Combination Therapies for Brain Injury

Abstract

The present invention relates to combination therapies for use in the treatment and prevention of brain injury, in particular ischemic and / or hypoxic brain injury, including but not limited to hypoxic-ischemic encephalopathy, preferably in term or preterm neonatal subjects.

Description

[Technical field]

[0001] The present invention relates to combination therapies for use in the treatment and prevention of brain injury, in particular ischemic and / or hypoxic brain injury, including but not limited to hypoxic-ischemic encephalopathy, preferably in term or preterm neonatal subjects. [Background technology]

[0002] Nerve growth factor (NGF or hNGF) is a neuroactive protein whose discovery was awarded the Nobel Prize in Medicine (1986, Rita Levi-Montalcini). For decades, its therapeutic potential has been widely investigated, especially in chronic / degenerative diseases. Recently, an ophthalmic formulation of recombinant human NGF (rhNGF) was approved by the EMA and FDA for the treatment of neurotrophic keratitis, a rare eye disease. There are few studies on the neuroprotective potential of hNGF in acute lesions of the central nervous system (CNS). Several scientific publications have shown that the underproduction of endogenous neurotrophic factors may play a role in determining the severity of long-term neurological impairments seen after neonatal hypoxic-ischemic injury (Wang et al., Int J Neurosci 2013; 123: 191-195) and suggest their administration during the acute phase of the lesion (Acta Cir Bras 2017; 32: 270-279, Int J Clin Exp Med 2013; 6: 951-955). In particular, NGF administration limits deficits in the development and maturation of complex behaviors assessed during early childhood. Unfortunately, hNGF also has a potent hyperalgesic effect (Nat Med 1995; 1: 774-780). Since (wild type) NGF is known to affect target cells by binding to two distinct receptors, (a) the receptor tyrosine kinase A (TrkA) that promotes neuronal survival, and (b) the p75 neurotrophin receptor that is involved in the control of cell death (Mesentier-Louro et al., 2017, Int. J. Mol. Sci., vol. 18(98)), the polypeptide sequence of wild type NGF after optic nerve crush injury may also have the effect of aggravating retinal degeneration by stimulating apoptosis. See Mesentier-Louro et al., 2018, Mol. Neurobiol., vol. 56, p. 1056-1069. It is also known that residue R100 of wild type human NGF is involved in the binding of NGF to p75, and mutation of this residue affects the binding to p75, see for example WO 2008 / 006893A1.In contrast to wild-type NGF, the polypeptide of the present invention, hNGF, has a lower binding affinity to p75 (see, for example, Malerba et al., 2015, PlosOne, 10(9): e0136425). Human NGF P61SR100E corresponds to the polypeptide of SEQ ID NO: 1, also named CHF6467 (SEQ ID NO: 1).

[0003] Hypoxic-ischemic encephalopathy (HIE) or neonatal asphyxia is a major cause of severe neurological disability and perinatal death. In Western Europe and the United States, its incidence is estimated at 1.5-2 cases per 1,000 live births, with a higher prevalence in premature infants (Pediatr Res 2013; 74, Suppl 1, 50-72). Neuronal death following HIE occurs by programmed cell death mechanisms mediated by autophagy or, most likely, by sequential apoptotic-necrotic cell death (Ann Neurol 2011; 69: 743-758), which is driven by release of excitotoxic glutamate, activation of NMDA and AMPA receptors, and increased intracellular Ca. 2+ It is triggered by a series of events including mitochondrial overload, mitochondrial dysfunction, oxidative stress, caspase activation and inflammation (Lancet Neurol 2011; 10: 372-382).

[0004] Undoubtedly, brain damage after HI insult is a complex process with multiple contributing mechanisms and pathways, resulting in both early and delayed damage (Patel, S. et al. (2014). Biochem. Soc. Trans. 42, 564-568). Therefore, novel interventions in addition to the current standard of care, therapeutic hypothermia, are urgently needed to enhance neuroprotection (Davidson, JO, et al., (2015). Front. Neurol. 6.). Furthermore, there is evidence that therapeutic hypothermia is more effective in moderate than severe hypoxic-ischemic encephalopathy (Sabir, H., et al., (2012). Stroke 43, 3364-3370). It is increasingly recognized that one of the major pathogenic factors in neonatal brain injury is inflammation induced by activation of the central and peripheral immune systems (Hagberg et al., (2015). Nat. Rev. Neurol. 11, 192-208). Immune responses can be induced within minutes and continue for weeks or even months after injury (Fleiss et al., 2015 Dev. Med. Child Neurol. 57, 17-28). Hypothermia, the gold standard intervention after hypoxic-ischemic injury, is not beneficial in all treated newborns (Bainbridge et al., 2012. Brain 136, 90-105). Nerve growth factor (NGF) has been shown to protect certain neurons expressing its signaling receptor, trkA, from a variety of insults (Holtzman et al., 1995 J. Neurosci. 15). Notably, there is data indicating that NGF has neuroprotective effects that extend beyond neurons in the developing brain (Fodelianaki et al., 2019 Exp. Cell Res. 377).Indeed, NGF has been previously suggested to have neuroprotective effects in animal models of newborn hypoxic-ischemic encephalopathy and to be a promising agent for protecting the neonatal brain from hypoxic-ischemic injury (Holtzman et al., 1996, Ann. Neurol. 39. doi:10.1002 / ana.410390117).

[0005] Therapeutic hypothermia is the standard of care to improve survival and reduce neurodevelopmental sequelae in neonates with moderate to severe hypoxic-ischemic encephalopathy (HIE). Selective head or whole-body cooling to achieve a core temperature of 33.5°C for approximately 72 hours, initiated within a therapeutic window of 6 hours after birth, improves survival and neurological outcomes in neonates with HIE (J Pediatr 2015; 91: S78-S83). Although there are relatively few preclinical studies comparing multiple therapeutic hypothermia temperatures, it has been recently suggested that cooling below 33.5°C does not appear to confer additional neuroprotection in a neonate rat model of HIE and that milder cooling should be considered in future clinical trials (Sci Rep 2016; 6: 23430). Therapeutic hypothermia is not associated with major complications but appears to benefit only a relatively small percentage of infants with HIE (Int J Mol. Sci 2015; 16: 22368-22401). Specifically, treatment with therapeutic hypothermia is currently reserved for term or near-term newborns (>1800g) and is typically limited to reference centers with specific expertise and facilities.

[0006] WO2018087656 relates to a pharmaceutical composition comprising NGF or a molecule having NGF-like activity for use in the treatment of traumatic brain injury, in particular traumatic brain injury or ischemic hypoxic brain injury, including but not limited to hypoxic-ischemic encephalopathy, by intranasal administration.

[0007] Hou et al. (Chinese Journal of Primary Medicine and Pharmacy 2017;24(3):338-341) report that the combination of cranial hypothermia and mouse nerve growth factor therapy for the treatment of moderately severe HIE in neonates provides protection. As noted above, the use of hypothermia in the treatment of neonatal asphyxia is well documented in the prior art (see, e.g., Volpe, 2001; Gunn et al, 2000). However, to date, there has been no teaching or suggestion in the art that hypothermia and administration of the mutant NGF, CHF6467, could be used in combination. Nor has there been any suggestion that such combined therapy could result in such a surprising and unexpected enhancement in the neuroprotective effects obtained.

[0008] Thus, there is an ongoing need for agents for the treatment of HIE. In particular, agents that can enhance the effects of hypothermia could add extremely valuable to current clinical practice. Summary of the Invention

[0009] CHF6467 (hNGFp, SEQ ID NO:1) has neurotrophic activity similar to wild-type human nerve growth factor (NGF), but its pain sensitizing activity is 10-fold lower (Cattaneo, A., and Capsoni, S. (2019). Pharmacol. Res. 139. doi:10.1016 / j.phrs.2018.10.028.). [ka]

[0010] The present invention provides a combination of a polypeptide of SEQ ID NO:1 and hypothermia for use in the treatment and / or prevention of traumatic brain injury or ischemic-hypoxic brain injury in a mammalian subject.

[0011] In a preferred embodiment, the polypeptide is administered at a dose of 20 μg / kg.

[0012] In a preferred embodiment, the traumatic brain injury is hypoxic-ischemic encephalopathy.

[0013] In a preferred embodiment, the mammalian subject is a human, preferably the subject is a neonate or newborn or premature neonatal subject, preferably the neonate or newborn subject suffers from moderate to severe hypoxic-ischemic encephalopathy.

[0014] In a preferred embodiment, the polypeptide is administered intranasally or the polypeptide is administered repeatedly, preferably for a period of 1 to 30 days, preferably 2 to 14 days. Repeated cycles of treatment are also envisaged.

[0015] In a preferred embodiment, the polypeptide is administered repeatedly, 1 to 7 times per day, or the polypeptide is administered to one or both nostrils.

[0016] In a preferred embodiment, the polypeptide is administered at a dose of 0.01 to 1.00 mg / day, preferably at a dose of 0.06 to 0.12 mg / day, preferably at a dose of 0.06 mg / day.

[0017] Preferably, the amount of 0.06 mg / day is for a neonate or newborn weighing approximately 3 kg.

[0018] A preferred dose is 3-333 μg / kg, especially for heavier subjects.

[0019] A preferred dose is about 20 μg / kg to about 40 μg / kg, preferably 20 μg / kg.

[0020] In a preferred embodiment, when the polypeptide is administered repeatedly, each dose of the polypeptide is 0.01-1.00 mg / day, preferably each dose of the polypeptide is 0.06-0.12 mg / day, preferably each dose of the polypeptide is 0.06 mg / day.

[0021] In a preferred embodiment, the polypeptide is administered within 1 to 24 hours after brain injury.

[0022] In a preferred embodiment, the polypeptide is contained in a composition which includes an acetate buffer and / or methionine.

