., any dose above the unsafe dose was considered unsafe). Patients enrolled in Phase Ib who received either placebo or one of the two selected doses of ApTOLL were combined with patients in Phase IIa. To successfully combine patients from both 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 exception of sequential blood and urine sampling for pharmacokinetic analysis, which was performed only in Phase Ib.

[0282] Categorical variables were summarized by means of counts and percentages. Continuous variables were summarized by means, standard deviations (SD), medians, and corresponding interquartile ranges. Where informative, these summaries were reported by treatment and visit. Infarct volumes showed a skewed distribution and were reported as medians (IQR).

[0283] The primary outcome of the study was compared between groups based on the absolute difference in proportions and 95% asymptotic Wald confidence intervals. When both proportions for comparison were zero, a general linear model was used. For confidence intervals of odds ratios, 95% asymptotic Wald confidence limits based on the logarithmic transformation of the odds ratio were used. Differences in continuous secondary outcomes (final infarct volume and NIHSS score at 72 hours) between groups were tested using one-way analysis of variance after adjustment by Scheffe's method, and the mean difference and 95% confidence interval between placebo and ApTOLL doses are reported. To evaluate shift analysis for mRS scores at 90 days, a proportional odds original logistic regression model was constructed using placebo as the reference category to estimate the unadjusted common odds ratio and 95% confidence interval for better outcome at 90 days.

[0284] To account for potential imbalances in baseline patient characteristics due to the lack of stratified randomization, post-hoc binomial logistic regression models were performed to estimate odds ratios and 95% confidence intervals, adjusting for predicted infarct core volume at admission, age, and baseline NIHSS score. A fully specified statistical analysis plan was reported prior to study completion. The trial is registered under EudraCT:2020-002059-38 and ClinicalTrials.gov:NCT04734548.

[0285] 1.12. Results. Study population Between November 2020 and June 2021, 32 patients were enrolled in Phase 1b and randomly assigned to receive ApTOLL or placebo (Figure 1), completing four dose-escalation arms. ApTOLL plasma concentrations were obtained from 22 patients in Phase 1b. The DSMB did not find any safety concerns and selected the 0.05 mg / kg and 0.2 mg / kg doses to be used in Phase 2a. The 0.05 mg / kg dose was selected because it should be sufficient to produce some benefit and all patients achieved sufficient concentrations of ApTOLL in pharmacokinetic analyses. The 0.2 mg / kg dose was expected to achieve the greatest therapeutic benefit, as this dose reached maximum bioavailability in the first-in-human study (Hernandez-Jimenez M et al., 2022). Additionally, no toxicity risks were observed that limited this choice.

[0286] Between July 2021 and April 2022, 119 patients were assigned to receive either ApTOLL 0.05 mg / kg (n = 36), ApTOLL 0.2 mg / kg (n = 36), or placebo (n = 47). The pooled 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 groups (Table 3). Patient adherence to eligibility criteria and treatment administration, major protocol nonadherence, patient withdrawals and reasons for withdrawal (e.g., AEs, protocol nonadherence, loss of follow-up, withdrawal of consent, and other reasons), and assignment to each analysis population were reported. Major protocol deviations occurred in 10 patients.

[0287] Of the 151 patients in the pooled Ib / IIa population, all received their assigned intervention (either the active dose or placebo) and no crossover occurred. All patients received their assigned intervention, but three received the incorrect volume or duration: two (4.8%) with ApTOLL 0.05 mg / kg and one (1.82%) with placebo. All but one patient (99.3%) in the ApTOLL 0.2 mg / kg group had EVT attempted. IV thrombolysis was administered in 29 (69.1%) patients in the ApTOLL 0.05 mg / kg group, 27 (64.3%) patients in the ApTOLL 0.2 mg / kg group, and 29 (52.7%) patients in the placebo group. The overall workflow (start to randomization, start to study drug administration, and study drug to reperfusion) and quality of reperfusion (on the expanded eTICI scale) were similar in all groups (Table 3).

[0288] 1.13. Results. Outcome At 90 days, primary safety outcome data were missing for two patients (1.3%; two patients withdrew consent) (Figure 1). Death from any cause occurred in 10 patients (18.2%) assigned to placebo, 11 patients (26.2%; absolute difference vs. placebo 8%; 95% CI: -9% to 25%) assigned to ApTOLL 0.05 mg / kg, and 2 patients (4.8%; absolute difference vs. placebo -13%; 95% CI: -25% to -1%) assigned to ApTOLL 0.2 mg / kg. Table 4 shows the distribution of primary and secondary endpoints across study groups.

