A polypeptide for use in the therapeutic management of acute ischaemic stroke

Abstract

The invention relates to a polypeptide of SEQ ID NO: 1 for use in the treatment of acute ischaemic stroke (AIS) or in the prevention of the complications of AIS in an adult mammalian subject. The polypeptide can be used in patients in whom AIS is followed by reperfusion of ischemic brain tissue, or in patients in whom AIS is not followed by reperfusion of ischemic brain tissue (patients with permanent cerebral ischemia).

Description

[0001] A polypeptide for use in the therapeutic management of Acute Ischaemic Stroke.

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a therapy for use in the treatment of acute ischaemic stroke, the prevention of complications associated with acute ischaemic stroke, and / or the reduction of post-stroke mortality in adult mammalian subjects, preferably human subjects.

[0004] BACKGROUND OF THE INVENTION

[0005] Cerebral ischemia is the second leading cause of death. Although mortality is decreasing in Western countries, the increasing incidence of stroke associated with population ageing remains one of the most serious health and social challenges [GBD 2021 Nervous System Disorders Collaborators. Global, regional, and national burden of disorders affecting the nervous system, 1990-2021 : a systematic analysis for the Global Burden of Disease Study 2021. Lancet Neurol 2024 Apr; 23 (4) : 344-381 ] . Ischemic stroke triggers angiogenesis, and vascular remodeling plays a key role in the complex process of brain regeneration following ischemic injury, including enhancing synaptic plasticity and restoring cerebral blood flow. Clinical data indicate that patients with cerebral infarction exhibit a significant increase in microvessel density within the penumbra, and higher microvessel density may be associated with longer post-stroke survival [Tiedt, Steffen et al. “The neurovascular unit and systemic biology in stroke - implications for translation and treatment.” Nature Reviews Neurology vol. 18,10 (2022): 597- 612],

[0006] Nerve growth factor (NGF) is a 120-amino acid protein primarily involved in the regulation of growth, maintenance, proliferation, and survival of cholinergic neurons and on homeostasis of glial cells [Lorenzini, Luca et al. “Nerve Growth Factor: The First Molecule of the Neurotrophin Family.” Advances in experimental medicine and biology vol. 1331 (2021): 3-10], Because of its high molecular weight, NGF does not readily cross the blood-brain barrier (BBB), thus limiting the use of NGF as a treatment for central nervous system diseases. A valid alternative approach, that bypasses the BBB, appears to be the intranasal administration and recent studies showed that a significant amount of NGF can reach the central nervous system through this administration route [Colitti, Nina et al. “Long-Term Intranasal Nerve Growth Factor Treatment Favors Neuron Formation in de novo Brain Tissue.” Frontiers in cellular neuroscience vol. 16 871532. 19 Jul. 2022], The olfactory route seems to be a viable option as it is relatively simple, not invasive, safe, and less likely to cause systemic side effects. To exploit the neuroprotective potential of NGF while improving its therapeutic window and reducing the known algogenic side effects, a recombinant mutated form of human NGF, also known as “painless human NGF” (hNGFp). hNGFp presents a mutation in position 100 (R100E) to reduce algogenic activity and another mutation in position 61 (P61S), to analytically distinguish it from the endogenous wild-type NGF. Compared to human NGF, hNGFp exhibits similar affinity for the “neurotrophic” TrkA receptors (1.35 vs 0.94 nM) while significantly lower affinity for the “apoptotic” p75NTR receptors (200 vs 1.53 nM) [Covaceuszach, Sonia et al. “In vitro receptor binding properties of a "painless" NGF mutein, linked to hereditary sensory autonomic neuropathy type V.” Biochemical and biophysical research communications vol. 391,1 (2010): 824-9],

[0007] The current treatment of acute ischemic stroke is based on reperfusion therapies, allowing the restoration of a blood flow in the insulted area; amongst these therapies, endovascular thrombectomy and intravenous thrombolytic therapy are found. Mechanical removal of the blood clot causing the ischemic stroke, called mechanical thrombectomy, is a potential treatment for occlusion of a large artery, such as the middle cerebral artery. However, as with thrombolytic pharmacologic therapy, many people are deemed ineligible for endovascular treatment due to the numerous contraindications and the short time window for intervention [Nogueira, Raul G, and Marc Ribo. “Endovascular Treatment of Acute Stroke.” Stroke vol. 50,9 (2019): 2612-2618. doi: 10.1161 / STROKEAHA.119.023811; Berkhemer, (Divert A et al. “A randomized trial of intraarterial treatment for acute ischemic stroke.” The New Engl and journal of medicine vol. 372,1 (2015): 11-20.; Goyal, Mayank et al. “Randomized assessment of rapid endovascular treatment of ischemic stroke.” The New England journal of medicine vol. 372,11 (2015): 1019-30], A study found that only 7 to 13% of patients with ischemic stroke were eligible candidates for EVT [Chia, Nicholas H et al. “Determining the Number of Ischemic Strokes Potentially Eligible for Endovascular Thrombectomy: A Population-Based Study.” Stroke vol. 47,5 (2016): 1377-80], In 2015, one review demonstrated the safety and efficacy of EVT, if performed within 12 hours of the onset of symptoms (Saver et al. Time to Treatment With Endovascular Thrombectomy and Outcomes From Ischemic Stroke: A Meta-analysis. JAMA 2016; 316: 1279-88). It did not change the risk of death but did reduce disability compared to the use of intravenous thrombolysis, which is generally used in people evaluated for mechanical thrombectomy. Certain cases may benefit from thrombectomy up to 24 hours after the onset of symptoms.

[0008] Thrombolysis in acute ischemic stroke, such as the thrombolytic therapy using recombinant tissue plasminogen activator (rtPA), when given within three hours of symptom onset, results in an overall benefit of 10% with respect to living without disability. It does not, however, improve chances of survival. Benefit is greater the earlier it is used. Between three and four and a half hours the effects are less clear. After four and a half hours, thrombolysis results in unfavorable risk / benefit ratio. It was found that treatment with thrombolysis results in an increase in the number of people living without disability at three to six months; however, there was an increased risk of death in the short term (deaths occurring within the first 7 to 10 days). These benefits or lack of benefits occurred regardless of the age of the person treated. [Wardlaw JM, Murray V, Berge E, del Zoppo GJ. Thrombolysis for acute ischaemic stroke. Cochrane Database Syst Rev 2014; 2014: CD000213], The use of alteplase is endorsed by the American Heart Association, the American College of Emergency Physicians and the American Academy of Neurology as the recommended treatment for acute stroke within three hours of onset of symptoms as long as there are no other contraindications (such as abnormal lab values, bleeding risk, high blood pressure, or recent surgery). Amongst patients with a proximal vessel occlusion in the anterior circulation, 60 to 80% of patients either die within 90 days after stroke onset or do not regain functional independence despite alteplase treatment [Broderick, JP, Palesch, YY, Demchuk, AM, et al. Endovascular therapy after intravenous t-PA versus t-PA alone for stroke. N Engl J Med 2013; 368: 893- 903 [Erratum, N Engl J Med 2013;368: 1265],

[0009] Effective drugs for the treatment of acute ischemic stroke remain limited. In recent decades, there has been significant progress in understanding the molecular and cellular mechanisms underlying progressive damage in infarcted and peri-lesional brain areas. Excitotoxicity, inflammation, oxidative stress, and spreading depolarizations are key pathways involved in acute ischemic stroke injury represent potential targets for promoting neuroprotection and preventing neuronal dysfunction. As a result, several promising neuroprotective targets have been identified and explored in various stroke models [Gonzalez-Nieto, D.; Fernandez- Serra, R.; Perez-Rigueiro, J.; Panetsos, F.; Martinez -Murillo, R.; Guinea, G.V. Biomaterials to Neuroprotect the Stroke Brain: A Large Opportunity for Narrow Time Windows. Cells 2020, 9, 1074],

[0010] There are in vitro studies suggesting that NGF may induce neuroprotection against oxygen and glucose deprivation (OGD) insult through upregulation of heme oxygenases- 1 [Sun, Zhitang et al. “NGF protects against oxygen and glucose deprivation-induced oxidative stress and apoptosis by up-regulation of HO-1 through MEKZERK pathway.” Neuroscience letters vol. 641 (2017): 8-14. doi: 10.1016 / j .neulet.2017.01.046]. To the best of the inventors’ knowledge, there are no reports describing the efficacy of intranasal human NGF or its muteins in the treatment of acute ischemic stroke. However, murine NGF (mNGF) has been reported to reduce brain infarct when administered 30 minutes and 24 hours after transient middle cerebral artery occlusion (tMCAO) in rats [Zhao, Hong-mei et al. “Intranasal delivery of nerve growth factor to protect the central nervous system against acute cerebral infarction.” Chinese medical sciences journal = Chung-kuo i hsueh k'o hsueh tsa chih vol. 19,4 (2004): 257-61], Conflicting results were obtained in this study, as no differences were observed among groups in the postural reflex test and adhesive tape tests, although balance function improved compared to the vehicle group at 24 and 48 hours after the onset of tMCAO. In other words, this study could not demonstrate that mNGF had a significant effect on neurological disability. On the other hand, intranasal mNGF was reported to induce neurogenesis in the ischemic core when administered 4 hours after tMCAO in rats, despite not reducing brain infarct volume [Zhu, Wusheng et al. “Intranasal nerve growth factor enhances striatal neurogenesis in adult rats with focal cerebral ischemia.” Drug delivery vol. 18,5 (2011): 338-43],

[0011] W02022200550 describes the combined use of intranasally administered hNGFp and therapeutic hypothermia for the treatment and prevention of brain injury, in particular hypoxic- ischemic encephalopathy, preferably in a neonatal subject either at term or at preterm.