[0023] In a preferred embodiment, the polypeptide is administered simultaneously with and / or within 24 hours following hypothermia.

[0024] In a preferred embodiment, hypothermia is initiated within 1 to 10 hours after hypoxic-ischemic (HI) injury, preferably, hypothermia is initiated within 2 to 8 hours after HI injury, preferably, hypothermia is initiated within 4 to 6 hours after HI injury.

[0025] In a preferred embodiment, hypothermia is maintained for about 1 hour to about 72 hours, preferably at least about 4 hours, more preferably at least about 6 hours, more preferably at least about 12 hours, following the hypoxic-ischemic (HI) insult.

[0026] Preferably, hypothermia is maintained for at least about 4 hours, more preferably at least about 6 or 12 hours, after birth.

[0027] In a preferred embodiment, the body temperature of the mammal is maintained at a temperature of about 32°C to about 36°C, preferably at a temperature of about 32°C to about 35°C.

[0028] Another aspect of the present invention is: (a) administering to a mammalian mother a therapeutically effective amount of CHF6467 prior to and / or during parturition; and (b) subjecting a mammal to hypothermia after birth; This invention relates to the treatment of neonatal asphyxia in a mammal in need thereof by

[0029] Hypothermia can occur passively, by allowing body temperature to drop rather than intentionally maintaining it (newborns are subject to temperature fluctuations and will rapidly reach the temperature of their surroundings), or the patient can be actively made hypothermic by intentionally lowering the temperature of the environment.

[0030] Further aspects of the present invention include: (a) administering a therapeutically effective amount of CHF6467 to a mammal; and (b) subjecting a mammal to hypothermia or hypothermic conditions; This invention relates to the treatment of neonatal asphyxia in a mammal in need thereof by

[0031] In a preferred embodiment, the mammal is a newborn subject within the first four weeks of age. More preferably, the mammal is within the first two weeks of age, and even more preferably, within the first week of age.

[0032] Preferably, the mammal is a human.

[0033] Preferred embodiments of the second aspect of the invention are similar to those described above for the first aspect.

[0034] Another aspect of the invention relates to the treatment of neonatal asphyxia in a mammal by administering a therapeutically effective amount of xenon in combination with hypothermia to a mammal in need of treatment.

[0035] Yet another aspect of the present invention relates to the use of CHF6467 in the manufacture of a medicament for the treatment of neonatal asphyxia, said treatment comprising administering CHF6467 in combination with hypothermia simultaneously, sequentially or separately to a subject.

[0036] A further aspect of the invention relates to the use of CHF6467 in combination with hypothermia to treat neonatal asphyxia.

[0037] In a preferred embodiment, in a combination for use in the treatment and / or prevention of traumatic brain injury or ischemic hypoxic brain injury in a mammalian subject, the polypeptide is administered at a dose of 0.06 to 0.12 mg / day and the body temperature of the mammal is maintained at about 32°C to about 35°C.

[0038] In a preferred embodiment, in a combination for use in the treatment and / or prevention of traumatic brain injury or ischemic hypoxic brain injury in a mammalian subject, the polypeptide is administered at a dose of about 20 μg / kg to 40 μg / kg and the body temperature of the mammal is maintained at a temperature of about 32°C to about 35°C.

[0039] The following figures and non-limiting examples illustrate the invention without limiting its scope. [Brief description of the drawings]

[0040] [Figure 1] Effect of CHF6467 on baseline transmission at CA1 hippocampal synapses. [Diagram 2] CHF6467 restored the OGD-induced depression (preventative measure). Normalized pooled data show the effect of OGD on excitatory field postsynaptic potentials (fEPSPs) and the protective effect of CHF6467 perfused at different concentrations (0.03 and 0.1 μg / mL) during OGD. [Diagram 3] The OGD-induced EPSC depression is restored by CHF6467 perfusion (therapeutic treatment). Panel A shows the time course of normalized EPSC data after treatment with OGD alone (closed circles) and OGD+CHF6467 (open circles). The histograms show the average normalized EPSC data over the last 10 min of recording after treatment with OGD alone and OGD+CHF6467. Panel B shows that in the absence of OGD, CHF6467 does not induce any change in baseline transmission. [Figure 4] Synergistic effect of CHF6467 and hypothermia on OGD. Normalized pooled data showing the synergistic effect of hypothermia and CHF6467 perfusion after 15 min of OGD. [Diagram 5] Diagram of the experimental protocol for the rat model of neonatal hypoxic-ischemic encephalopathy. TTC = 2,3,5-triphenyltetrazolium chloride. [Figure 6] Effect of intranasally administered CHF6467 (20 and 40 μg / kg) alone and in combination with hypothermia on log-transformed cerebral infarct volume in juvenile rats 72 hours after hypoxic-ischemic injury at 10 days of age. Numbers of animals are indicated above each column. [Figure 7] Hippocampal mRNA levels of a panel of rat cytokines and chemokines in sham-operated, vehicle-treated hypoxic-ischemic encephalopathy (HIE), HIE treated with CHF6467 20 μg / kg (HIE+CHF 20 μg / ml), HIE treated with hypothermia (HIE+IPO), and HIE treated with CHF6467 and hypothermia (HIE+IPO+CHF). Scatter plots shown with mean ± standard error (SEM). **p<0.01 and *p<0.05 vs. HIE. [Figure 8] Cortical mRNA levels of cytokines and chemokines in rat populations with sham, hypoxic-ischemic encephalopathy (HIE), HIE treated with CHF6467 20 μg / kg (HIE+CHF20 μg / ml), HIE treated with hypothermia (HIE+IPO), and HIE treated with CHF6467 and hypothermia (HIE+IPO+CHF). Scatter shown with mean ± standard error (SEM). **p<0.01 and *p<0.05 vs. HIE. [Figure 9] Statistical analysis of mean log10 transformed cerebral infarct volumes by treatment group. [Figure 10] Brain penetration of CHF6467 after intranasal administration in control juvenile rats as determined by quantification of the SSSHPIFHR (SEQ ID NO:2) peptide. [Figure 11]Brain penetration of CHF6467 after intranasal administration in juvenile rats with hypoxic-ischemic injury. Light-to-heavy ratios of CHF6467-specific peptides, SSSHPIFHR (SEQ ID NO:2) (A) and QAAWEFIR (SEQ ID NO:3) (B) and the non-specific peptide, QYFFETK (SEQ ID NO:4) (C) in various rat brain regions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Detailed Description of the Invention The neuroprotective effects of CHF6467 or the polypeptide of SEQ ID NO:1 were tested using in vitro and in vivo experimental models of hypoxic-ischemic encephalopathy (HIE). In acute hippocampal slices subjected to oxygen-glucose deprivation (OGD), an established in vitro model of hypoxic-ischemic brain injury, CHF6467 significantly suppressed abnormal excitatory postsynaptic currents (EPSCs). Unexpectedly, the combination of CHF6467 and hypothermia completely restored the abnormal EPSCs (Figure 4). In neonatal rats subjected to hypoxia after permanent left common carotid artery occlusion, an established model of neonatal hypoxic-ischemic encephalopathy, intranasal administration of CHF6467 (20 and 40 μg / kg) significantly reduced cerebral infarct volume compared to vehicle-treated animals. Remarkably, the combination of CHF6467 and hypothermia dramatically reduced cerebral infarct volume by 86% compared to normothermic vehicle-treated animals. Unexpectedly, the neuroprotective effect of the combination of CHF6467 and hypothermia was significantly higher than that of either single agent (CHF6467 or hypothermia) alone, demonstrating a synergistic effect (Figure 6). Notably, the effect of CHF6467 at the preferred dose (20 μg / kg) in vivo was synergistic with hypothermia in providing neuroprotective effects. No mortality, clinical signs, or weight loss due to CHF6467 treatment was observed, suggesting that CHF6467 was well tolerated.

[0042] Thus, the present invention provides a combination of CHF6467 and hypothermia in the treatment of brain injury, traumatic brain injury or ischemic hypoxic brain injury, preferably hypoxic-ischemic encephalopathy.In particular, the combination of CHF6467 and hypothermia can significantly reduce mortality and severe neurodevelopmental disorders in infants and neonates with moderate to severe hypoxic-ischemic encephalopathy compared with hypothermia alone, especially when appropriate dosage is considered, as reported in the experimental section below.

[0043] In addition, a panel of neuroinflammatory markers was also examined in the cortex and hippocampus of rat pups subjected to hypoxic-ischemic brain injury with CHF6467 alone or in combination with hypothermia.

[0044] A significant increase in the mRNA levels of several cytokines and chemokines in the hippocampus and frontoparietal cortex was measured in rats subjected to hypoxic-ischemic injury compared to sham-operated rats. CHF6467 (20 μg / kg, intranasal) significantly counteracted the upregulation of several neuroinflammatory markers in the hippocampal region of rats subjected to hypoxic-ischemic injury, but hypothermia itself was not as effective as CHF6467. In the cortex, CHF6467 (20 μg / kg) itself was effective in counteracting the upregulation of several neuroinflammatory markers induced by hypoxic-ischemic injury, and such effects were enhanced in some cases by combination with hypothermia.

[0045] Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, presentations, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein shall be construed as an admission that the invention is not entitled to antedate any particular teaching, and / or that any particular document, other than common general knowledge, contains information sufficiently clear and complete for one of ordinary skill in the art to practice the invention.

[0046] The expression "and / or", e.g., "X and / or Y", shall be understood to mean "X and Y" or "X or Y" and shall be considered to provide clear disclosure of "and", "or" and both meanings ("and" or "or").

[0047] Unless otherwise indicated, the terms "about," "ca.," and "substantially" all mean "approximately" or "nearly," and in the context of numerical values ​​or ranges described herein, preferably specify ±10%, more preferably ±5%, of the stated or claimed numerical value or range.