[0289] Final infarct volumes were 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 vs. placebo -12%; 95% CI: -49%-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 vs. placebo -42%; 95% CI: -66%-1%). The NIHSS assessed at 72 hours was 7 (IQR 3 to 17) in patients assigned to placebo, 8 (IQR 3 to 17) in patients assigned to ApTOLL 0.05 mg / kg (mean difference in log-transformed 72-hour NIHSS vs. placebo -1%; 95% CI: -41% to 40%), and 3 (0 to 11) in patients assigned to ApTOLL 0.2 mg / kg (mean difference in log-transformed 72-hour NIHSS vs. placebo -45%; 95% CI: -67% to -10%). The proportion of patients with an mRs score of 0 to 2 at 90 days was 47.1% in patients assigned to placebo, 37.5% in patients assigned to ApTOLL 0.05 mg / kg (common odds ratio for better outcome versus placebo 0.76, 95% CI 0.37 to 1.56), and 64.3% in patients assigned to ApTOLL 0.2 mg / kg (common odds ratio for better outcome versus placebo 2.44, 95% CI 1.76 to 5.00). Analysis of multiple blood biomarkers was not significantly different between study groups. Figures 2–4 show graphical representations of secondary efficacy outcomes.

[0290] 1.14. Discussion The APRIL study demonstrated that infusion of ApTOLL 0.2 mg / kg (SEQ ID NO: 1) in combination with EVT in selected stroke patients was safe and reduced mortality at 90 days. Furthermore, the efficacy of ApTOLL as a neuroprotective agent in acute cerebral ischemia is supported by consistent positive results for most predefined secondary endpoints, including final infarct volume, early neurological dysfunction, and long-term disability.

[0291] ApTOLL demonstrated (1) effective reach to tissues at risk exhibiting a direct protective effect in the region of the ischemic penumbra, potentially extending the therapeutic window of reperfusion therapy; and (2) limiting the phenomenon of hemorrhagic transformation and reperfusion injury that can occur after recanalization.

[0292] A key strength of the clinical trial is that its design closely mirrored the preclinical ischemia / reperfusion model of transient middle cerebral artery occlusion. Patients had a median time from stroke onset to study drug administration of 210 minutes and a high reperfusion rate after EVT (final eTICI 2b-3 score of 87%). The observed pharmacokinetic curve indicates that ApTOLL can exert its neuroprotective effects for up to 12 hours after administration, including the early period of ischemia and the subsequent post-reperfusion period. The median time from drug infusion to recanalization (if achieved) was approximately 180 minutes, corresponding to the potential neuroprotective time during ischemia.

[0293] Subgroup analysis suggests similar treatment effects in patients who received the drug in an early time frame (<3 hours from symptom onset) and in patients treated in a later time frame (3–6 hours).

[0294] Although this study identified a safe dose in these patients, it was underpowered to reach conclusions regarding the effectiveness of ApTOLL in improving outcomes in acute stroke patients. Nevertheless, promising results were observed in this phase. More results are shown in Table 5 and Figure 4.

[0295] 1.15. Conclusion In AIS, 0.2 mg / kg ApTOLL administered within 6 hours of disease onset in combination with EVT was safe and associated with meaningful clinical outcomes, reducing mortality and disability at 90 days compared with placebo.

[0296] 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 time appearing healthy; ASPECTS: Alberta Stroke Program Early CT score; ICA: internal carotid artery; MCA: middle cerebral artery; EVT: endovascular therapy; eTICI: extended thrombolysis for cerebral ischemia). [Table 3] TIFF2026506313000005.tif164170

[0297] Table 4. Primary and secondary endpoints. IQR: interquartile range. Efficacy In the placebo vs. ApTOLL 0.2 mg / kg column, % positive values ​​represent a decrease, while % negative values ​​represent an increase. [Table 4] TIFF2026506313000007.tif118170

[0298] [Table 5]

[0299] Example 2: Comparison of ApTOLL (APRIL Study) vs. Nerinetide (ESCAPE-NA1 Study) Nerinetide (NA-1) is a neuroprotective drug focused on inhibiting postsynaptic density protein 95, which is involved in excitotoxic processes occurring after stroke. The overall results of the ESCAPE-NA1 clinical trial, involving 1,105 patients, were negative because nerinetide did not improve the overall proportion of patients achieving a favorable clinical outcome after EVT compared with patients receiving placebo. This negative effect could be explained by the fact that the EVT and EVT + tPA-treated populations were combined in the final analysis, but in a subsequent subanalysis, investigators observed evidence of nerinetide effect modification, resulting in an inhibition of nerinetide's effect in patients receiving alteplase (Hill M et al., 2020). Results considering only patients not treated with tPA were summarized in Figure 7, with a 19% increase in mRS of 0 to 2 and a 37% reduction in mortality.