[0012] WO20 18087656 discloses pharmaceutical compositions comprising NGF (extracted or recombinant mouse NGF or human recombinant NGF), or molecules with NGF -like activity for use in the treatment of traumatic brain injury in the paediatric population, by means of intranasal administration.

[0013] EP4342485 describes the use of NGF, administered intranasally, to restore the activity of motor neurons in the brain, thereby reducing spasticity and improving the patient's motor function. The document reports that rhNGF restores motor activity in an animal model of mechanically induced brain damage (TBI model) and in a rat chemogenetic model that reflects neuronal defects observed in hereditary spastic paraplegia (HSP).

[0014] WO2024 / 028487 describes the use of NGF, administered intranasally, for the prevention or treatment of sensorineural hearing loss in a subject. The document states that rhNGF, delivered via the intranasal route, has a protective effect against age-related hearing impairment in the SAMP8 mouse model of accelerated senescence, which is used to study preclinical age-related hearing loss.

[0015] In sum, AIS is a challenging disease, which needs urgent comprehensive management. Up until now there have been no alternative therapies approved for use in clinical practice. Therefore, there is pressing need to develop new neuroprotective agents for the treatment of AIS and to halt or limit irreversible damage in the ischemic penumbra which is at risk of progressing to infarction but is still salvageable [Fisher, M., Savitz, S.I. Pharmacological brain cytoprotection in acute ischaemic stroke — renewed hope in the reperfusion era. Nat Rev Neurol 18, 193-202 (2022)].

[0016] SUMMARY OF THE INVENTION

[0017] In one aspect, the present invention refers to a polypeptide of SEQ ID NO: 1 for use in the treatment of acute ischemic stroke (AIS) and / or in the prevention of the complications of AIS in an adult mammalian subject, wherein the polypeptide is preferably administered intranasally. Preferably, the present invention refers to a polypeptide of SEQ ID NO: 1 for use in the treatment of AIS in an adult mammalian subject; or for use in the treatment of AIS and in the prevention of the complications associated with AIS in an adult mammalian subject. In one embodiment, the polypeptide according to the invention is preferably used in patients in whom AIS is followed by reperfusion of ischemic brain tissue.

[0018] In another embodiment, the polypeptide according to the invention is preferably used in patients in whom AIS is not followed by reperfusion of ischemic brain tissue (also referred to herein as permanent cerebral ischemia).

[0019] DESCRIPTION OF FIGURES

[0020] Figure 1: Schematic representation of the protocol followed: the day before tMCAO rats were tested with adhesive removal test (ART) for basal values. The first administration of NGF P61SR100E or Vehicle (0,9% saline) was performed 15 min after tMCAO induction (filament insertion). After 45 min of occlusion, the filament was removed to allow reperfusion. Twenty-four hours after tMCAO, animals were tested for baseline behavioral score (BBS) and ART, and then the second intranasal administration of NGF P61SR100E or vehicle was performed. Forty-eight hours after tMCAo animals were tested with ART and then were sacrificed for sample collection.

[0021] Figure 2: Evaluation of neurological deficit with BBS 24h after tMCAO. Data are expressed as median with interquartile range. Mann-Whitney rank sum test; *p<0.05 NGF P61SR100E vs Vehicle.

[0022] Figure 3: ART in ipsilateral (right) (A) and contralateral (left) (B) forepaw. Data are expressed as mean ± SEM. Two-way analysis of variance for repeated measures with fixed factors for “time” (Baseline, 24h, 48h), “treatment” (NGF P61SR100E and Vehicle), and their interaction; p < 0.05, p < 0.05, p < 0.05 and p < 0.05 NGF P61SR100E vs. Vehicle.

[0023] Figure 4: Brain ischemic volume expressed as median with interquartile range. Mann- Whitney rank sum test; * p < 0.05 NGF P61SR100E vs. Vehicle.

[0024] Figure 5: Representative coronal brain slices stained with TTC of Vehicle and NGF P61SR100E treated animals.

[0025] Figure 6: Mean brain infarct areas (A panel) and mean infarct volume (B panel) measured 24 hours after MCAo in the vehicle and NGF P61SR100E (7, 20 pg / kg) groups.

[0026] Figure 7: Mean general neurological score measured 24 hours after MCAo in the vehicle and NGF P61SR100E (7, 20 pg / kg) groups.

[0027] DETAILED DESCRIPTION OF THE INVENTION

[0028] DEFINITIONS

[0029] The terms “NGF mutein” and “mutein of NGF”, or, with reference to NGF “mutein thereof’, are used herein interchangeably to refer to a polypeptide which is characterized by at least one mutation, compared to wild-type NGF, as further described in detail herein. Unless the context dictates otherwise, the term nerve growth factor stands for wild-type NGF (wtNGF) only, preferably human wild type NGF, and does not refer to the NGF muteins. The terms “NGF mutein” and “mutein of NGF”, or, with reference to NGF “mutein thereof’” refers to human NGF muteins. Preferably a mutein of NGF has 80 to 99.5% sequence identity with wtNGF, particularly human wtNGF, more preferably a mutein has 90 to 99% sequence identity with wtNGF, particularly human wtNGF.

[0030] The terms “subject” or “patient”, as used herein, relate to a mammal. For example, mammals in the context of the present invention are humans, non-human primates, domesticated animals including but not limited to dogs, cats, sheep, cattle, goats, pigs, horses etc., laboratory animals including but not limited to mice, rats, rabbits, etc., as well as animals in captivity such as animals of zoos. Preferably the subject is an adult mammalian individual, wherein by adult is meant a patient of at least the age past puberty, wherein the physical changes through which a body matures to reach sexual maturity has already occured (18 years old in human beings and equivalent age in other mammalian species).

[0031] Preferably, the term “subjects” or “patients” as used herein refer to humans, preferably adult mammalian subjects. More preferably in the context of the present invention, the term “patient” refers to a subject which suffers from a condition, is at risk of suffering from a condition, has suffered from a condition, or is predicted to suffer from a condition, and which may be subjected to therapy, e.g. by administration of an agent. The patient’s condition may be chronic and / or acute. Thus, a “patient” can also be described as a subject subjected to a therapy and / or or in need of a therapy.

[0032] According to the present invention, an "effective amount" or “therapeutically effective amount” is the amount or dose which achieves a desired reaction or a desired effect, either alone or together with further doses.

[0033] Stroke is defined as a medical condition in which poor blood flow to the brain causes cell death. There are two main types of stroke: ischemic, due to lack of blood flow, and hemorrhagic, due to bleeding. Ischaemic strokes are caused by acute ischemia of an area of the brain supplied by one artery (focal ischemia). Stroke onset can be accompanied by sign or symptoms often appearing soon after the stroke has occurred. The symptoms of stroke can be transient or permanent. Diagnosis is typically based on a physical exam and supported by medical imaging such as a CT scan or MRI scan. Other tests such as an electrocardiogram (ECG) and blood tests are done to determine risk factors and rule out other possible causes. Stroke is a medical emergency. Ischemic strokes, if detected within three to four-and-a-half hours, may be treatable with medication that can break down the clot (Donnan GA, Fisher M, Macleod M, Davis SM. Stroke. Lancet 2008; 371 : 1612-23).

[0034] Ischemic stroke may be followed by reperfusion or not. In the absence of reperfusion, blood flow remains interrupted for a prolonged or permanent period. The resulting damage is primarily due to sustained deprivation of oxygen and nutrients, leading to cellular necrosis and functional loss of the affected tissue. However, secondary complications typically associated with reperfusion do not occur in this context.

[0035] When the ischemia is followed by reperfusion, the temporary interruption of blood flow to a tissue or organ is subsequently restored. Although reperfusion is essential to preserve ischemic tissue, if it occurs too late, it can lead to additional cellular damage known as reperfusion injury. This injury is mediated by free radicals, inflammation, oxidative stress, and endothelial dysfunction.

[0036] When the expression “and / or” connects two or more elements / aspects / embodiments it means that each of the elements / aspects / embodiments identified thereof is disclosed in isolation and can be singled out as such; and that each element / aspect embodiment is disclosed in combination with one or more of the remaining elements / aspects / embodiments and each of those combination can be singled out.

[0037] “AIS onset” means the first appearance of the signs or symptoms of the stroke. Symptoms of AIS are preferably selected from at least one of the elements the following group consists of numbness, weakness or pain of the face, arm or leg, preferably on one side of the body; confusion; trouble speaking or understanding; trouble seeing in one or both eyes; trouble walking; dizziness, loss of balance or coordination; sudden, severe headache with no known cause; hiccups or nausea; chest pain or palpitations; shortness of breath and combinations thereof. The skilled person would be capable to identify and acknowledge other symptoms or signs of AIS onset, even though they were not listed here above.

[0038] “Last seen well”, also referred to as “last known well” (LKW), is the date and time at which the patient was last known to be without the signs and symptoms of the current stroke or at their baseline.

[0039] In the context of the present invention, “treatment of AIS” means to treat a subject with AIS, using the polypeptide according to the invention, to promote revascularization, modulate inflammation and avoid or limit the occurrence of complications. The use of the polypeptide according to the invention in the treatment of AIS provides for / equates to an acute treatment of AIS.