[0048] Unless otherwise indicated, the term "comprise" or variations such as "comprises" or "comprising" are used in the context of this specification to indicate that additional elements may optionally be present in addition to the listed elements introduced by "comprising". However, it is contemplated as a specific embodiment of the present invention that the term "comprising" encompasses the possibility that no additional elements are present, i.e., this embodiment is understood as "comprising" having the meaning of "consisting of".

[0049] Unless otherwise indicated, the indication of relative amounts in the present invention is on a weight / weight basis. The indication of the relative amount of a component characterized by a generic term is meant to indicate the total amount of all specific variants or members included in said generic term. If a specific component defined by a generic term is present in a specific relative amount and is further characterized as a specific variant or member included in the variant, it means that there is no additional presence of other variants or members included in the variant such that the total relative amount of the components included in the variant exceeds the specific relative amount; more preferably, it means that there is no presence of the specific variant or member included in the variant at all.

[0050] Unless the context indicates otherwise, the term nerve growth factor refers only to wild-type NGF and does not include the polypeptide of SEQ ID NO:1.

[0051] The terms "NGF mutein" and "mutein of NGF", or "mutein thereof" for NGF, are used interchangeably herein and refer to a polypeptide characterized by at least one mutation compared to wild-type NGF, as described in more detail herein. The polypeptide of SEQ ID NO: 1 is a mutein of NGF. It refers to any species, preferably a mammalian species, although a mutein of human NGF is always preferred. Preferably, the mutein of NGF has 80-99.5% sequence identity with NGF, in particular human NGF, more preferably the mutein has 90-99% sequence identity with NGF, in particular human NGF.

[0052] The term "hypothermia" as used herein refers to subjecting a particular subject (e.g., a neonatal subject) to hypothermic conditions by passive or active techniques, for example by lowering the body temperature, preferably by 3-5° C. The preferred hypothermia temperature is 32° C.-36° C., preferably 32° C.-35° C., preferably 32° C. or 33° C.

[0053] As used herein, the term "concurrently" is used to mean that CHF6467 is administered simultaneously with hypothermia, while the term "in combination" is used to mean that CHF6467 is administered "sequentially" if not simultaneously, within a time frame in which both CHF6467 and hypothermia are therapeutically effective, i.e., both are available to act therapeutically in the same time frame. Thus, when neonate subjects are exposed to hypothermic conditions, "sequential" administration may be within 5 minutes, 10 minutes, or hours before or after hypothermia, provided that the circulating half-life or target tissue residence time of CHF6467 is such that CHF6467 is present in a therapeutically effective amount.

[0054] The terms "patient" and "subject" are used interchangeably herein, particularly with respect to patients / subjects characterized by an ocular disorder as described herein.

[0055] In the context of the present invention, the term "prevent" is understood in a broad sense, which includes not only the initiation of prevention of brain damage, but also the prevention of the progression of brain damage. In particular, in the context of disorders involving brain damage and / or brain damage, the term "prevent" also includes preventing further progression of brain damage.

[0056] In the context of the present invention, the term "treating" is understood broadly and includes, but is not limited to, the amelioration of symptoms of a disorder.

[0057] According to the present invention, an "effective amount" is an amount or dose that alone or together with further doses achieves a desired reaction or a desired effect.

[0058] The term "pharmaceutical acceptable" generally indicates that a particular substance can be administered to a subject, optionally and preferably in combination with a drug, without causing intolerable adverse effects at the dosages used.

[0059] The terms "pharmaceutical acceptable carrier" and "pharmaceutical acceptable excipient" are used to refer to any one or more of solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are physiologically compatible and suitable for administration to a subject as described herein or that do not interfere with such administration. Such pharmaceutical acceptable carriers include, but are not limited to, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, polysorbate 80, etc., and combinations thereof. For liquid pharmaceutical compositions in particular, it may be preferable to include an isotonic agent in the composition, for example, sugars, polyalcohols such as mannitol or sorbitol, or sodium chloride.

[0060] The pharma- ceutically acceptable carrier may further contain auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the drug. A pharma- ceutically acceptable carrier is typically included in the composition according to the present invention.

[0061] The term "pharmaceutical active agent" refers to an agent that can be used for administration to a subject, for example, when it is beneficial to improve symptoms of a disease or disorder. Furthermore, a "pharmaceutical active agent" can have a positive or beneficial effect on the condition or disease state of the subject when administered to a subject in a therapeutically effective amount. Preferably, a pharmaceutical active agent has curative properties and can be administered to improve, alleviate, reduce, regress, delay the onset or reduce the severity of one or more symptoms of a disease or disorder. A pharmaceutical active agent can have preventive properties and can be used to delay the onset of a disease or reduce the severity of such a disease or condition. For example, the agent of the present invention is considered as a pharmaceutical active ingredient for the treatment of cystic fibrosis, as claimed. In another example, a pharmaceutical active protein can be used to treat cells or individuals that do not normally express a protein, or do not express a protein at a desired level, or do not express a protein correctly, for example, a pharmaceutical active protein can compensate for a mutation or lack of sufficiently high expression by providing the desired protein. The term "pharmacologically active peptide or protein" includes the entire protein or polypeptide and may also refer to pharma- ceutical active fragments thereof. It also includes analogs of pharma- ceutical active peptides or proteins.

[0062] The terms "subject" and "patient" as used herein relate to mammals. For example, a mammal in the context of the present invention is a human, a non-human primate, a domestic animal, including but not limited to a dog, a cat, a sheep, a cow, a goat, a pig, a horse, etc., a laboratory animal, including but not limited to a mouse, a rat, a rabbit, etc., and a captive animal, such as a zoo animal. The terms "subject" and "patient" as used herein specifically include a human. A subject (human or animal) has two pairs of chromosomes; that is, the subject is diploid. The term "patient" refers to a subject who has a condition, is at risk of having a condition, has a condition, or is expected to have a condition, and who may be subjected to a treatment, such as the administration of a drug. The condition of the patient may be chronic and / or acute. Thus, a "patient" may be described as a subject who is subjected to a treatment and / or who needs treatment.

[0063] The term "treatment" is understood in a broad sense and refers to the treatment of a subject with the goal of preventing or treating a condition in the subject. In a preferred embodiment, treatment specifically involves the administration of a drug to the subject.

[0064] The agent according to the invention, also referred to herein as "polypeptide of SEQ ID NO:1", is described in more detail. The term "polypeptide of SEQ ID NO:1" and similar terms are used herein to refer to a polypeptide comprising an amino acid sequence defined in SEQ ID NO:1 and / or an agent having equivalent biological activity. Thus, these terms also include functionally equivalent portions or analogs of such polypeptides. An example of a biologically equivalent portion of a polypeptide may include a domain or subsequence of the polypeptide of SEQ ID NO:1 that comprises a binding site that allows it to exert substantially the same biological activity as the full-length polypeptide of SEQ ID NO:1, or a gene encoding such a polypeptide. The term "substantially the same biological activity" refers to an equivalent portion or analog polypeptide having at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% and most 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 Examples 1-3. An example of a biologically equivalent analog of a polypeptide would be a fusion protein that contains at least a portion of the amino acid sequence of the polypeptide of SEQ ID NO: 1, but could also be a homologous analog of the polypeptide. Also, completely synthetic molecules that mimic the specific biological activity of the polypeptide of SEQ ID NO: 1 are considered to constitute "biologically equivalent analogs."

[0065] More preferably, the term "polypeptide of SEQ ID NO:1" and similar terms herein refer to a polypeptide comprising the amino acid sequence defined in SEQ ID NO:1; such an agent may optionally be a fusion protein comprising, inter alia, the amino acid sequence defined in SEQ ID NO:1. Preferably, the term "polypeptide of SEQ ID NO:1" and similar terms herein refer to a polypeptide consisting of the amino acid sequence defined in SEQ ID NO:1; in this embodiment, the agent consists of a polypeptide consisting of 118 amino acid residues in the order defined in SEQ ID NO:1. In this and other embodiments, the polypeptide may optionally have one or two or three cysteine ​​bonds, such 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.

[0066] The polypeptide of the present invention may be characterized by further post-translational modifications, if necessary. Such post-translational modifications may include glycosylation and / or phosphorylation if necessary. However, preferably, the polypeptide of the present invention is not glycosylated and / or phosphorylated. Indeed, the test examples herein show beneficial effects and beneficial benefit-adverse effect ratios on the healing of brain injury, particularly traumatic brain injury or ischemic hypoxic brain injury (including but not limited to hypoxic-ischemic encephalopathy), whereby the polypeptides used are typically obtained by cytoplasmic recombinant expression in bacteria, which does not result in glycosylation and / or phosphorylation, so it is reasonable to consider that the beneficial effects of the present invention are not dependent on such types of post-translational modifications.

[0067] Typically, a polypeptide according to the invention is a non-naturally occurring polypeptide that is not spontaneously produced by the subject to which the polypeptide is administered.

[0068] Preferably, the polypeptide according to the invention is an isolated polypeptide. More preferably, the polypeptide according to the invention is essentially free of host cell proteins, degradation products (e.g., des-nona variants) and proteases (e.g., trypsin, etc.). When the polypeptide according to the invention is essentially free of host cell proteins, degradation products (e.g., des-nona variants) and proteases (e.g., trypsin, etc.), it can also be referred to as a "pure polypeptide". Preferably, the polypeptide according to the invention is administered as a 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, based on the total protein in the composition. Most preferably, the pure polypeptide according to the invention has a purity that complies with Good Manufacturing Practice (GMP).