[0300] It should be noted that ESCAPE-NA1 was a Phase III clinical trial involving 1,105 patients, with very limited results, as the effect was observed only in patients not treated with tPA. Therefore, the applicability of nerinetide to future clinical practice will be limited to patients who are not eligible for tPA treatment. In contrast, the APRIL study, conducted in only 151 patients, resulted in a 36% increase in mRS of 0 to 2 and a 72% reduction in mortality compared with patients treated with or without tPA.

[0301] Example 3: Mutation analysis of ApTOLL The percentage of sequence identity of the ApTOLL sequence (SEQ ID NO: 1) and the effect of 5' and 3' end extensions were tested experimentally.

[0302] Initially, six variants of the ApTOLL sequence presented in Table 6 were obtained and tested. The variants ranged in sequence identity to SEQ ID NO: 1 from 89.83% to 81.53% and included mutations to the aptamer sequence of SEQ ID NO: 1, such as additions at the 5' end of the sequence, additions at both the 5' and 3' ends of the sequence, internal deletions, conservative substitutions, and non-conservative substitutions.

[0303] [Table 6]

[0304] Antagonist activity against TLR4 was assessed using a secreted embryonic alkaline phosphatase (SEAP) assay protocol in HEK-Blue hTLR4 cells (InvivoGen, catalog code Hkb-htlr4). LPS-EK up (InvivoGen catalog code tlrl-peklps) was the TLR4 agonist control. HEK-Blue hTLR4 cells were obtained by cotransfection of human TLR4, MD-2, and CD14 coreceptor genes and an inducible SEAP reporter gene into HEK293 cells. The SEAP reporter gene was placed under the control of the IL-12 p40 minimal promoter fused to five NF-κB and AP-1 binding sites. Stimulation with TLR4 ligands activates NF-κB and AP-1, which induces the production of SEAP. HEK-Blue hTLR4 cells seeded in P96 plates had their TLR4 receptors activated by the addition of LPS-EK up. After 1 h, the aptamer was added at two final concentrations (20 nM and 200 nM), and expression of the reporter protein SEAP was quantified after 16–20 h.

[0305] Figure 8 shows the activity of the SEAP reporter protein produced by TLR4 receptor activation. The natural agonist ligand for TLR4 is LPS (bacterial lipopolysaccharide), and LPS-EK up was used in this assay. The agonist control bar on the left represents 100% activation of the TLR4 receptor by the listed agonist. When aptamers (control ApTOLL and ApTOLL-Mut 1-6) were added to activated cells, TLR4 activity decreased, and the aptamer bars became shorter than the agonist control bars. Therefore, the aptamers have concentration-dependent TLR4 antagonist activity, except for ApTOLL-Mut 4, which is not concentration-dependent. The tested mutants had similar behavior, and thus Figure 8 shows that all tested ApTOLL variants (ApTOLL-Mut, SEQ ID NOs: 17-22) had TLR4 antagonist activity equivalent to that of the ApTOLL aptamer (SEQ ID NO: 1).

[0306] Furthermore, a competition assay for the TLR4 receptor against these aptamers was also evaluated. An ELONA cell assay was performed using the ApTOLL aptamer (SEQ ID NO: 1) modified with a digoxigenin tag (ApTOLL-Dig) and HEK-Blue hTLR4 cells. Equimolar mixtures containing ApTOLL-Dig (100 nM) and each of ApTOLL-Mut 1-6 (100 nM) were used to determine the potential for digoxigenin-related signal reduction relative to the positive ApTOLL-Dig control (100 nM). The positive ApTOLL-Dig control and the respective ApTOLL-Dig / ApTOLL-Mut mixture were added to the wells and incubated for 15 min at 37°C in a CO2 incubator, then the wells with cells were washed with PBS, anti-dig antibody was added, and finally ApTOLL-Dig was detected using the ABTS protocol (Roche ref. 11684302001).