[0040] In the context of the present invention, “prevention of the complications of AIS” means to treat a subject with AIS, using the polypeptide according to the invention, to reduce the incidence of or to reduce the gravity of post-stroke events selected from at least one of the elements the following group consists of: medical complications, neurological complications, functional disabilities and combinations thereof. The use of the polypeptide according to the invention in the prevention of the complications of AIS provides for / equate to a chronic treatment of AIS. A non-exhaustive list of neurological complications include, for example, one or more among new infarcted areas, extension of the volume of previously infarcted areas, brain edema, intraparenchymal hemorrhage, carotid stenosis, brain herniation, diminished level of consciousness, seizure, dizziness, depression, transient ischemic attack, hydrocephalus, acute toxic encephalopathy, hallucination, intracranial hypertension. A non-exhaustive list of medical complications include, for example, one or more among nausea / vomiting, fever, constipation, hypokalemia, hyperglycemia, bradycardia, dysphagia, skin rash, urinary incontinence, diarrhea, anemia, dyspepsia. Functional disabilities generally include physical impariments (such as, for example, weakness or paralysis, sensory loss, hearing loss, balance and coordination problems, mobility issues, spsticity, continence problems), speech and language impariments (such as, for example, aphasia and dysarthria), and cognitive impairments (such as, for example, memory loss, cognitive efficiency, spatial attention).

[0041] In the context of intracranial circulation, there is a general consensus on the definitions to be given to the terms “large”, “medium” and “small” vessels based on the lumen diameter of the artery: small vessels are defined as cerebral arteries with lumen diameter < 0.75 mm (e.g., lenticulostriate artery); medium vessels are cerebral arteries with lumen diameters of 0.75-2.0 mm (e.g., M3 / 4-, A2-5, and P2-5- segments of middle, anterior and posterior cerebral arteries; posterior inferior, anterior inferior and superior cerebellar arteries; M2- and Al / Pl -segments are usually also considered medium sized); large vessels are cerebral arteries with lumen diameter > 2.0 mm (e.g., Internal carotid, vertebral and basilar artery, Ml -segment of middle cerebral artery) [Saver, Jeffrey L et al. “Thrombectomy for Distal, Medium Vessel Occlusions: A Consensus Statement on Present Knowledge and Promising Directions.” Stroke vol. 51,9 (2020): 2872-2884. doi: 10.1161 / STROKEAHA.120.028956],

[0042] The “anterior circulation” is the blood supply to the anterior portion of the brain, including most of the supratentorial structures excluding the occipital lobes. The anterior circulation is supplied by the internal carotid arteries which each divide into two large terminal branches, the anterior cerebral and middle cerebral arteries. Due to the anastomotic circle of Willis, the anterior circulation connects via the posterior communicating arteries to the posterior circulation (https: / / radiopaedia.org / articles / anterior-circulation.

[0043] The “posterior circulation” is the blood supply to the posterior portion of the brain, including the occipital lobes, cerebellum and brainstem. The posterior circulation is supplied by the vertebral arteries that combine to form the basilar artery which then divides into the posterior cerebral arteries. From these main vessels, many smaller vessels supply the posterior structures of the brain, including: posterior inferior cerebellar artery; anterior inferior cerebellar artery; pontine branches; superior cerebellar artery (https: / / radiopaedia.org / articles / posterior-cerebral-circulation). Approximately 20-25% of all acute strokes occur in the posterior circulation (Go S. Posterior Circulation Ischemic Stroke. Mo Med. 2015 May-Jun; 112(3): 192-6. PMID: 26168589; PMCID: PMC6170115).

[0044] EVT is a treatment option to facilitate the recanalization / reperfusion of occluded blood vessels. The term ‘endovascular therapy’ encompasses multiple pharmacological and / or mechanical procedures, from intra-arterial administration of thrombolytic drugs to mechanical thrombectomy that uses various devices to disrupt, catch, or aspirate a thrombus from a patient’s bloodstream. Patient eligibility criteria may include several factors, such as patient factors (age, gender, comorbidities, baseline NIHSS score, baseline ASPECTS, location of vessel occlusion, and baseline collateral status) and clinical factors (time intervals from symptom onset to randomization, groin puncture, or recanalization / reperfusion; patient transportation method (mothership versus drip-and-ship); and use of IV tPA prior to EVT). There are other general factors that could impact clinical outcomes, such as the type of anaesthesia [Bing Guo et al. SECTION THREE Technological Safety and Effectiveness, Endovascular Therapy for Acute Ischemic Stroke, Edmonton (AB): Institute of Health Economics; 2017 Aug],

[0045] In the context of the present invention, terms like “formulation”, “composition” or “medicament” refer to a combination of ingredients, including pharmaceutically active agents and pharmaceutically acceptable excipients and / or carriers, constituting a finished product, suitable for marketing and distribution, or an intermediate product that is suitable, as such, for the administration to the patient, but may require the addition of further ingredients and / or packaging to be stored long-term without loosing its properties.

[0046] The term “pharmaceutically acceptable” generally describes that a certain substance can be administered to a subject, optionally and preferably in combination with an agent, without the agent causing intolerable adverse effects, at the dosage used.

[0047] The terms “pharmaceutically acceptable carrier” and “pharmaceutically acceptable excipient” are used to refer to any one or more of solvents, buffers, dispersion media, coatings, pH adjusting agents, antimicrobial, bacterial and antifungal agents, isotonic and absorption delaying agents, rheologic agents and the like that are physiologically compatible and are suitable for administration to a subject as described herein, or do not otherwise interfere with such administration. Examples of such pharmaceutically acceptable carriers comprise without limitation one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, polysorbate 80 and the like, as well as combinations thereof. Particularly for the case of liquid pharmaceutical compositions, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the agent.

[0048] The term “pharmaceutically active agent” or simply “agent” or “active agent” refers to an agent that can be used in the administration to a subject where the agent would be of benefit, e.g., in ameliorating the symptoms of a disease or disorder. In addition, a “pharmaceutically active agent” can have a positive or advantageous effect on the condition or disease state of a subject when administered to the subject in a therapeutically effective amount. Preferably, a pharmaceutically active agent has therapeutic properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder. A pharmaceutically active agent may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease or pathological condition.

[0049] In the context of the present invention, the terms “at least one administration” or “administered at least once” shall refer to the frequency with which the active agent is administered to the patient; more precisely, the terms shall refer to both a first and only (unique) administration to the patient, within a pre-determined time unit (for example, a day); or to a first administration of a series of administrations to the patient, within that same time unit.

[0050] The polypeptide according to the present invention is also termed herein as “polypeptide of SEQ ID NO: 1”, “NGF P61SR100E”, “mutant human NGF P61 SR100E”, “hNGFp”, “painless human NGF” or similars. The polypeptide of SEQ ID NO: 1 is a mutein of NGF as previously defined. The term “polypeptide of SEQ ID NO: 1” and similar terms denote herein a polypeptide comprising the amino acid sequence defined by SEQ ID NO: 1 and / or an agent with equivalent biological activity.

[0051] Thus, within these terms are also included functionally equivalent parts or analogues of such polypeptides. One example of a biologically equivalent part of the polypeptide could be a domain or subsequence of the polypeptide of SEQ ID NO: 1, which includes the binding site to enable the domain or subsequence to exert substantially the same biological activity as the full-length polypeptide of SEQ ID NO: 1 or alternatively a gene coding for such a polypeptide. The term "substantially the same biological activity" refers to an equivalent part or analogues polypeptide having at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95% and more preferably at least 97%, at least 98% or at least 99% of the activity of the polypeptide of SEQ ID NO: 1 in the assays described in the Examples herewith appended. An example of a biologically equivalent analogue of the polypeptide could be a fusion protein which includes at least a part of the amino acid sequence of the polypeptide of SEQ ID NO: 1, but it can also be a homologous analogue of the polypeptide. Also, completely synthetic molecules that mimic the specific biological activity of the polypeptide of SEQ ID NO: 1 would constitute "biologically equivalent analogues".

[0052] More preferably, the term “polypeptide of SEQ ID NO: 1” and similar terms denote herein a polypeptide comprising the amino acid sequence defined by SEQ ID NO: 1; such agents are optionally fusion proteins which comprise inter alia the amino acid sequence defined by SEQ ID NO: 1. Most preferably, the term “polypeptide of SEQ ID NO: 1” and similar terms denote herein a polypeptide consisting of the amino acid sequence defined by SEQ ID NO: 1; in this case, the agent consists of a polypeptide consisting of the 118 amino acid residues in sequential order as defined by SEQ ID NO: 1.

[0053] As previously stated, the present invention is directed to the therapeutic use of a mutein of NGF, i.e. a polypeptide of SEQ ID NO: 1, for the treatment of AIS and / or for the prevention of the complications of AIS in an adult mammalian subject. Preferably, the polypeptide of SEQ ID NO: 1 is for use in the treatment of AIS in an adult mammalian subject; or in the treatment of AIS and in the prevention of the complications of AIS in an adult mammalian subject. Even more preferably, the polypeptide of SEQ ID NO: 1 is for use in the treatment of AIS in an adult mammalian subject.

[0054] Another aspect of the invention is a therapy using the polypeptide of SEQ ID NO: 1, such therapy including either (i) a method of treating an adult mammalian subject with AIS (having AIS); (ii) a method of preventing complications of AIS in an adult mammalian subject with AIS; or (iii) both, the therapy in any case comprising the administration of a polypeptide of SEQ ID NO: 1, preferably in a therapeutically effective amout. Preferably, the therapy includes either (i) a method of treating an adult mammalian subject with AIS; or (iii) a method of treating an adult mammalian subject with AIS and a method of preventing complications of AIS in an adult mammalian subject with AIS.