[0069] The polypeptide consisting of SEQ ID NO:1 has two different amino acid sequences from human nerve growth factor (NGF, also called wild-type human NGF or wild-type NGF). The difference between the polypeptide according to the present invention and the wild-type polypeptide, as described in detail herein and supported by the test examples herein, has a significant effect on the treatment or prevention of brain injury, particularly traumatic brain injury or ischemic hypoxic brain injury, including but not limited to hypoxic-ischemic encephalopathy, and has no side effects.

[0070] Among several variants of human NGF (hNGF variants), variant human NGF P61SR100E is considered to be the most promising for the treatment of brain injury.However, the specific use of treatment and / or prevention according to the present invention is not indicated by those documents, and those documents also do not indicate that the respective polypeptides are available in a sufficiently high purity to allow their medical use in humans.Therefore, the polypeptides for use according to the present invention described and provided herein provide some unexpected advantages over wild-type human NGF.Administration of the agent according to the present invention causes a reduction in cerebral infarct volume (see, for example, Example 2), and said administration is not accompanied by pain.

[0071] The polypeptide of SEQ ID NO: 1 does not occur in nature and may also be referred to as a non-naturally occurring polypeptide.The agent according to the invention is not wild-type NGF, in particular is not wild-type human NGF.

[0072] Preferably, the non-natural polypeptides according to the present invention are provided in high purity. Optionally, the polypeptides may be soluble in aqueous media.

[0073] Preferably, the treatment and / or prophylaxis does not result in side effects or adverse effects in the subject being or to whom the polypeptide is administered. Preferably, administration of the polypeptide of the present invention does not cause any undesired effects in a mammalian subject.

[0074] Certain side effects or adverse effects that are preferably absent in this context are hyperalgesia or pain. It is particularly preferred that the treatment and / or prevention according to the invention does not result in hyperalgesia in the mammalian subject. Thus, preferably, administration of the agent of the invention does not induce any hyperalgesia reaction syndrome (pain).

[0075] It is important to note that the absence of pain does not merely result in a more pleasant (or less unpleasant) treatment than administration of a painful reference compound (such as wild-type NGF), but is at least partially causally related to the success of treating or preventing brain injury, particularly traumatic brain injury or ischemic hypoxic brain injury, including but not limited to hypoxic-ischemic encephalopathy itself. Given that the polypeptide according to the present invention is preferably administered intranasally, the absence of pain allows the subject to accept administration of the polypeptide without adverse reactions, such as scraping, washing off, or otherwise removing the polypeptide to avoid pain, so that the polypeptide exerts a therapeutically beneficial effect, such as treating or preventing brain injury, particularly ischemic brain injury, including but not limited to hypoxic-ischemic encephalopathy. Thus, the absence of pain associated with the polypeptide of the present invention is suitable to overcome the hesitation of consumers' caregivers and the concerns of regulators. In other words, the absence of pain is associated with a significantly increased benefit-risk ratio compared to painful drugs.

[0076] In particular, preferably, the treatment and / or prevention does not cause hyperalgesia in mammalian subjects.In some embodiments, the subject administered with the polypeptide of the present invention does not have mechanical allodynia.More particularly, mechanical allodynia is not induced in the subject administered with the polypeptide, and as a result, the subject administered with the polypeptide does not have mechanical allodynia.

[0077] Typically, the agent of the present invention is well tolerated by the subject to which it is administered.In particular, preferably, administration of the polypeptide of the present invention is not associated with the formation of anti-drug antibodies that neutralize the drug in the subject.In fact, since the amino acid sequence of the polypeptide of the present invention differs from wild-type human NGF at only two amino acid positions, it is reasonable to consider that the immunological tolerance in humans is particularly advantageous, and it is reasonable to consider that administration of the polypeptide of the present invention is not associated with the formation of anti-drug antibodies in humans.

[0078] Preferably, administration according to the present invention positively affects one or more of the following: inflammation, innervation and angiogenesis.

[0079] The pathology according to the present invention is generally characterized by mechanical and / or pathological destruction (e.g., degeneration) of interneuronal connections, and the death of nerve cells, glial cells, and possibly vascular tissue at the site affected by such destruction.Non-limiting examples of pathologies or conditions according to the present invention are constituted by traumatic events involving the central nervous system, such as brain injury or traumatic brain injury (TBI) or pathologies / conditions related thereto, or pathologies / conditions directly derived therefrom, such as hypoxic / ischemic brain injury (HIBI) or hypoxic-ischemic encephalopathy (HIE), preferably especially in neonate patients, infantile cerebral palsy, or other pathologies / conditions caused by mechanical trauma or hypoxic / ischemic encephalopathy.

[0080] Also included are injuries such as brain injury caused by various types of cerebral ischemia including hypoxic / ischemic encephalopathy (primary or secondary injury), birth injury, infantile cerebral palsy, ruptured cerebral aneurysm, cerebral arteriovenous malformation, and cerebral venous sinus thrombosis.

[0081] According to the present invention, pathologies caused by changes in the vascular / circulatory system include, for example, traumatic injury; hemorrhagic injury; atherosclerotic injury; cardiogenic injury; hypoxic-ischemic syndrome, including hypertensive injury.

[0082] In accordance with the present invention, pathologies caused by inflammatory damage due to infectious causes, such as meningitis / encephalitis, whether fungal, bacterial, viral, prion or other etiology, are of interest.

[0083] At the pediatric level, such conditions may be, for example, fetal and / or neonatal hypoxia / asphyxia; major head trauma; acute cerebral hemorrhage, toxicosis (carbon monoxide, cyanide); cerebral venous sinus thrombosis; clotting factor deficiencies; or diseases, including genetic disorders.

[0084] According to the present invention, the polypeptide of SEQ ID NO: 1 can be administered to a subject in need of such administration.The subject in need of such administration can be a subject having a disorder described herein, a subject at risk of having such a disorder, or a subject otherwise suffering from such a disorder.The agent is administered to the subject in a therapeutically effective amount.The therapeutically effective amount can be determined by a physician in light of the disclosure herein.

[0085] In particular, the polypeptides according to the present invention are administered to a mammalian subject. The subject may also be referred to as a "patient." Most preferably, the mammalian subject is a human.

[0086] The subject may be a newborn, defined as a child from birth to 12 weeks of age, while a neonate is defined as a child from birth to 4 weeks of age. Treatment of newborns and neonates is contemplated in the present invention. A premature infant refers to an infant born before the full term of pregnancy, and a premature infant refers to an infant born 3 weeks or more before the full term of pregnancy.

[0087] In the context of the present invention, prevention, such as (partial) reduction of cerebral infarction or other improvement or amelioration of a condition or disorder, is preferably a preferred and integral part of the present invention as claimed herein and is also shown in the experimental examples described herein. In fact, achieving the claimed therapeutic effect is a functional technical feature of the present invention. The examples herein make it convincing that said functional technical feature is achievable as a direct result of administration of the polypeptide of the present invention. In other words, the inventors have identified that the polypeptide of the present invention, especially in combination with hypothermia, is a factor for achieving improvement in subjects with brain damage. The brain damage is preferably characterized by brain injury and / or brain damage or brain dysfunction.

[0088] In particular, administration to the brain may be accomplished by taking advantage of the unique anatomical relationships and functions of the nasal cavity. It has long been recognized that therapeutic agents (including biologics) applied to the cribriform plate region of the superior anterior nasal cavity can reach the central nervous system via the perforations that pass the olfactory nerve from the skull to the nasal cavity. Furthermore, for drugs taken up by neural tissue and transported along axonal projections, auxiliary pathways exist via the olfactory nerve itself and its associates, as well as the nasal branch of the trigeminal nerve, by which therapeutically relevant concentrations of drugs may reach many regions of the brain.

[0089] In the present invention, intranasal administration is preferably performed by a device suitable for topical administration which acts to absorb the drug through the nasal mucosa, preferably by a microparticle formulation having a diameter of 10-50 micrometers.

[0090] An example of a suitable device for intranasal administration of the compositions of the present invention is constituted by a MAD (mucosal atomizer device) type device or a commercially available device that operates in a similar manner. The MAD is attached to a syringe, allowing atomization of the entire drug, ensuring optimal absorption in the nasal mucosa and minimizing diffusion to the external environment and lung structures. Its structure makes the MAD an easy to use and highly effective device.

[0091] Intranasal administration of the polypeptides of the invention by a MAD device is an effective and convenient method to render this active ingredient accessible at the CNS level.

[0092] Thus, devices loaded with the compositions described herein can be used to administer the polypeptides of the present invention to the nasal mucosa and olfactory epithelium for delivery through the olfactory pathway and / or nerve endings towards the brain parenchyma. Thus, devices in combination with drugs can be used in clinical practice to induce neuronal regeneration in the brain, for example, to treat the consequences of hypoxic-ischemic brain injury or disease, and to treat pathologies and conditions that are not currently pharmacologically treatable. The compositions described herein can be administered one or more times per day, with the total amount ranging from 0.01 to 1.00 mg / day per patient.

[0093] Generally, the polypeptides according to the invention will be administered repeatedly or in a single dose.

[0094] In some embodiments, the polypeptide is administered in a single dose. In some embodiments, the polypeptide is administered in a single dose and administration does not continue after the single dose.

[0095] In another embodiment, the polypeptide is administered repeatedly for 1, 2 or 3 to 30 days, preferably 7 to 14 days. Repeated administration for up to 30 days, followed by an appropriate rest period, is also envisaged to allow treatment of chronically progressive or relapsing pathologies.

[0096] In some embodiments, the polypeptide is administered repeatedly. In some embodiments, the polypeptide is administered repeatedly, for example, until brain injury is completely healed, or at least until an improvement in symptoms of injury is observed. Alternatively, the polypeptide is administered repeatedly for a period of 3 to 30 days, preferably 7 to 14 days. Optionally, administration is not continued after said period. In a particularly preferred embodiment, the polypeptide is administered repeatedly at least three times a day.