[0307] Figure 9 shows the results of the competition assay, showing that the binding percentage of the ApTOLL control aptamer (SEQ ID NO: 1) to the TLR4 receptor was 100% when it was added to cells. In contrast, when the ApTOLL-Mut aptamers (1-6) in an equimolar mixture were added individually to cells, the binding percentage of the ApTOLL aptamer decreased, indicating that the ApTOLL-Mut aptamers (1-6) competed for the same TLR4 receptor binding site as the ApTOLL control aptamer. ApTOLL-Mut 6 was the aptamer with the highest binding affinity (35.9%) for the TLR4 receptor, while ApTOLL-Mut 2 was the aptamer with the lowest binding affinity. ApTOLL-Mut 1 and ApTOLL-Mut 4 obtained similar binding percentages (20% and 21%, respectively). Thus, the experimental data presented in Figure 9 show that the ApTOLL-Mut aptamers (SEQ ID NOs: 17-22) compete with ApTOLL (SEQ ID NO: 1), which would indicate that they are structurally similar and therefore bind to the same binding site on TLR4 as the original ApTOLL aptamer.

[0308] Furthermore, the effect of lengthening the 5' and 3' ends of ApTOLL (SEQ ID NO: 1) and simultaneously introducing mutations was also tested. Three variants of SEQ ID NO: 1 were tested (see Table 7 below):

[0309] Aptamer 4F (SEQ ID NO: 4): This has 13 additional nucleotides at the 5' end and 4 additional nucleotides at the 3' end compared to the ApTOLL sequence (SEQ ID NO: 1). The sequence identity to SEQ ID NO: 1 would be 100% if only the central region were considered. If the entire sequence were considered, the percentage of sequence identity would be 77.6%.

[0310] Aptamers 4F-Mut2 and 4F-Mut3 (SEQ ID NO: 23 and 24): These two aptamers correspond to the most divergent sequences relative to the ApTOLL sequence (SEQ ID NO: 1).

[0311] To test whether any 5' or 3' extension works, the additional 5' and 3' regions of 4F-Mut2 and 4F-Mut3 are highly divergent from the extension in aptamer 4F. The 5' extensions in 4F-Mut2 and 4F-Mut3 have 0% sequence identity to the 5' extension of aptamer 4F. Similarly, the 3' extensions in 4F-Mut2 and 4F-Mut3 have 0% sequence identity to the 3' extension of aptamer 4F.

[0312] 5' additional sequence 3' additional sequence 4F GCGGATGAAGACT CAAC 4F-Mut2 TTTTTCTTTTTTC TTTT 4F-Mut3 ATAAGCAGGAGTC TGGT

[0313] Table 7. Regions of 4F, 4F-Mut2 and 4F-Mut3 that are homologous to ApTOLL (SEQ ID NO: 1) are shaded grey, while mutations in 4F-Mut2 and 4F-Mut3 relative to ApTOLL (SEQ ID NO: 1) are shown in bold. [Table 7]

[0314] The ability of the above aptamers to inhibit TLR4 and to bind to TLR4 (antagonist activity against TLR4) was determined by the means disclosed above, and the results are shown in Table 8.

[0315] [Table 8]

[0316] Therefore, the addition of nucleotides at the 5' and 3' ends of SEQ ID NO: 1 does not affect the formation of an effective binding structure and its activity, as experimentally confirmed in Table 8. The results from Table 8 confirm that both the 4F-Mut2 and 4F-Mut3 aptamers, which have 90% sequence identity with SEQ ID NO: 1 and also have an addition of 13 nucleotides at the 5' end and 4 nucleotides at the 3' end of the aptamer of SEQ ID NO: 1, maintain the ability to inhibit and bind to TLR4 (antagonist activity against TLR4) similar to their original aptamer of SEQ ID NO: 1.

[0317] Thus, variant aptamers having at least 90% sequence identity (or at least 69% sequence identity when considering the entire sequence) with the original nucleic acid sequence (SEQ ID NO: 1), 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).