[0055] An additional aspect of the invention relates to the use of the polypeptide of SEQ ID NO: 1 in (a) the preparation of a medicament for the treatment of AIS or the preparation of a medicament for the prevention of the complications of AIS, or (b) in the preparation of a medicament for the treatment of AIS and for the prevention of complications of AIS, wherein said treatment / prevention of complications comprises administering to an adult mammalian subject the aforementioned polypeptide, preferably in a therapeutically effective amount. Preferably, the use of the polypeptide is either in (a) the preparation of a medicament for the treatment of AIS; or (b) in the preparation of a medicament for the treatment of AIS and for the prevention of complications of AIS, wherein said treatment / prevention of complications comprises administering to an adult mammalian subject the aforementioned polypeptide.

[0056] According to one embodiment, the use of the polypeptide of SEQ ID NO: 1 is preferably designed to be used in subjects in which the AIS epidode / s is / are due to an occlusion of a cerebral artery followed by reperfusion.

[0057] The polypeptide of SEQ ID NO: 1 is intended to “freeze” the penumbra area and reduce the final infarct size, thereby limiting the further expansion of the infarct core before or during the achievement of definitive or successful reperfusion. This action on the penumbra area is believed to mediate the neuroprotective effect of the polypeptide, minimizing the final infarct size and thereby reducing the medical, neurological and / or functional complications associated with infarct expansion and the resulting damage. In the absence of neuroprotective therapy, the final infarct size may be larger, even in the case of successful reperfusion.

[0058] In this context, when the polypeptide of SEQ ID NO: 1 is administered in combination with EVT, the neuroprotective therapy is effective if reperfusion occurs in close temporal proximity to the administration of hNGFp, while its protective effect is still active. If neuroprotective therapy is not administered within a certain time window following the ischemic event and / or from EVT, the final infarct size may be as large as it would have been without neuroprotection [Fisher M, Savitz SI. Pharmacological brain cytoprotection in acute ischemic stroke - renewed hope in the reperfusion area. Nat Rev Neurol. 2022; 18: 193-202],

[0059] The inventors have now found that, compared to the therapies or the intervention strategies already available at the state of the art, the use of the polypeptide of SEQ ID NO: 1 provide for a AIS therapy that, other than being effective, does not have the contraindications that thrombolysis and endovascular thrombectomy present, especially in the determination of the eligible population. In fact, when investigating the neuroprotective effects of intranasal administration of the polypeptide of SEQ ID NO: 1 in transient (45 min) middle cerebral artery occlusion (tMCAO) - a well established animal model of acute ischaemic stroke - the agent not only resulted in a significant reduction of the infarct volume and of the neurological deficit in rats, but it also unexpectedly reduced mortality compared to vehicle-treated animals (7.7% vs 27.3%), according to the present experimental part. As previously commented, based on the available bibliography, endovascular thrombectomy and thrombolysis, despite being currently used as intervention strategies for the management of AIS, did not managed to impact on the mortality of AIS patients, thus distinguishing from the invention herewith disclosed.

[0060] According to another embodiment, the use of the polypeptide of SEQ ID NO: 1 is preferably designed to be used in subjects in which the AIS episode / s is / are due to an occlusion of a cerebral artery which is not followed by reperfusion.

[0061] The inventors have found that the polypeptide of SEQ ID NO: 1 effectively reduces cerebral ischemic volume and improves the neurological status in an animal model of permanent cerebral ischemia (Example 3; Figures 6-7).

[0062] This is a remarkable outcome, as most preclinical studies currently rely on cerebral ischemia-reperfusion model. However, the majority of patients with ischemic stroke either do not achieve successful recanalization or are not eligible for it. rtPA is the only thrombolytic agent currently approved for the treatment of stroke, but its narrow therapeutic window and associated risk of hemorrhage significantly limit its clinical use.

[0063] Although the use of rtPA has steadily increased in recent years, recanalization rates remain suboptimal, and only a fraction of patients with failed rtPA thrombolysis go on to receive successful mechanical thrombectomy. Overall, patients with permanent cerebral ischemia who do not undergo recanalization still represent the majority of stroke cases. Therefore, positive outcomes obtained in an animal model of permanent cerebral ischemia are highly relevant for evaluating the primary efficacy of therapeutic agents and supporting their clinical translation to the majority of real -world stroke cases.

[0064] Preferably, the polypeptide of SEQ ID NO: 1 can be used in subjects in whom the AIS episode(s) result from occlusion of a cerebral artery, whether or not followed by reperfusion.

[0065] Without wishing to be bound by any theory, the inventors believe that the polypeptide according to the invention will be safe to be administered to all patients with AIS, with minor or no restriction in terms of subject eligibility.

[0066] Thus, the therapy exploitable by the use of the polypeptide of the present invention provides for an efficacious and easy to use method of treatment of AIS and / or prevention of the complications thereof, particularly in adult human patients, said method comprising the administration of the polypeptide of SEQ ID NO: 1, preferably shortly after AIS onset or from when the patient was last seen well. Preferably, the preferred way of administration of the polypeptide is through the nasal route.

[0067] Preferably, the polypeptide of SEQ ID NO: 1 optionally carries one or two or three internal cysteine bonds, so that cysteine (Cys, C) residues are covalently linked to each other to form intramolecular disulfide bridges. The cysteine bonds are preferably equivalent to those in wild-type human NGF.

[0068] The polypeptide of the present invention may optionally be characterized by further post-translational modifications. Such post-translational modifications optionally include glycosylation and / or phosphorylation. Preferably, however, the polypeptide according to the present invention is free of glycosylation and / or phosphorylation. Indeed, considering that the experimental examples attached herein demonstrate a beneficial effect on animal models of acute ischeamic stroke (including both an animal model of acute ischaemic stroke due to a transient occlusion of a cerebral artery followed by reperfusion, and an animal model of permanent ischemic stroke not followed by reperfusion) caused by a large vessel occlusion in the anterior circulation, whereby the polypeptide used was obtained by cytosolic recombinant expression in bacteria, which typically does not result in glycosylation and / or phosphorylation, it is plausible that the beneficial effect of the present invention is not contingent on such type of post-translational modification.

[0069] Typically, the polypeptide according to the present invention is a non-natural polypeptide which is not naturally produced by the subject to which the polypeptide is administered.

[0070] Preferably the polypeptide according to the present invention is an isolated polypeptide. More preferably, the polypeptide according to the present invention is essentially free of host cell proteins, degradation products (such as des-nona variant, for example), and protease (such as trypsin, for example). When the polypeptide according to the present invention is essentially free of host cell proteins, degradation products (such as des-nona variant, for example), and protease (such as trypsin, for example) it may also be referred to as “pure polypeptide”. Preferably, the polypeptide according to the present invention is administered as pure polypeptide. More preferably, the pure polypeptide consisting of SEQ ID NO: 1 has a weight percentage of 90% or more, preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, more preferably 99.2% or more, more preferably 99.4% or more, more preferably 99.6% or more, more preferably 99.8% or more, more preferably 99.9% or more, with respect to the total protein in the composition. Most preferably, the pure polypeptide according to the present invention has a purity grade compatible with Good Manufacturing Practices (GMP).

[0071] Methods for obtaining the polypeptide of SEQ ID NO: 1 are known to the state of the art and are described in the international application No. WO2019207106.

[0072] The polypeptide consisting of SEQ ID NO: 1, differs in two positions from the amino acid sequence of human nerve growth factor (NGF, also referred to as wild-type human NGF or wild-type NGF).

[0073] The polypeptide of SEQ ID NO: 1 is not found in nature and can also be referred to as a non-natural polypeptide. Thus, the agent according to the present invention is not wildtype NGF, and in particular not wild-type human NGF.

[0074] Preferably, the polypeptide can be solubilised in an aqueous medium.

[0075] Preferably, the complications of AIS are selected from at least one in the group consisting of: medical complications, neurological complications, functional disabilities and combinations thereof. Preferably, the complications of AIS are selected in the group consisting of: neurological complications and functional disabilities. Preferably, the complications of AIS consist of functional disabilities. More preferably, the functional disabilities comprise or consist of, in the context of the invention, physical impariments, preferably balance and coordination problems; and cognitive impairments, preferably cognitive efficiency. Even more preferably, the functional disabilities comprise or consist of, in the context of the invention, cognitive impairments, preferably cognitive efficiency.

[0076] According to one embodiment, the polypeptide of SEQ ID NO: 1 is preferably administered in cases of AIS followed by reperfusion of the ischemic brain tissue.

[0077] According to another embodiment, the polypeptide of SEQ ID NO: 1 is preferably administered in cases of AIS not followed by reperfusion of the ischemic brain tissue (alternatively referred to herein as permanent cerebral ischemia).

[0078] Preferably, the polypeptide according to the invention can be used in both patient populations: those experiencing AIS followed by reperfusion and those with AIS without reperfusion (permanent cerebral ischemia)

[0079] Preferably, the polypeptide is administered as soon as possible after the onset of AIS or from the time the subject was last seen well.

[0080] The polypeptide can be administered only once or multiple times to the subject, with at least a first or the first administration occurring as soon as possible after AIS or from the time the subject was last seen well.