[0097] Preferably, the polypeptide is administered after brain injury. Alternatively, the polypeptide may be used prophylactically in situations where brain injury is anticipated or is likely (such as neurosurgical intervention or the onset of an inflammatory brain disease).

[0098] Preferably, the polypeptide is administered as soon as possible after the diagnosis of brain injury to minimize the extent of brain cell death. "As soon as possible" includes the embodiment of within one day after the diagnosis of brain injury or damage. However, the polypeptide is neuroprotective even when administered several days after the occurrence of brain injury. Preferably, the polypeptide is administered at least 3 days after the induction of brain injury.

[0099] The agents and compositions described herein are administered in effective amounts.For the treatment of certain disorders, the desired response is preferably related to the inhibition of disease progression.This includes delaying the progression of the disease, and preferably inhibiting or reversing the progression of the disease.The desired response to the treatment of a disease or condition can also include delaying or preventing the onset of said disease or condition.In some embodiments, the desired response includes complete healing of the symptoms of the disorder locally and / or systemically.

[0100] The effective amount of the agent or composition described herein depends on the condition or disorder to be treated, the severity of the disorder, the individual parameters of the subject to which the agent is administered, such as age, physiological condition, concomitant condition (if any), size and weight, duration of treatment, type of concomitant treatment (if any), specific administration route and other parameters.Therefore, the dosage of the agent described herein varies according to such parameters.If the response of the patient to the initial dose is insufficient, a higher dose (or an effective high dose achieved by another, more local administration route) can be used.

[0101] In accordance with the present invention, an appropriate and therapeutically effective dosage of a therapeutic agent for administration to a human subject for the treatment and / or prevention of brain injury, particularly traumatic brain injury, or ischemic hypoxic brain injury, including but not limited to hypoxic-ischemic encephalopathy, can be determined based on an appropriate and therapeutically effective dosage of a therapeutic agent for administration to a rodent subject, particularly a mouse, for the treatment and / or prevention of brain injury, particularly traumatic brain injury, or ischemic hypoxic brain injury, including but not limited to hypoxic-ischemic encephalopathy.

[0102] The animal models (Examples 2 and 3) are useful for establishing the pharmacological response and evaluating the potential toxicity of the treatment. In some embodiments, the dose administered to the subject is the dose described in Example 2 or Example 3.

[0103] Preferably, the dosage of the polypeptide is determined at or before the start of treatment.In some embodiments, the dosage is adjusted for subsequent administrations according to the progression of the condition being treated.In other embodiments, the dosage is not adjusted for subsequent administrations, so that the subsequent dosage corresponds to the initial dosage.

[0104] The polypeptide is active both in nasal and systemic administration. For nasal administration, the preferred dose / each dose per subject has an amount of 0.10-1.00 mg / day of the polypeptide per nostril, preferably 0.06-0.12 mg / day, more preferably about 0.06 mg / day of the polypeptide per nostril. These doses can be administered separately to one or both nostrils, depending on the recipient's preference, tolerance and access to the nostrils, etc. Most preferably, these indicated doses are specifically for administration to humans.

[0105] The following describes the composition comprising the polypeptide of the present invention.Such composition is also part of the present invention in the specific context of its use in the prevention and / or treatment of brain injury, particularly traumatic brain injury, or ischemic hypoxic brain injury, including but not limited to hypoxic-ischemic encephalopathy.Therefore, the present invention relates to the medical use of such composition.

[0106] In the composition according to the invention, the polypeptide of SEQ ID NO: 1 is included as an active ingredient. Further ingredients may also be included.

[0107] In certain embodiments, the polypeptide is contained in an aqueous medium, the aqueous medium being for administration to a mammalian subject.

[0108] A particular aqueous composition for use according to the invention is a liquid composition suitable for intranasal use. The formulation has a concentration of 2 mg / mL, 20 mM acetate buffer, 20 mM methionine, pH 5.5. It is dissolved in 0.9% saline and can be delivered intranasally, preferably at a dose of 20 μg / kg or 40 μg / kg (total volume 4 μL, 2 μL / nostril), alternately to each nostril, preferably with a dosing interval of 1-5 minutes, preferably 2 minutes. Methods for producing such compositions are known in the art.

[0109] In some embodiments, the polypeptide of SEQ ID NO:1 described herein is included in a composition, such as a liquid composition, that further comprises one or more carriers and / or one or more excipients.The term "carrier" as used herein refers to a natural or synthetic organic or inorganic component that is combined with active ingredient to enable, enhance, for example, facilitate the application of active ingredient.The term "excipient" as used herein is intended to refer to any substance that can be present in the pharmaceutical composition of the present invention and is not the active ingredient itself.

[0110] Preferably, the composition of the present invention comprises at least water as an excipient. In some embodiments, the composition of the present invention comprises an aqueous medium, and more preferably, the composition of the present invention is in the form of an aqueous solution. In some embodiments, the polypeptide is comprised in an aqueous medium, and the aqueous medium is administered to a mammalian subject. The aqueous medium is, for example, an aqueous solution.

[0111] Thus, the polypeptide of SEQ ID NO:1 described herein may be present in a composition, for example a pharmaceutical composition. The compositions described herein are preferably sterile and preferably contain the polypeptide of SEQ ID NO:1 as a pharma- ceutically active peptide or protein, and may optionally contain additional agents described or not described herein. The composition may be in any state, for example liquid, frozen, lyophilized, etc.

[0112] The compositions described herein may contain salts, buffer substances, preservatives, carriers, diluents and / or excipients, all of which are preferably pharma- ceutically acceptable. The term "pharmaceutically acceptable" refers to a substance that is non-toxic and / or does not interact with the action of the active ingredients of the pharmaceutical composition.

[0113] Suitable buffers for use in the present invention include 20 mM acetate buffer.

[0114] The compositions (formulations) according to the invention are protected by one or more of the following embodiments, but are not limited thereto: - First embodiment: the formulation can be stored frozen at -70°C or -20°C (data not shown) and thawed prior to administration. - Second embodiment: the formulation can be stored in a refrigerator, preferably at a temperature range of +2 to +8°C (data not shown). - Third embodiment: the formulation can be dried or lyophilized and then stored, for example, at room temperature or at 2-8° C. (data not shown). It can be reconstituted before administration.

[0115] Suitable preservatives for use in compositions according to the invention include those known in the art, including, by way of example and not limitation, benzyl alcohol, benzalkonium and its salts, m-cresol, phenol, chlorobutanol, parabens and thimerosal. These and other preservatives may optionally be included in compositions according to the invention.

[0116] Thus, the present invention provides a polypeptide of SEQ ID NO: 1 for therapeutic use, i.e. for use in a method of treating the human or animal body by therapy. The treatment may include prevention and / or treatment of a condition. In view of the potential therapeutic use, said polypeptide may also be referred to as a pharma- ceutically active protein or peptide.

[0117] Optionally, administration according to the invention may involve administration of at least another therapeutic agent, such as, for example, an agent intended to combat inflammation, edema / swelling, elevated intracranial pressure, infection, seizures, pain, psychological sequelae, etc. The other therapeutic agent may be part of a composition comprising a polypeptide according to the invention or may be administered separately to the subject, to the same or a different site, and by the same or a different route of administration.

[0118] The carrier may be an isotonic, optionally buffered solution, such as saline solution or phosphate buffer, containing, if necessary, preservatives or other suitable excipients suitable for the formulation of a composition for intranasal administration.

[0119] Administration can be performed, for example, once daily, twice daily, three times daily, four times daily, five times daily, six times daily, seven times daily or more, depending on the needs of the patient, with each dose being 0.01 to 1.00 mg / day of CHF6467, as described above.

[0120] In certain embodiments, for example, the composition can be administered two to four times per day, eg, three times per day, at a dose of 0.02-0.04 mg of CHF6467 per administration.

[0121] For example, the composition may be administered at a dose of 0.02 mg of CHF6467 and / or molecule having NGF-like activity per administration.

[0122] The compositions may be administered in any of the methods and dosages described above for one or more treatment cycles.

[0123] Such a treatment cycle may last, for example, one day to two weeks.

[0124] By way of example, the pharmaceutical composition may be provided in the form of a pre-prepared number (e.g., any number between 10 and 20) of aliquots, each of which contains, by way of non-limiting example, 1 ml of physiological solution in which 0.5 to 2.0 mg of CHF6467 is diluted, or any other suitable carrier. Such aliquots may be contained in vials, or in particularly easy-to-use embodiments, may also be contained in pre-dosed syringes suitable for connection to the MAD device. The aliquot embodiments are provided to allow for the administration of doses of 0.01 to 1.00 mg / day of CHF6467. According to a possible embodiment, the composition of the invention contains, for example, a 1 ml aliquot containing 1 mg of CHF6467, this type of formulation being particularly suitable for children weighing around 10 kg. Alternatively, the composition contains, for example, a 1 ml aliquot of a suitable carrier containing 2 mg of CHF6467, for children weighing around 20 kg. With knowledge of the dosages advised herein, preparing appropriate aliquots of the pharmaceutical composition according to body weight ranges will be readily accomplished by one of ordinary skill in the art.

[0125] The possibility of administering drugs intranasally in order to deliver them to the brain, bypassing the blood-brain barrier, was already proposed more than ten years ago and is currently used for the administration of several types of sedatives and anesthetics, the intranasal administration of which allows the dose to be reduced and limited, thus reducing the possible side effects.

[0126] According to a further embodiment, the one or more sub-doses may be pre-formulated for pediatric or adult administration and further divided into single doses for each weight range containing an appropriate amount of CHF6467.

[0127] In another embodiment, the one or more sub-doses are multiple dose sub-doses, such as "daily" or "weekly" sub-doses, which may be further divided into appropriate doses based on the patient's weight.