[0318] References WO2015197706A1(AptaTargets SL) WO2020230108A1(AptaTargets SL) WO2020230109A1(AptaTargets SL) Hernandez-Jimenez M, et al. First-in-human phase I clinical trial of a TLR4-binding DNA aptamer, ApTOLL: Safety and pharmacokinetics in healthy volunteers. Mol Ther Nucleic Acids. 2022;28:124-135. doi:10.1016 / j.omtn.2022.03.005 Garcia-Culebras A, et al. Toll-Like Receptor 4 Mediates Hemorrhagic Transformation After Delayed Tissue Plasminogen Activator Administration in In Situ Thromboembolic Stroke. Stroke. 2017 Jun;48(6):1695-1699. doi: 10.1161 / STROKEAHA.116.015956 Berge E, et al. European Stroke Organisation (ESO) guidelines on intravenous thrombolysis for acute ischaemic stroke. European Stroke Journal. 2021;6(1):I-LXII. doi:10.1177 / 2396987321989865 Jovin TG, et al. Thrombectomy within 8 Hours after Symptom Onset in Ischemic Stroke. N Engl J Med 2015; 372:2296-2306. doi: 10.1056 / NEJMoa1503780 Lyden P, et al. Improved reliability of the NIH Stroke Scale using video training. NINDS TPA Stroke Study Group. Stroke. 1994;25(11):2220-2226. doi:10.1161 / 01.str.25.11.2220 Van Swieten JC, et al. Interobserver agreement for the assessment of handicap in stroke patients. Stroke. 1988;19(5):604-607. doi:10.1161 / 01.str.19.5.604 Chen S, et al. Cerebral edema formation after stroke: emphasis on blood-brain barrier and the lymphatic drainage system of the brain. Front. Cell. Neurosci. 15:716825. doi: 10.3389 / fncel.2021.716825 Pexman JH, et al. Use of the Alberta Stroke Program Early CT Score (ASPECTS) for assessing CT scans in patients with acute stroke. AJNR Am J Neuroradiol. 2001;22(8):1534-1542 Olive-Gadea M, et al. Defining a target population to effectively test a neuroprotective drug. Stroke. 2021;52(2):505-510. doi:10.1161 / STROKEAHA.120.032025 Turc G, et al. European Stroke Organisation (ESO)- European Society for Minimally Invasive Neu-rological Therapy (ESMINT) guidelines on mechanical thrombectomy in acute ischemic stroke. J Neurointerv Surg. 2019;11(6):535-538. doi:10.1136 / neurintsurg-2018-014568 Hill M et al. Efficacy and safety of nerinetide for the treatment of acute ischaemic stroke (ES-CAPE-NA1): a multicentre, 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 sequence number 1 for reducing the risk of intracranial hemorrhage after acute ischemic stroke in a subject, wherein the aptamer is administered at a dose of at least about 0.2 mg / kg after the acute ischemic stroke, and the 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 of claim 1, wherein the intracranial hemorrhage is symptomatic intracranial hemorrhage and the administration of the aptamer reduces the risk of the symptomatic intracranial hemorrhage by at least about 34% relative to a subject not treated with the aptamer.

3. 1 for improving neurological recovery after acute ischemic stroke in a subject, wherein said improved neurological recovery is mediated by a reduced risk of intracranial hemorrhage, said aptamer being administered at a dose of at least about 0.2 mg / kg after said acute ischemic stroke, and said aptamer is administered at a dose of at least about 0.2 mg / kg after said acute ischemic stroke, relative to a subject not treated with said aptamer. (i) reduce NIHSS scores, and (ii) reducing the mRS score to 0-2; Aptamers.

4. 4. The aptamer of claim 3, wherein the reduction in the NIHSS score at 72 hours is at least about a 57% reduction relative to a subject not treated with the aptamer.

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

6. The aptamer of any one of claims 1 to 5, wherein the aptamer reduces cerebral edema by at least about 67% relative to a subject not treated with the aptamer.

7. The aptamer according to any one of claims 1 to 6, wherein the aptamer is administered within about 8 hours from the onset of stroke, in particular within about 4 hours from the onset of stroke.

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

9. The aptamer of claim 8 , wherein the aptamer is administered in combination with an intravascular treatment.

10. The aptamer of claim 9 , wherein the aptamer is administered prior to or concurrently with the intravascular treatment.

11. The aptamer of claim 10 , wherein the aptamer is administered less than about 4 hours prior to intravascular treatment.

12. The aptamer according to any one of claims 8 to 11, wherein the aptamer is administered in combination with pharmacological thrombolysis, in particular with the administration of tissue plasminogen activator.

13. The aptamer of claim 12 , wherein the aptamer is administered simultaneously with or after administration of the pharmacological thrombolysis.

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

15. The subject, at the time of stroke onset, i) is between about 18 and about 90 years old; ii) have a baseline NIHSS score of about 8 points to about 25 points; iii) have a pre-stroke mRS score of 0 points to about 2 points; iv) has an infarct volume of about 5 cc to about 70 cc; v) having occlusion in the M1 or M2 segment of the terminal internal carotid artery, middle cerebral artery; vi) have an mTICI score of 0 or 1; vii) having a volume of the restricted region of the diffusion-weighted image of about 5 mL to about 70 mL; viii) having ASPECTS of about 6 to about 10; or ix) any combination thereof; An aptamer according to any one of claims 1 to 14.

Images