[0081] Preferably, the polypeptide is administered at least once within about 24 hours after AIS onset or from the time the subject was last seen well; preferably at least once within about 12 hours from AIS onset or from the time the subject was last seen well; preferably at least once within about 8 hours from AIS onset or from the time the subject was last seen well; preferably at least once between about 6 hours and about 3 hours from AIS onset or from the time the subject was last seen well; most preferably at least once between about 4,5 hours and about 3 hours from AIS onset or from the time the subject was last seen well. Preferably the at least one administration occurring within about 24h, or 12h, or 8h, or 6 to 3 h, or 4,5 to 3 hours from AIS onset or from the time the subject was last seen well is the first administration, and in certain cases the first and only administration.

[0082] As it can be seen from the appended Example section, the use of the polypeptide of SEQ ID NO: 1 can prevent death in AIS subjects or reduce (post-stroke) mortality in AIS patients. Thus, preferably, the administration of the polypeptide of the invention reduces the mortality of about 20% in AIS subjects.

[0083] Based on the animal data described in the Example section, we believe that the administration of the polypeptide of the invention may reduce the mortality of about 10% in AIS patients undergoing endovascular thrombectomy but not receiving thrombolysis, preferably alteplase, and by 5% in AIS patients undergoing endovascular thrombectomy and receiving thrombolysis, preferably alteplase.

[0084] Preferably, the therapy using the polypeptide of SEQ ID NO: 1 is applied in cases of a vessel occlusion either in the anterior or in the posterior circulation. Preferably, the vessel occlusion interests small, medium or large vessels in the anterior or posterior circulation. The vessels that could be occluded in the anterior circulation in AIS are preferably selected from at least one of the arteries in the group consisting of internal carotid artery (ICA); first segment of the middle cerebral artery (MCA Ml), second segment of the middle cerebral artery (MCA M2) and branches thereof. The vessels that could be occluded in the posterior circulation in AIS are preferably selected from at least one of the arteries the following group consists of basilar artery, intracranial vertebral artery, posterior cerebral arteries and branches thereof.

[0085] Even more preferably, the therapy using the polypeptide of SEQ ID NO: 1 is applied in cases of AIS due to large or medium vessel occlusion in the anterior circulation, wherein the large or medium vessels of the anterior circulation are preferably selected from at least one of the arteries in the group consisting of: internal carotid artery (ICA); first segment of the middle cerebral artery (MCA Ml), and second segment of the middle cerebral artery (MCA M2).

[0086] According to one embodiment, the subjects with AIS that are administered with the polypeptide of SEQ ID NO: 1 preferably are eligible to undergo or has undergone endovascular thrombectomy therapy (EVT). In other words, these subjects are patients in whom AIS is followed by reperfusion, the reperfusion occuring thanks to EVT. Within the scope of the present invention, patients eligible for endovascular thrombectomy are preferably patients wherein the vessel occlusion interests medium or large cerebral vessels, located either in the anterior or in the posterior circulation, and more preferably located in the anterior circulation.

[0087] In this embodiment, the polypeptide of SEQ ID NO: 1 can be administered before and / or after EVT.

[0088] EVT can be performed using a variety of techniques, such as mechanical thrombectomy, and stent-retriever technology; in the context of the present invention, endovascular therapy is preferably mechanical thrombectomy. Mechanical thrombectomy is generally preferred in cases of patients with AIS due to large or medium vessels occlusion in the anterior circulation.

[0089] Preferably, when the polypeptide of SEQ ID NO: 1 is used in combination with EVT, i.e. in patients receiving EVT and preferably receiving mechanical thrombectomy, the patients could also be receiving a thrombolytic / fibrinolytic agent or not (with or without thrombolysis). Thrombolytics / fibrinolytics can be divided into two different categories: 1) fibrin-specific thrombolytics and 2) nonfibrin-specific thrombolytics. Some examples of fibrin-specific drugs are: alteplase, reteplase, and tenecteplase. Nonfibrin-specific drugs include, for example, streptokinase or staphylokinase. Preferably, thrombolysis is intravenous and it is preferably performed prior to EVT (commonly referred to as “bringing thrombolysis”). Bridging thrombolysis is meant to permit a more successful removal / dissolution of the clot during EVT.

[0090] Preferably, when the polypeptide of SEQ ID NO: 1 is used in combination with EVT, i.e. in patients receiving EVT and preferably receiving mechanical thrombectomy, the patients are not receiving any thrombolytic / fibrinolytic agent (without thrombolysis). More generally, independently from the fact the patients under the therapy using the polypeptide according to the present invention receive or not EVT, it is preferred that the subjects are not receiving any thrombolytic / fibrinolytic agent.

[0091] When the polypeptide of SEQ ID NO: 1 is used in combination with EVT, i.e. in patients receiving EVT and preferably mechanical thrombectomy, such polypeptide is preferably administered before EVT is performed. Preferably, the polypeptide of SEQ ID NO. 1 is administered within 3 hours from EVT performance; preferably within 2 hours from EVT performance; preferably within 1 hour from EVT performance. Most preferably, the polypeptide of SEQ ID NO. 1 is administered within 1 hour from EVT performance.

[0092] Alternatively, when the polypeptide of SEQ ID NO: 1 is used in combination with EVT, i.e. in patients receiving EVT and preferably mechanical thrombectomy, such polypeptide is preferably administered after EVT is completed. Preferably, the polypeptide of SEQ ID NO. 1 is administered within 6 hours after EVT is completed; preferably within 4 hours after EVT is completed; preferably within 2 hours after EVT is completed; preferably within 1 hour after EVT is completed. Most preferably, the polypeptide of SEQ ID NO. 1 is administered within 6 hours after EVT is completed.

[0093] Optionally, when the polypeptide of SEQ ID NO: 1 is used in combination with EVT, i.e. in patients receiving EVT and preferably mechanical thrombectomy, such polypeptide can be administered before and after EVT, preferably according to the timings previously indicated for the administration before or after EVT and in any combination deriving therefrom.

[0094] According to another embodiment, the subjects with AIS that are administered with the polypeptide of SEQ ID NO. 1 preferably are not eligible to undergo EVT or has undergone EVT but it was not successful in recanalization of the occluded vessel(s). In other words, these patients are patients in which no reperfusion / recanalization is possible after AIS.

[0095] Preferably, the polypeptide of SEQ ID NO: 1 is for use in therapy of adult human subjects. Preferably, the adult human subjects that are treated with the polypeptide of SEQ ID NO: 1 are at least 18 years old; preferably the adult human subjects have an age comprised between 18 and 85 years; preferably the adult human subject have an age comprised between 50 and 85 years; most preferably the adult human subjects have an age comprised between 60 and 80 years.

[0096] Preferably, the treatment of AIS and / or prevention of the complications related to AIS does not cause side effects or adverse effects in the subject to which the polypeptide is administered or has been administered. Thus, preferably, the administration of the polypeptide of the invention is not causative for any undesired effects in the mammalian subject. One side effect or adverse effect that is preferably absent in this context is hyperalgesia or pain. It is specifically preferred that the administration of the polypeptide according to the present invention does not cause hyperalgesia in the mammalian subject. Thus, preferably, administration of the agent according to the present invention does not induce any hyperalgesic syndrome (pain).

[0097] It is important to point out that the absence of pain does not merely cause a more pleasant (or less unpleasant) treatment than the administration of a reference compound associated with pain (such as wild-type NGF), but is at least in part causative for the success of the treatment or prevention of complications of AIS: considering that the polypeptide according to the present invention is preferably nasally administered, the absence of pain will enable the treated subj ect to accept the administration of the polypeptide without adverse reactions such as scraping it off or washing it off or otherwise removing it in order to avoid to pain, and as a result of that, the polypeptide will exert is therapeutically beneficial effect. Thus, the absence of pain associated with the polypeptide of the present invention will be suitable to overcome consumer carer’s reluctance and concerns of the regulatory authorities. In other words, the absence of pain is associated with a significant increase in the benefit- to-risk ratio compared to agents that are associated with pain.

[0098] In particular, preferably, the treatment and / or prevention does not cause hyperalgesia in the mammalian subject. Preferably, the subject to which the polypeptide of the invention is administered does not suffer from mechanic allodynia. More precisely, mechanic allodynia is not induced in the subject to which the polypeptide of the invention is administered, so that the subject to which the polypeptide is administered does not suffer from mechanic allodynia.

[0099] Typically, administration of the agent according to the present invention is well tolerated by the subject. In particular, preferably, administration of the polypeptide according to the present invention is not associated with the formation of drug-neutralising, anti-drug antibodies in the subject. Indeed, as the amino acid sequence of the polypeptide according to the present invention differs in only two amino acid positions from wild type human NGF, it is plausible that the immunological tolerability in humans is particularly advantageous, and it is plausible that administration of the polypeptide of the present invention is not associated with the formation of anti-drug antibodies in humans.

[0100] The polypeptide of SEQ ID NO: 1 is preferably administered intranasally. Therefore, the polypeptide of SEQ ID NO: 1 is preferably in the form of a formulation suitable for intranasal administration. Another object of the present invention preferably concerns formulations suitable for the intranasal administration and including at least the polypeptide of SEQ ID NO: 1. The present invention is also preferably directed to the medical use of such formulations.

[0101] Preferably, the polypeptide of SEQ ID NO: 1 is comprised in the formulation as an active ingredient, preferably in combination with pharmaceutically acceptable excipients and / or carriers.

[0102] The formulations described herein are preferably sterile and preferably contain the polypeptide of SEQ ID NO: 1 as a pharmaceutically active agent. The compositions may be in any state, e.g. liquid, frozen, lyophilized, etc.