[0128] As already mentioned above, according to certain embodiments, said one or more devices for administration of the composition may be a MAD (mucosal atomizer device) type device.

[0129] As a non-limiting example, CHF6467 may be in the form of a kit. The kit of the invention may be provided in a number of pre-adjusted aliquots (e.g., any number between 10 and 20), each of which contains, as a non-limiting example, 1 ml of physiological solution in which 0.01-1.00 mg of CHF6467 has been diluted, allowing for the administration of 0.01-1.00 mg / subject or 3-333 μg / kg of CHF6467 for the pre-filled syringes. Such aliquots may already be contained in a sterile syringe, possibly already equipped with a MAD device for intranasal administration of the polypeptide. In a possible embodiment, the kit of the invention may contain, for example, 10 syringes containing 0.01-1 mg of CHF6467 for a neonate weighing around 3 kg. With knowledge of the doses reported herein, adjusting the appropriate pharmaceutical kit for the weight range is easily performed by the skilled artisan. In this way, it is possible to secure intranasal administration of an appropriate amount of CHF6467, which can stimulate the brain receptors of NGF (TrkA and p75) present in most of the serotonergic and cholinergic regions of the brain. Moreover, this kind of kit allows intranasal administration of CHF6467 to be performed at home by the parents of neonates (considering the simplicity and safety of the method), significantly improving the quality of life of neonates.

[0130] Furthermore, the compositions, kits and methods of the present invention may be used not only in patients (including those of pediatric or neonatal age) with neurological deficits caused by major head trauma, but also in all children affected by brain damage caused by hypoxic-ischemic encephalopathy resulting from birth injury, and in patients generally affected by pediatric cerebral palsy and secondary brain injuries and cerebral ischemia of various natures (e.g., ruptured cerebral aneurysm, cerebral arteriovenous malformation and cerebral venous sinus thrombosis).

[0131] In another preferred embodiment of the invention, the neonate is subjected to hypothermia prior to treatment with CHF6467.

[0132] In a preferred embodiment, CHF6467 is administered sequentially with hypothermia.

[0133] In a preferred embodiment, CHF6467 is administered sequentially before or after hypothermia.

[0134] In preferred embodiments, CHF6467 is administered simultaneously with hypothermia, and in certain preferred embodiments of the invention, CHF6467 is administered in a therapeutically effective amount.

[0135] In another preferred embodiment, CHF6467 is administered in a sub-therapeutically effective amount.

[0136] In other words, CHF6467 is administered in an amount insufficient to provide the desired therapeutic effect if administered without hypothermic conditions.

[0137] Even more preferably, the combination of CHF6467 and hypothermia has a synergistic effect, i.e., the combination is synergistic.

[0138] In certain particularly preferred embodiments, CHF6467 is administered prior to the hypoxic / ischemic insult itself, or prior to definitive diagnosis of hypoxic / ischemic insult that is otherwise suspected to have occurred. Thus, in certain preferred embodiments, CHF6467 is administered to the neonate prenatally via the mother, for example by administration to the mother before or during delivery.

[0139] Preferably, CHF6467 is administered up to about 48 or 24 hours prior to birth, more preferably up to about 12 hours prior to birth, more preferably up to about 6 or 3 or 1 hour prior to birth. After birth, the neonate is subjected to hypothermic conditions.

[0140] In another preferred embodiment, CHF6467 is administered to neonates or newborns shortly after birth when there is clinical reason to suspect that clinically significant hypoxic / ischemic injury may occur, but before a diagnosis has been confirmed. This situation is facilitated by the fact that CHF6467 can be administered quickly and easily without interfering with other important clinical activities or interventions. EXAMPLES

[0141] Example 1: Acute Oxygen-Glucose Deprived (OGD) Hippocampal Slices method C57Bl6 / J mice (30–50 days old) were sacrificed by decapitation, and brains were rapidly removed and placed in ice-cold oxygenated (95% O2-5% CO2) artificial CSF (aCSF) (0°C). Parasagittal hippocampal slices (250–350 μM) were cut with a vibratome and allowed to recover in aCSF for 1 h at room temperature. Single slices were placed on a nylon mesh, fully immersed in a recording chamber, and perfused with continuously oxygenated aCSF (3 mL / min, approx. 30°C). Extracellular electric field recordings were performed with a bipolar electrode placed on Schaffer collateral fibers, and evoked excitatory field postsynaptic potentials (fEPSPs) were recorded with aCSF-filled glass microelectrode placed in the CA1 region of the stratum radiatum. Using whole-cell patch-clamp recordings, we assessed the neuroprotective effects of CHF6467 on single-neuron parameters altered by OGD. Specifically, we measured spontaneous excitatory postsynaptic potentials (sEPSCs) and membrane potentials (Vm). We also evaluated the time window of neuroprotective effects of CHF6467 during and after OGD recording excitatory postsynaptic currents (EPSCs). In vitro oxygen / glucose deprivation (OGD) was achieved by perfusing slices with glucose-free CSF and gassing with 95% N2-5% CO2. After OGD, slices were reperfused with CSF containing glucose and oxygen. Hypothermia was achieved by perfusing slices with glucose-containing, oxygenated (95% O2-5% CO2) aCSF (+4°C).

[0142] result We tested the neuroprotective effects of CHF6467 and hypothermia, alone or in combination, in acute mouse hippocampal slices exposed to OGD.

[0143] First, we tested the effect of CHF6467 on basal neurotransmission in the hippocampus. We performed test recordings of fEPSCs for 20 min in control conditions, and then added CHF6467 to the bath solution for 15 min at various concentrations (0.001-0.1 μg / mL). CHF6467 was able to increase the amplitude of fEPSCs at a concentration of 0.1 μg / mL (Figure 1).

[0144] The ability of hippocampal slices to recover synaptic function upon return to normoxia depends on the duration of OGD. In our test conditions, application of ODG for 10 min induced an irreversible fEPSP depression (data not shown). Application of CHF6467 to the bath solution during OGD rescued the fEPSP depression observed in control conditions. Slices treated with CHF6467 during OGD showed a complete recovery of fEPSP amplitude (Figure 2).

[0145] To explore the therapeutic window in an in vitro model, we performed a whole-cell patch clamp study to evaluate the neuroprotective effect of CHF6467 on single neurons. We recorded evoked excitatory postsynaptic potentials (EPSCs), which were irreversibly reduced 15 min after OGD. We observed the neuroprotective effect of CHF6467 when it was applied after OGD (Figure 3A), whereas there was no change in basal neurotransmission when CHF6467 was applied under normal conditions (Figure 3B).

[0146] To investigate the possible synergistic effect between CHF6467, the only treatment currently approved for HIE, and hypothermia, we performed a study in which we applied CHF6467 and hypothermia after OGD. In our test conditions, hypothermia was able to partially restore the decrease in EPSCs induced by OGD (Figure 4). Co-application of CHF6467 during hypothermia was able to completely restore the decrease in EPSCs (Figure 4).

[0147] The in vitro and in vivo doses and administration protocols of CHF6467 were selected in a dose-response study to support potential clinical applications. Maximal attenuation of neuronal damage in vitro was observed at 0.03 μM CHF6467. Surprisingly, the neuroprotective effect of CHF6467 in vitro was dramatically enhanced by hypothermia at 4°C, leading to almost complete recovery of neuronal electrophysiological damage.

[0148] Overall, the present electrophysiological data support the neuroprotective potential of CHF6467 against several functional changes induced by ischemia.

[0149] Given these in vitro results (data not shown) showing that CHF6467 protects against ischemia-mediated excitotoxicity, without being bound by any theory, CHF6467, in combination with hypothermia, may serve as a potential neuroprotective therapy for the treatment of brain injury or traumatic brain injury or ischemic-hypoxic brain injury, including but not limited to hypoxic-ischemic encephalopathy.

[0150] Example 2: Rat model of neonatal hypoxic-ischemic encephalopathy method Animals. Test and animal use procedures were in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals (NIH publication no. 80-23, revised 1996). Study protocols were approved by the Animal Care and Use Committee, Department of Health Sciences, University of Florence, in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS no. 123) and the European Communities Council Directive of November 24, 1986 (86 / 609 / EEC). All efforts were made to minimize the number of animals used and their suffering.

[0151] Procedure. Surgery was performed on 7-day-old Wistar rats (Harlan, MI, Italy) according to the Rice-Vannucci model (Ann Neurol 1981; 9: 131-141). Briefly, pups were anesthetized with isoflurane gas (induction, 4%; maintenance, 1%) in oxygen:nitric oxide (1:1). A midline incision was made and the left common carotid artery was ligated with 4-0 silk. After surgery, all pups were returned to their dams for 1.5 h to recover and nurse. Pups were then placed in an enclosed, ventilated Plexiglas chamber (W20, D20, H17) partially immersed in a water bath and exposed to heated, humidified gas (8% oxygen 92% nitrogen) for 120 min. After injury, pups were returned to their dams for 1 h and then placed in normothermic (37°C) or hypothermic (32°C) chambers for 4 h. CHF6467 was administered intranasally immediately and 24 h after hypoxia at a dose of 20 μg / kg, which had been well tolerated in previous experiments. All pups were then maintained with their dams and sacrificed by decapitation under isoflurane anesthesia 72 h later. To measure infarct area and volume, 1-mm-thick coronal sections were cut with a blade and incubated in 2% 2,3,5-triphenyl-tetrazolium chloride (TTC) for 20 min at 37°C, then fixed in 4% paraformaldehyde for 24 h. Infarct area was measured using a computerized image analysis system (Image-Pro Plus 3.0, Silver Spring, MD, USA). Infarct volumes were measured as previously described (Ann Neurol 1981; 9: 131-141).