[0103] Preferably, the polypeptide of SEQ ID NO: 1 is comprised in an aqueous formulation; in other words, the polypeptide is dispersed or solubilized in an aqueous medium comprised in the formulation, the aqueous medium serving the purpose of carrier within the formulation.

[0104] The aqueous medium can be an isotonic solution, optionally buffered, such as saline solution or phosphate buffered saline solution, and can further comprise, if necessary, appropriate preservatives or other excipients suitable for the formulation of compositions for intranasal administration.

[0105] Suitable preservatives for use in the formulation according to the present invention include those known in the art; examples of preservatives suitable within the aim of the invention are benzyl alcohol, benzalkonium and its salts, M-cresol, phenol, chlorobutanol, paraben and thimerosal. These and other preservatives are optionally included in the composition according to the present invention.

[0106] Preferably, the carrier is an aqueous medium suitable for administration to the mammalian subject and it is preferably an isotonic solution, preferably a saline solution. Preferably, the formulation including the polypeptide of SEQ ID NO: 1 is in the form of a liquid or a viscous formulation suitable for intranasal use in a mammalian subject.

[0107] Preferably, the polypeptide of SEQ ID NO: 1 is contained in the formulation at a concentration comprised between about 0,5 and about 10.0 mg / ml, preferably comprised between about 0,5 and about 8 mg / ml, more preferably comprised between about 0,5 mg and about 6 mg / ml. The concentration of the polypeptide can be ranging between about 0.5 and about 10 mg / ml both when only one or multiple admini strati on(s) per day is / are contemplated, more preferably when only one administration per day is contemplated. Optionally, the polypeptide of SEQ ID NO: 1 is contained in the formulation at a concentration comprised between about 0,5 and about 3.0 mg / ml, preferably between about 0,5 and about 2.5 mg / mL. Preferably, the concentration of the polypeptide is ranging between 0.5 and 3.0 mg / ml both when only one or multiple administration(s) per day is / are contemplated, more preferably when multiple administrations eer day are contemplated.

[0108] Amongst the suitable excipients for the preparation of the formulation containing the polypeptide of SEQ ID NO: 1, stabilizers can be found. Examples of suitable stabilizers within the aim of the invention are sucrose, phosphate, sodium chloride, methionine and polysorbate-80 or combinations thereof. More preferably, the stabilizers are selected between methionine, polysorbate 80 or combinations thereof.

[0109] Amongst the suitable excipients for the preparation of the formulation containing the polypeptide of SEQ ID NO: 1, buffers can be found. Examples of suitable buffers within the aim of the invention are acetate or phosphate buffers.

[0110] The formulation is preferably characterized by a pH ranging from about 5.0 and about 7.0, preferably ranging from about 5.0 and about 6.5, preferably equal to about 5.5. Optionally, the administration of the polypeptide of SEQ ID NO: 1 or formulations thereof according to the present invention is accompanied by administration of at least another therapeutic agent, such as agents intended to counter inflammation, oedema / swelling, raised intracranial pressure, infection, seizure, pain, psychological sequelae, etc. The other therapeutic agent may be part of the composition comprising the polypeptide according to the present invention, or alternatively may be administered to the subject separately, to the same or a different site, by the same or a different route of administration.

[0111] Preferably, the polypeptide of SEQ ID NO: 1 is administered at doses ranging from 2 to 250 pg / kg / day, preferably from 7 to 250 pg / kg / day, preferably ranging from 7 to 200 pg / kg / day, preferably ranging from 7 to 180 pg / kg / day, preferably ranging from 7 to 150 pg / kg / day, preferably ranging from 7 to 100 pg / kg / day, preferably at doses of 7, 10, 20, 30, 40, 50, 60 pg / kg / day. Preferably the polypeptide of SEQ ID NO: 1 is administered at doses ranging from 10 to 250 pg / kg / day, preferably ranging from 20 to 250 pg / kg / day, preferably ranging from 40 to 250 pg / kg / day, preferably ranging from 60 to 250 pg / kg / day, preferably ranging from 80 to 250 pg / kg / day, preferably ranging from 100 to 250 pg / kg / day, more preferably ranging from 120 to 250 pg / kg / day.

[0112] In the context of the present invention, the dose ranging from 7 to 250 pg / kg / day and any range or nominal value included therein represents the therapeutically effective amount.

[0113] According to the invention, the polypeptide of SEQ ID NO: 1 can be administered multiple times (i.e., repeatedly) over the day or over a period of time lasting days (several days). Preferably, the polypeptide of SEQ ID NO: 1 can be administered for a number of times ranging from 1 to 7 times, preferably from 2 to 7 times, preferably 2, 3, 4, 5, 6 or 7 times after AIS onset or last seen well, wherein the first administration occurs preferably within at least 24 h, or 12 h, or 8h, or 6 to 3 h, or 4,5 to 3 h from AIS onset or from the time the subject was last seen well.

[0114] Preferably, the polypeptide of SEQ ID NO: 1 is administered between 1 and 7 times per day, preferably from 1 to 5 times per day, preferably from 2 to 5 times per day, preferably from 2 to 4 times per day, preferably from 2 to 3 times per day. Most preferably, the polypeptide of SEQ ID NO: 1 is administered only once a day, in a single administration. The administrations can be performed either in the same nostril at all times or divided across both nostrils. In case the administration is performed in both nostrils, the administration in one or the other nostril is established according to prevailing clinical circumstances such as recipient preference, tolerable volume load and nostril access.

[0115] Preferably, the polypeptide of SEQ ID NO: 1 is administered between 1 and 30 days in patients with AIS (after AIS or last seen well), more preferably for a period comprised between 1 and 14 days after AIS or last seen well, and equally more preferably for a period equal to any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 days after AIS onset or last seen well.

[0116] Preferably, the dose of the polypeptide is quantitatively determined at or before the start of the therapy. Preferably, the dose is adjusted over time, preferably over the days of treatment, depending on the progression of the subject under therapy. In this case, therefore, the dose administered on the first day could be at the highest value of the dose range (> 100 mcg / kg / day) and be lowered by the physician over the 1-30 days, preferably 1-14 days. Alternatively, the dosing is not adjusted over the days of therapy, so that subsequent dosages correspond to the first dose.

[0117] The possibility of administering drugs intranasally, with the objective of carrying them towards the brain, bypassing the blood-brain barrier, was already suggested more than a decade ago and is currently used for the administration of some types of sedative or anesthetic drugs, for which, by means of intranasal administration, it is possible to reduce the dose and limit the possible side effects.

[0118] Preferably, the formulation including the polypeptide of SEQ ID NO: 1 can be provided in the form of a kit including a pre-determined number (for example, any number from 10 to 20) of pre-prepared aliquots of formulations, wherein each aliquot contains, by way of nonlimiting example, 1 ml of physiological solution or any other suitable carrier, in which 0.5- 3.0 mg, preferably 0.5-2.0 mg of the polypeptide of SEQ ID NO: 1 are diluted. Such aliquots can be contained in phials, pre-dosed syringes or also, in an embodiment offering particular ease of use, in pre-dosed syringes designed to be equipped or equipped with an atomizer for intranasal delivery, such as a MAD device for intranasal delivery.

[0119] In accordance with a further embodiment, said one or more aliquots can be precalibrated for administration in adults and can be subdivided into single doses per body weight range comprising a suitable amount of the polypeptide of SEQ ID NO: 1.

[0120] In an alternative embodiment, said one or more aliquots can be multi-dose aliquots, for example “daily” or “weekly” aliquots which can be subdivided into a suitable dose based on patient weight.

[0121] As already mentioned above, in accordance with one embodiment said one or more devices for administration of the composition can be devices of the MAD (mucosal atomizer device) type.

[0122] A kit of the kind described herein would advantageously allow the intranasal administration of the polypeptide of SEQ ID NO: 1 at home and by the patients, significantly improving the quality of life of these subjects.

[0123] Any embodiment or aspect specifically and explicitly recited herein may be combined with one or more further embodiments or aspect disclosed separately in this application; even the combination between aspects and embodiment is encompassed.

[0124] When the description does not specify to which embodiment or aspect the feature(s) disclosed thereto pertain, the feature(s) at hand is / are understood to be applicable to any of the disclosed embodiment / aspect.

[0125] Subject headings herein are employed to divide the document into sections and are not intended to be used to construe the meaning of the disclosure provided herein, nor to isolate information from one another rendering them non-combinable.

[0126] The present invention is further described by way of illustration, without limiting its scope, by the following examples.

[0127] EXAMPLES

[0128] 1. IN VIVO STUDY: NGF P61SR100E EFFECTS IN AN ANIMAL MODEL OF CEREBRAL ISCHEMIA (FOLLOWED BY REPERFUSION)

[0129] In this study, the effects of intranasal administration in rats of the polypeptide of SEQ ID NO. 1 subjected to transient (45 min) middle cerebral artery occlusion (tMCAO) were investigated.

[0130] Male Wistar rats were treated with intranasal NGF P61SR100E or vehicle (0.9% saline). Each rat underwent two intranasal administrations: the first administration was given 15 minutes after transient Middle Cerebral Artery occlusion (tMCAO) onset and the second 24h later. The neurologic behavioral tests were performed 24 h prior to tMCAO and then 24 and 48 h after tMCAO. The infarct volume was determined 48 h after tMCAO (Figure 1).

[0131] The NGF P61SR100E used was obtained using the methodology described in the international application No. W02019207106.

[0132] The study was performed in five different experimental sessions (Table 1). A total of 59 animals (33 in the Vehicle-treated group and 26 in the NGF P61SR100E group) underwent surgery, but 8 animals administered with Vehicle died 48h after tMCAO and one rat was euthanized at 24h, because of profound suffering. Two rats administered with NGF P61SR100E died 48h after tMCAO. Table 1

[0133] / . / MA TERIALS AND METHODS

[0134] The investigators responsible for infarct volume measurement and behavioral assessment were unaware of the treatment assignment.

[0135] Exclusion criteria: (1) development of subarachnoid hemorrhage or hemorrhagic transformation after treatment determined at the moment of sacrifice, and (2) major violations of the experimental protocol: surgical errors or complications (e.g., major arterial or venous hemorrhage, vagus nerve section, carotid artery dissection, entrapment or filament displacement) during the MCA occlusion procedure; errors in the timing of ischemia.

[0136] Animals: All animal experiments were performed and reported following the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines. Animal care and experimental procedures were carried out following the guidelines of the Italian Ministry of Health (DL 26 / 2014), following the European Directive 2010 / 63 / UE. The protocol was approved by the Committee set by the Ministry of Health at the National Institute of Health (N°98 / 2018-PR). All the experiments were performed on male Wistar rats (N = 59; 9-10 weeks of age), weighing 280-320 g (Charles River, Calco, Como, Italy). All other reagents were purchased from Sigma-Aldrich, unless specified otherwise.

[0137] Focal cerebral ischemia: The experimental procedure followed in the transient middle cerebral artery occlusion (tMCAO) model is shown in Figure 1. tMCAO was achieved using the intraluminal filament technique (Greco et al., 2014). The right MCA was occluded for 45 minutes before withdrawal of the thread to allow reperfusion for the remainder of the experiment (48 hours). Briefly, rats were anesthetized (tiletamine, 100 mg / kg, i.m., xylazine 4mg / kg i.p.) and their body temperature monitored with a rectal probe - maintained at 37 °C throughout the surgical operation with a heating pad. A neck incision was done, and under an operating microscope, the external and internal right carotid arteries were isolated. A silicone-coated nylon filament (diameter: 0.37 mm, Doccol Corporation, Redlands, CA, USA) was introduced through the external carotid artery and advanced approximately 19 mm from the carotid bifurcation in order to occlude the origin of the right MCA. To allow reperfusion, rats were re-anaesthetized and then were kept in their cages with free access to food and water.

[0138] Intranasal administration: After anesthesia, rats were placed in a supine position. NGF P61SR100E was dissolved in 0.9% saline and intranasally delivered at doses of 20 pg / kg / 40 pl, alternating nostrils with an interval of 2 min between doses. Specifically, rats were treated with intranasal NGF P61 SR100E (20 pg / kg / 40pL) or Vehicle (0.9% saline, 40pL / kg) 15 minutes after tMCAO onset and then 24h later. The second administration (at 24h) was performed after behavioral testing. Rats of the Vehicle group received 0.9% saline in the same manner. Animals were assigned to the following experimental groups of treatment:

[0139] Table 2

[0140] 1.1.1 Behavioral tests

[0141] To assess behavioral deficit after stroke, the basic behavioral scale and the adhesive removal test were performed. The basic behavioral score was recorded in all animals 24h after tMCAo. The adhesive removal test was performed at the following times: 24h prior to tMCAO, 24 and 48h after tMCAO in 24 rats treated with Vehicle and 18 rats treated with NGF P61SR100E.

[0142] At the end of the last behavioral test, at 48h post-surgery, animals were sacrificed and brains collected for the analysis of the infarct volume.

[0143] Basic behavioral scale: Bederson scores were used to evaluate neurological function in all rats after tMCAO, as previously described (Bederson et al., 1986, Li et al., 2018). The basic behavioral scale assignment ranged from 0 to 4, in which 0: no observable deficit; 1 : forelimb flexion; 2: decreased resistance to lateral push and forelimb flexion; 3: same behavior as 2 with circling; 4: cannot walk spontaneously or seizure activity.

[0144] Adhesive removal test: The adhesive removal test has been used as a measure of motor coordination and sensory neglect after stroke in experimental studies (Freret et al., 2009). Since it was previously reported that most animals are unable to perform the test on the first day of testing, we evaluated the adhesive removal test on the second day (48 h) as well. All animals were acclimatized to the test procedure in the week prior to surgery via 3 training sessions of 3 tests. A baseline test was recorded prior to surgery. Each rat was lightly restrained to allow the attachment of a 10 mm circular office stickers to the palm of each forepaw. The order of attachment (left or right applied first) was alternated at each test. Immediately after the placement of both stickers, a gentle pressure was simultaneously made on both forelimbs to minimize bias. The time necessary to remove each sticker was recorded. Then the animal was placed in the observation box and their behavior was recorded with a video camera for offline analysis. Three trials were undertaken in each test session, with a rest period of at least 1 minute between trials. A maximum time of 180 seconds was assigned for each trial. If the stickers could not be removed within this period, a maximum time was recorded.

[0145] 1.1.2 Quantification of ischemic damage

[0146] Cerebral infarct volume was evaluated 48 h after reperfusion in all rats (experimental sessions 1-5) subjected to 45 min tMCAO (treated with saline or NGF P61 SR100E), using the triphenyltetrazolium chloride (TTC) vital staining method. Rats were sacrificed and their brains were rapidly dissected to obtain eight serial coronal sections, cut at 2-mm intervals from the frontal pole, using a rat brain matrix (RBMA-300C, 2 Biological Instruments, Besozzo, Italy). Brain slices were stained in a solution containing 2% TTC in saline, at 37°C for 10 min, and then fixed in 10% neutral -buffered formaldehyde at 4°C. Images of TTC- stained sections were captured using a digital scanner and analyzed using an image analysis software (Imaged, version 1.30). The infarct volume (mm3) was calculated by summing the infarcted area (unstained) of the eight sections and multiplying the obtained value by the interval thickness between sections (Amantea et al., 2011).

[0147] 1.1.3 Statistics and analysis

[0148] With the assumption of a type 1 error of 5% and a power of 80%, a sample size of at least 56 animals was required. Statistical analysis was conducted using SigmaPlot version 11.0. A value of P < 0.05 was considered significant.

[0149] Since the data did not show a normal distribution, brain infarct volumes were analyzed with the Mann-Whitney rank sum test. Missing values due to premature death (9 in the Vehicle group and 2 in the NGF P61SR100E group) were imputed with the maximum possible stroke volume as defined by the largest infarct volume observed in the experiment.

[0150] Neurological scores were also analyzed with the Mann-Whitney rank sum test. Missing values due to premature death were imputed with the maximum possible neurological deficit score being 4. Times to remove the ipsilateral and contralateral tape were analyzed with two- way analysis of variance for repeated measures with fixed factors for “time” (Baseline, 24 h, 48 h), “treatment” (NGF P61SR100E and Vehicle), and their interaction. Missing values due to premature death were imputed with the maximum possible time to remove the tape of 180 seconds. Due to technical problems (video recording problems; compromised video) and the inability of some rats to perform the test properly in the training session, data from the adhesive removal test of 17 animals were missing; therefore, only 24 rats treated with Vehicle and 18 rats treated with NGF P61SR100E were considered for the analysis of the adhesive removal test.

[0151] Hazard ratio was used to compare mortality rate in the two treatment groups. Differences between treatment groups in the rate of mortality were analyzed with the one- side Fisher’s exact test. The Holm-Sidak test was used for making pairwise comparisons versus the reference group. One-tailed tests were adopted. Data are expressed as mean ± SEM.

[0152] 1.2 RESULTS

[0153] 1.2.1 Mortality

[0154] Nine rats died in the Vehicle group (27.3%) and two in the NGF P61SR100E group (7.7%), the difference being statistically significant (p = 0.05, one-tailed test).

[0155] 1.2.2 Behavioural tests

[0156] Compared to Vehicle-treated animals, rats treated with NGF P61SR100E showed a significant lower median neurological deficit score as evaluated with the Bederson scale (p < 0.05, Mann-Whitney rank sum test) (Figure 2).

[0157] In the adhesive removal test, prior to surgery all animals were able to remove the tape rapidly (4,2±2,44 sec; 4,36±3,9 sec ipsilateral (right) and contralateral (left) forepaw, respectively) and did not show preference in the order in which tapes were removed. Following tMCAO, the time required to remove the tape increased in both groups, 24 and 48h post-surgery, contralaterally and, to a lesser extent, ipsilaterally to the lesioned hemisphere. However, such increase was significantly less pronounced in the animals that received NGF P61SR100E, both at 24h and 48h post-tMCAO (p<0.05 and p<0.05 vs. controls) (Figure 3A and 3B).

[0158] 1.2.3 Quantification of brain ischemic volume

[0159] At 48 h, treatment with intranasal NGF P61SR100E induced a significant reduction (-

[0160] 39.3 ± 9.3%) of the brain infarct volume compared to the vehicle group 48 h after the lesion (p < 0.05, Mann-Whitney rank sum test, Figure 4). Representative coronal brain slices stained with 2,3,5-triphenyltetrazolium chloride (TTC) of the vehicle- and NGF P61SR100E- treated animals are shown in Figure 5.

[0161] 1.3 CONCLUSIONS

[0162] The results obtained in this study show that intranasal administration of NGF P61SR100E reduces mortality, brain infarct volume and improves the neurological (motor) deficit in a rodent model of transient cerebral ischemia.