[0152] CHF6467 Intranasal Formulation and Administration. After the end of hypoxia, rats were placed in supine position. CHF6467 (batch no. NGF183704-TR1, concentration 2 mg / mL, 20 mM acetate buffer, 20 mM methionine, pH 5.5) was dissolved in 0.9% saline and delivered intranasally at a dose of 20 μg / kg (total volume 4 μL, 2 μL / nostril) alternating between each nostril with a 2 min interval between doses.

[0153] Statistical analysis. Data are presented as the mean ± SEM of the study. The statistical significance of differences between propidium iodide fluorescence intensity or TTC staining was analyzed using two-way ANOVA, Holm-Sidak test for multiple comparisons. All statistical calculations were performed using SigmaPlot (version 11.0). Probability values ​​(P) < 0.05 were considered significant.

[0154] result We tested the neuroprotective effect of CHF6467 alone or in combination with hypothermia in a rat model of neonatal hypoxic-ischemic encephalopathy (HIE) (Rice-Vannucci model; Ann Neurol 1981; 9: 131-141). The study protocol is shown in the figure (Figure 5). In this model, exposure to hypoxia (8% O2 and 92% nitrogen, 2 hours) after left carotid artery occlusion leads to the development of ipsilateral cerebral infarction in approximately 80% of vehicle-treated rats as previously described (Landucci et al., Neurosci Lett. 2018; 668:103-107).

[0155] Observations in the fresh brain hemisphere ipsilateral to the carotid artery ligation included pallor, atrophy, and tissue loss in the striatum, hippocampus, cerebral cortex, and thalamus (data not shown). When brain slices were stained with 2,3,5-triphenyltetrazolium chloride (TTC), we found a volume of 70.4 ± 7.8 mm in the HIE group. 3 Severe cerebral infarction was observed (Figure 6).

[0156] After the end of hypoxia, rats were placed in a supine position. CHF6467 was dissolved in 0.9% saline and delivered intranasally at a dose of 20 μg / kg or 40 μg / kg (total volume 4 μL, 2 μL / nostril) alternately to each nostril with a 2 min interval between doses. Intranasal administration of 20 and 40 μg / kg (equivalent to approximately 60-120 μg for a human neonate weighing 3 kg) of CHF6467 immediately and 24 h after hypoxia significantly reduced the extent of infarction compared to the vehicle group.

[0157] Hypothermia (32°C, 4 h initially and 1 h after termination of hypoxia) significantly reduced the extent of infarct volume compared to normothermic vehicle-treated animals (Figure 6). The combination of 20 μg / kg CHF6467 and hypothermia significantly reduced infarct volume compared to hypothermia alone (Figure 6). Interestingly and surprisingly, the neuroprotection of the combination of hypothermia and CHF6467 superseded the sum of the neuroprotective effects of the single agents.

[0158] The mean percent reduction in mean Log-transformed cerebral infarct volume in the vehicle+normothermic group after the various treatments was as follows: - Normothermia+CHF6467 20μg / kg:-13.6% - Normothermia+CHF6467 40μg / kg:-24.3% - Hypothermia + Vehicle: -13.4% - Hypothermia + CHF6467 20μg / kg: -47.9%.

[0159] Animals were monitored once daily for clinical signs and twice daily for mortality and morbidity.

[0160] Analysis of interaction effects Statistical analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, North Carolina, USA) in the following. Statistical analyses of the interaction effect of CHF6467 and hypothermia were based on log10-transformed data, using an ANOVA model assuming separate variances for each treatment group. Fixed effects of hypothermia (yes / no), CHF6467 (yes / no) and their interaction were included in the model. Data from the group treated with normothermia + CHF6467 40 μg / kg were excluded, as they do not contribute to the evaluation of the interaction effect (the study does not include a hypothermia + CHF6467 40 μg / kg group).

[0161] The interaction between CHF6467 20 μg / kg and hypothermia was statistically significant (p=0.006). This interaction is evident in FIG. 9, which shows the mean log10-transformed cerebral infarct volumes by treatment group. Parallelism would be expected for the additive effects of CHF6467 20 μg / kg and hypothermia (no interaction). The absence of parallelism suggested an interaction between the effects of CHF6467 20 μg / kg and hypothermia. The neuroprotective effect of the combination of CHF6467 20 μg / kg and hypothermia was greater than the sum of the individual effects of CHF6467 and hypothermia.

[0162] Table 1 shows the effect of CHF6467 alone or in combination with hypothermia on body weight. There was no statistically significant difference in weight gain and no adverse clinical signs were observed. Table 1: Effect of CHF6467 alone or in combination with hypothermia on body weight [Table 1] TIFF2024511613000003.tif111161

[0163] Additionally, CHF6467 may be used in combination with hypothermia as a neuroprotectant in neonates with HIE.

[0164] Example 3: Effects of CHF6467 on neuroinflammatory marker expression in a juvenile rat model of hypoxic-ischemic brain injury method Study animal procedures. All procedures and conditions involving study animals were reviewed and approved by the local ethical committee and by the Italian Ministry of Health (authorization number 671 / 2019-PR).

[0165] The experimental procedure was performed as previously described (Rita Levi-Montalcini, 1986). Briefly, 7-day-old Wistar rat pups were anesthetized with isoflurane gas (induction, 4–5%; maintenance, 1%). A midline incision was made and the left common carotid artery was ligated with 4-0 silk. After surgery, all pups were returned to their dam for 1 h to recover and nurse. They were then placed in an enclosed, ventilated Plexiglas chamber (W20, D20, H17) partially immersed in a water bath and exposed to heated, humidified gas (8% oxygen, 92% nitrogen) for 120 min. After injury, pups were returned to their dam for 1 h and then placed in normothermic (37°C) or hypothermic (32°C) chambers for 4 h. CHF6467 was administered intranasally at a dose of 20 μg / kg immediately after hypoxia and 24 hours later, after which all pups were maintained with their dams and sacrificed by decapitation.

[0166] CHF6467 Intranasal Formulation and Administration. After termination of hypoxia, rats were placed in supine position. CHF6467 (batch no. NGF183704-TR1, concentration 2 mg / mL, 20 mM acetate buffer, 20 mM methionine, pH 5.5) was dissolved in 0.9% saline and delivered intranasally at a dose of 20 μg / kg or 40 μg / kg (total volume 4 μL, 2 μL / nostril) alternating to each nostril with a 2 min interval between doses.

[0167] RNA extraction from hippocampal and cortical brain regions. Lysis of samples with phenol / guanidine: For both hippocampus and cortex, 800 μl of QIAzol lysis reagent was used per 30 mg of tissue. Tissue samples were homogenized using beads and a Tissue Lyser (Qiagen) device. After addition of chloroform, the homogenate was centrifuged and separated into aqueous and organic phases. The upper aqueous layer was extracted with a QiaCube Connect automated system (Qiagen). To remove gDNA, a protocol including a DNase step was chosen. RNA samples were eluted in RNase-free water and stored at -80 °C until the reverse transcription step.

[0168] The concentration of the RNA samples was determined and adjusted with a Nanodrop (ThermoFisher) analyzer to transcribe 1000 ng of RNA per RT reaction for each sample. Additional ezDNase was added prior to the RT reaction to remove gDNA. TM The enzymatic step was carried out. The RT reaction was carried out according to the thermocycling conditions suggested by the manufacturer.

[0169] A panel of neuroinflammatory markers. cDNA samples were tested on custom-designed array plates (Taqman gene array, Thermofisher) containing primers and probes for target and housekeeping genes.

[0170] The following genes were targeted during the study (Thermofisher catalog numbers in parentheses): Housekeeping genes: 18s rRNA (Hs99999901_s1), B2m (Rn00560865_m1), Tbp (Rn01455648_m1); Target genes: Ccl2 (Rn00580555_m1), Ccl22 (Rn01536591_m1), Ccl12 (Rn01464638_m1), Tnf (Rn00562055_m1). , Cxcl2(Rn00586403_m1), Ccl7(Rn01467286_m1), Ccr5(Rn02132969_s1), Ccl20(Rn00 570287_m1), Il6(Rn01410330_m1), Cxcl10(Rn01413889_g1), Ccl6(Rn01456400_m1).

[0171] TaqMan® Fast Advanced Master Mix (Applied Biosystems) and diluted cDNA were added to the plate; PCR reactions were performed according to the temperature cycling conditions suggested by the manufacturer. Results were calculated using the 2-ΔΔCt method based on the amplification of a housekeeping gene.

[0172] Statistical analysis was performed with Graph Prism software; one-way ANOVA test followed by Dunnett's or Tukey's test for comparison was applied.

[0173] result A significant increase in the mRNA levels of a panel of cytokines and chemokines in the hippocampus and frontoparietal cortex was measured in rats subjected to hypoxic-ischemic injury compared to sham-operated rats (Figures 7 and 8).CHF6467 (20 μg / kg, intranasal) significantly inhibited the upregulation of several neuroinflammatory markers (ccl2, cxcl2, TNF-α, IL-6, ccl20) in the hippocampal region of rats subjected to hypoxic-ischemic injury, while hypothermia itself significantly inhibited ccl2 upregulation, but not as effectively as CHF6467 (Figure 7).

[0174] In the cortex, CHF6467 (20 μg / kg) inhibited the upregulation of several neuroinflammatory markers induced by hypoxic-ischemic injury (although only CXCL2 reached statistical significance), and such effects were enhanced for several neuroinflammatory markers (CCL7, CCL2, IL-6, TNF-α, CXCL2) by combination with hypothermia (Figure 8).

[0175] Furthermore, the combination of CHF6467 and hypothermia enhanced the effects of single treatments on ccl2, ccl7, TNF-α and IL-6, and CHF6467 may exert a major regulatory effect on the immunoinflammatory response caused by hypoxia-ischemia in the immature rodent brain, and such effects are not redundant when combined with hypothermia.