[0163] Rats treated with intranasal NGF show better neurological recovery, compared to untreated rats, already at day 1 after tMCAO (Li et al., 2018).

[0164] 2. AQUEOUS LIQUID FORMULATION.

[0165] An illustrative and non-limiting example of an aqueous liquid formulation containing the polypeptide of SEQ ID NO: 1, and suitable to be administered to patients for the therapy according to the present invention, is the provided herewith; the aqueous medium used for the preparation of the formulation is saline (isotonic) solution.

[0166] 3. IN VIVO STUDY: NGF P61SR100E EFFECTS IN AN ANIMAL MODEL OF CEREBRAL ISCHEMIA (PERMANENT OCCLUSION)

[0167] In this study, the effects of intranasal administration in rats of the polypeptide of SEQ ID NO. 1 subjected to permanent middle cerebral artery occlusion (MCAO) were investigated. The NGF P61SR100E used was obtained using the methodology described in the international application No. W02019207106. Experiments were performed on adult male Wistar rats, housed under controlled environmental conditions (i.e., temperature of 22°C, relative humidity of 65% and 12 h light: 12 h dark cycle), with free access to food and water. A total of 29 animals (11 in the Vehicle-treated group; and 9 in the both the 7 and 20 pg / kg groups) underwent surgery. 3.1 MATERIAL AND METHODS

[0168] Animals: The in vivo procedures were conducted following the guidelines of the Italian Ministry of Health (DL 26 / 2014), in accordance with the 2010 / 63 / UE European Directive, and the protocol was approved (n. 214 / 2025-PR) by the Committee set by the Ministry of Health at the National Institute of Health (Rome).

[0169] Surgical procedure: Focal cerebral ischemia was induced by proximal occlusion of the middle cerebral artery (MCAO). To this end, animals were anesthetized with 5% isoflurane vaporized in air, maintained at 2.5% during surgery. The external carotid artery was isolated and a silicone-coated nylon filament (diameter: 0.37 mm, Doccol Corporation, Redlands, CA, USA) was introduced into the internal carotid artery up to the Willis circle to occlude the origin of the middle cerebral artery (i.e., approximately 18 mm from carotid artery bifurcation). Successful occlusion was evaluated by the intra-ischemic clinical assessment, namely if the animals presented > 3 of the following deficits after filament insertion:

[0170] 1. ellipsoidal shape of the palpebral fissure

[0171] 2. lateral extension of one or both ears

[0172] 3. asymmetric body bending on the ischemic side

[0173] 4. laterally extending limbs that do not align to the body.

[0174] Intranasal administration: NGF P61SR100E was administered at two different doses (7, 20 pg / kg) at 10 pl / animal, included in a formulation including acetic acid, L-methionine, sodium chloride, polysorbate 80, sodium hydroxide, hydrochloric acid (pH 5.5; osmolarity 320 mOsm / kg;), or vehicle formulation (35 pl / kg; comprising the same ingredients as above, exception made for NGF P61SR100E) were administered 15 min after MCAO, by the intranasal route, in rats briefly (30-min) anesthetized with 5% isoflurane vaporized in air.

[0175] 3.1.1 Assessment of infarct size, edema.

[0176] Cerebral ischemic damage was assessed after 24 h of permanent MCAo. To this end, brains were rapidly dissected and sectioned into 2 mm-thick consecutive coronal slices using a rat brain matrix (Harvard Apparatus, Massachusetts, USA). To measure ischemic damage, brain slices were stained in a solution containing 2% 2,3,5-triphenyltetrazolium chloride (TTC) in saline, at 37°C. After 10 min incubation, the slices were transferred to 10% neutral buffered formaldehyde and stored at 4°C prior to analysis. Images of TTC-stained sections were captured using a digital scanner and analysed using an image analysis software (Imaged, version 1.30). Infarct volume (mm3) was determined by summing the infarcted (pale) areas and multiplying the obtained value by slice thickness (2 mm). Infarct edema (mm3) was calculated by subtracting the volume of the contralateral hemisphere from the volume of the ipsilateral hemisphere. 3.1.2 Neurological deficits (De Simoni composite neuroscore)

[0177] Neurological deficits were assessed 24h after MCAo, or 72h after PC by using the dichotomized De Simoni composite neuroscore that allows to evaluate the general and focal neurological dysfunctions caused by the ischemic insult (Orsini et al., 2012; Tettamanti et al., 2020; Valente et al., 2023; Valente Alessia, Multicentre translational Trial of Remote Ischaemic Conditioning in acute ischaemic Stroke (TRICS), MILANO-BICOCCA University, School of Medicine and Surgery, ACADEMIC YEAR 2021 / 2022). Each animal was assigned a score ranging from 0 (healthy) to 56 (the worst performance in all the 13 categories). This score represents the sum of

[0178] (i) 6 general deficits describing with the general well-being of the animal and including information on the physical appearance of the rats (fur [0-2], ears [0-2], eyes [0- 4], posture [0-4], spontaneous activity [0-4], and epileptic behavior [0-12]); and

[0179] (ii) 7 focal deficits describing the neurological damage and evaluated through observation on different parameters (body asymmetry [0-4], gait [0-4], climbing [0-4], circling behavior [0-4], forelimb symmetry [0-4], compulsory circling [0-4], and whisker response [0-4]).

[0180] 3.2 RESULTS

[0181] 3.2.1 Assessment of infarct size, edema.

[0182] At 24 hours after the lesion, histological examination revealed that treatment with 7 pg / kg and with 20 pg / kg of intranasal NGF P61SR100E induced a significant reduction (- 30.7 ± 20.8 and -25.7 ± 7.7% respectively) of the cerebral ischemic volume compared to the vehicle group (p < 0.05; Tukey’s multiple comparisons test, Figure 6B). Ischemia-induced brain edema was not affected by the treatment.

[0183] 3.2.2 Neurological deficits (De Simoni composite neuroscore)

[0184] Neuroprotection by intranasal administration of NGF P61SR100E was associated with improved neurological status in rats receiving 7 pg / kg or 20 pg / kg, as indicated by a reduction in general neurological deficits (p = 0.052, one-tailed analysis of variance; Figure 7).

[0185] Even though the effects on mortality will be evaluated in the following phases of the study (days after MCAo), it is worth mentioning that none of the rats under study died in the 24 hours following MCAo.

Claims

CLAIMS1. A polypeptide of SEQ. ID NO: 1 for use in the treatment of acute ischaemic stroke (AIS) and / or in the prevention of the complications of AIS in an adult mammalian subject, wherein the polypeptide is administered intranasally.

2. The polypeptide for use according to claim 1, wherein AIS is followed by reperfusion of ischemic brain tissue.

3. The polypeptide for use according to claim 1 or 2, wherein AIS is due to a vessel occlusion either in the anterior or in the posterior circulation.

4. The polypeptide for use according to any one of claims from 1 to 3, wherein AIS is due to large or medium vessel occlusion in the anterior circulation.

5. The polypeptide for use according to claim 4, wherein the subject is eligible to undergo or has undergone endovascular thrombectomy therapy (EVT), preferably endovascular mechanical thrombectomy.

6. The polypeptide for use according to claim 5, wherein EVT is performed in association or not with thrombolysis, wherein thrombolysis is preferably intravenous thrombolysis.

7. The polypeptide for use according to claim 5 or 6, wherein the polypeptide is administered before and / or after EVT.

8. The polypeptide for use according to any one of claims from 1 to 6, wherein the polypeptide is administered at least once within 24 hours after AIS onset or last seen well.

9. The polypeptide for use according to any one of claims from 1 to 8, wherein the polypeptide is administered at least once within 12 hours after AIS onset or last seen well.

10. The polypeptide for use according to any one of claims from 4 to 9, wherein the polypeptide is administered at least once within 6 hours after EVT.

11. The polypeptide for use according to any one of claims from 1 to 10, wherein the mammalian subject is a human subject.

12. The polypeptide for use according to claim 1, in the form of an aqueous formulation.

13. The polypeptide for use according to claim 12, wherein the polypeptide is contained in the formulation at a concentration ranging between 0.5 to 3.0 mg / mL, preferably ranging between 0.5 and 2.0 mg / mL.

14. The polypeptide for use according to any one of claims from 1 to 13, the polypeptide being administered between 1 and 30 days in patients with AIS, preferably for a period comprised between 1 and 14 days.

15. The polypeptide for use according to any one of claims from 1 to 14, wherein the polypeptide is administered only once a day.

16. The polypeptide for use according to claim 1, wherein AIS is not followed by reperfusion of ischemic brain tissue.

17. The polypeptide for use according to claim 16, wherein AIS is due to a vessel occlusion either in the anterior or in the posterior circulation.

18. The polypeptide for use according to any one of claims 16, 17, wherein AIS is due to large or medium vessel occlusion in the anterior circulation.

19. The polypeptide for use according to any one of claims from 16 to 18, wherein the polypeptide is administered at least once within 24 hours after AIS onset or last seen well.

20. The polypeptide for use according to any one of claims from 16 to 18, wherein the polypeptide is administered at least once within 12 hours after AIS onset or last seen well.

21. The polypeptide for use according to any one of claims from 16 to 20, wherein the mammalian subject is a human subject.

22. The polypeptide for use according to any one of claims from 16 to 21, wherein the polypeptide is administered between 1 and 30 days in patients with AIS, preferably for a period comprised between 1 and 14 days.

23. The polypeptide for use according to any one of claims from 16 to 22, wherein the polypeptide is administered only once a day.

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