[0176] Example 4: Quantification of CHF6467 in rat brain method Peptide selection and synthesis of heavy-labeled standards Four peptides exhibiting different specificities for CHF6467 and / or mature rat NGF were selected (Table 2) and their corresponding AQUA (absolute quantification) homologues were synthesized at Thermo Scientific. [Table 2]

[0177] Test animal procedures All procedures and conditions involving test animals were reviewed and approved by the local ethical committee and approved by the Italian Ministry of Health (authorization number 671 / 2019-PR). Seven-day-old Wistar rat pups were anesthetized with isoflurane gas (induction, 4–5%; maintenance, 1%) as previously described (Landucci et al., 2018, Neurosci. Lett. 668. doi:10.1016 / j.neulet.2018.01.023.). A midline incision was made and the left common carotid artery was ligated using 4-0 silk. After surgery, all pups were returned to their dam for 1 h of recovery and nursing. The pups were then placed in enclosed, ventilated Plexiglas chambers (W20, D20, H17) partially submerged in a water bath and exposed to heated, humidified gas (8% oxygen, 92% nitrogen) for 120 min. After injury, pups were returned to their dams for 1 h and then placed in normothermic (37°C) or hypothermic (32°C) chambers for 4 h. CHF6467 was administered intranasally at a dose of 40 μg / kg immediately after the end of hypoxia. Additionally, animals that had not received hypoxic-ischemic injury were administered 40 μg / kg intranasally.

[0178] CHF6467 Intranasal Formulation and Administration After the end of hypoxia, rats were placed in a supine position. CHF6467 (2 mg / mL, 20 mM acetate buffer, 20 mM methionine, pH 5.5) was dissolved in 0.9% saline and delivered intranasally at a dose of 20 or 40 μg / kg (total volume of 4 μL, 2 μL / nostril) alternately to each nostril with a 2 min interval between doses.

[0179] Protein extraction from hippocampal and cortical brain regions Thawed brain samples were cooled on ice and extraction buffer was added at a ratio of 10:1 (v / w) (50 mM ABC + 2% SDS + protease inhibitor cocktail, diluted 500x). Tissue homogenization was performed for 2 min at 30 / s using a TissueLyser II bead mill. Samples were then kept at 70°C for 5 min, cooled at room temperature, and treated with 25 U / mL benzonase for 15 min. Protein content was quantified by BCA assay.

[0180] Protein Digestion Brain homogenates are reduced (5 mM DTT, 95°C for 5 min) and alkylated (15 mM IAA, 24°C for 45 min in the dark). Detergent is removed from the sample buffer using a Pierce spin column. 20% ACN is then added to the digestion buffer, followed by trypsin at 1:20 (w / w) and incubation at 37°C for 5 h, followed by further trypsin at 1:20 (w / w) and incubation at 37°C overnight. All samples are titrated with 1.5 ng / g AQUA peptides, after which the reaction is quenched with 0.1% TFA.

[0181] Sample fractionation The digested sample is fractionated by size exclusion chromatography on an AKTA Pure 25M system equipped with a Superdex 30 Increase 3.2 / 300 column and an F9-R collector using a linear gradient of 30% ACN, 0.1% TFA.

[0182] CHF6467 detection and quantification Selected SEC fractions were collected, dried in a SpeedVac, redissolved in 3% ACN, 0.1% TFA, and then run on an Orbitrap Fusion TM Lumos TM Tribrid TM UltiMate with Mass Spectrometer TMNanoLC-MS analysis is performed with a 3000 RSLC Nano system. The nanoLC is operated in preconcentration mode and separation is performed with an EASY-Spray PepMap RSLC C18 column, 50 cm x 75 μm. The mobile phase gradient is 5-40% solution B in 100 min, solution B is 80% ACN and 0.1% FA. Mass spectrometry consists of full-scan and timed PRM to detect the CHF6467 peptides of interest and their corresponding heavy-labeled peptides. A temporal calibration curve was generated to quantify the levels of CHF6467 signal in the brain matrix. An eight-point concentration range between 0.09-12 ng / g was first tested for the analysis of tissues from control animals, followed by an eight-point concentration range between 0.06-13.5 ng / g prior to the analysis of HIE animals.

[0183] result By sequence analysis of natural mature human and rat NGF isoforms and CHF6467 variants, we identified long homology regions, and also some unique tryptic peptides (Table 2). Considering the animal model of this study, we selected two peptides that are proteotypes of CHF6467 variants: SSSHPIFHR (m / z 534.265) and QAAWEFIR (m / z 510.759). We also monitored the contribution of a peptide that may report both exogenous and endogenous isoforms: QYFFETK (m / z 481.727, SEQ ID NO: 4). Finally, we identified a rat-selective peptide, ALTTDDK (m / z 382.188, SEQ ID NO: 5), specifically reporting potential endogenous responses.

[0184] We preliminarily tested the distribution of CHF6467 after intranasal administration to control juvenile rats. This proof of principle was performed in a small number of samples: 3 olfactory bulbs, 2 hippocampi and 2 cerebral cortices. CHF6467 was detected in all samples, proving its ability to penetrate the brain regions of interest. Quantitative measurements (Figure 10) performed based on the SSSHPIFHR (SEQ ID NO: 2) peptide signal showed that on average several hundred picograms could be found in the cortical regions and hippocampus.

[0185] We followed up this study by confirming the degree of brain penetration of CHF6467 administered intranasally to juvenile rats undergoing hypoxic-ischemic injury (Landucci et al., 2018 Neurosci. Lett. 668. doi:10.1016 / j.neulet.2018.01.023.). Cerebral cortex and hippocampus from six animals were analyzed, comparing ipsilateral and contralateral tissue. With the exception of three ipsilateral hippocampi that were lost during sample preparation due to tube cracks, CHF6467 was detected in all samples analyzed, with high reproducibility between sample replicates within the same region (Figure 11). Based on all three peptides selected for quantification, the amount of CHF6467 detected was considered to be outside the quantification range tested (0.06-13.5 ng / g), suggesting levels below 60 pg / g within this group of animals undergoing hypoxic-ischemic injury.

[0186] This study demonstrated that CHF6467 could indeed enter the brain after intranasal administration, reach the hippocampus and cerebral cortex, and exert neuroprotective and anti-neuroinflammatory effects.

[0187] Mass spectrometry is an important tool for specifically detecting and quantifying proteins in complex matrices. Overall, the data show that intranasal administration of CHF6467 results in detectable levels of the protein in relevant brain regions, particularly the hippocampus and cortex.

Claims

1. A medicament comprising a polypeptide of SEQ ID NO: 1 for use in the treatment and / or prevention of traumatic brain injury or ischemic hypoxic brain injury in a mammalian subject, the medicament being used in combination with hypothermia.

2. The method of claim 1, wherein the polypeptide is administered at a dose of 20 μg / kg.

3. The pharmaceutical composition according to claim 1 or 2, wherein the traumatic brain injury is hypoxic-ischemic encephalopathy.

4. 4. The medicament according to any one of claims 1 to 3, wherein the mammalian subject is a human, preferably the subject is a neonate or newborn subject or a premature newborn subject, preferably the neonate or newborn subject is affected by moderate to severe hypoxic-ischemic encephalopathy.

5. The pharmaceutical composition of claim 1, which is administered intranasally or repeatedly, preferably for a period of 1 to 30 days, preferably 2 to 14 days.

6. A pharmaceutical composition described in any one of claims 1 to 5, which is administered repeatedly 1 to 7 times daily or administered to one or both nostrils.

7. The method according to any one of claims 1 to 6, wherein the polypeptide is administered at a dose of 0.01 to 1.00 mg / day.

8. The method of claim 7, wherein the polypeptide is administered at a dose of 0.06 to 0.12 mg / day.

9. The method of claim 7, wherein the polypeptide is administered at a dose of 0.06 mg / day.

10. A pharmaceutical composition according to any one of claims 1 to 9, which is administered repeatedly, each dose of the polypeptide being 0.01 to 1.00 mg / day.

11. The method of claim 10, wherein the dose of each of the polypeptides is 0.06 to 0.12 mg / day.

12. The pharmaceutical composition of claim 10, wherein the dose of each of the polypeptides is 0.06 mg / day.

13. A pharmaceutical agent described in any one of claims 1 to 12, which is administered within approximately 1 hour to 24 hours after brain injury.

14. The pharmaceutical composition of any one of claims 1 to 13, further comprising acetate buffer and / or methionine.

15. A pharmaceutical agent described in any one of claims 1 to 14, administered simultaneously with hypothermia and / or within 24 hours after hypothermia.

16. The medicament according to any one of claims 1 to 15, wherein the hypothermia is initiated within 1 to 10 hours after the hypoxic-ischemic (HI) injury, preferably the hypothermia is initiated within 2 to 8 hours after the hypoxic-ischemic (HI) injury, preferably the hypothermia is initiated within 4 to 6 hours after the hypoxic-ischemic (HI) injury.

17. The method of any one of claims 1 to 16, wherein the hypothermia is maintained for about 1 to about 72 hours after the hypoxic-ischemic (HI) injury.

18. The method of any one of claims 1 to 17, wherein the body temperature of the mammal is maintained at a temperature of about 32°C to about 36°C.

19. The method of claim 18, wherein the body temperature of the mammal is maintained at a temperature of about 32°C to about 35°C.

20. The method of any one of claims 1 to 19, wherein the polypeptide is administered at a dose of 0.06 to 0.12 mg / day and the body temperature of the mammal is maintained at a temperature of about 32°C to about 35°C.

21. The method of any one of claims 1 to 20, wherein the polypeptide is administered at a dose of 20 µg / kg and the body temperature of the mammal is maintained at a temperature of about 32°C to about 35°C.

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