Combination therapy 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 a combination therapy for use in the treatment and prevention of ischemic and / or hypoxic brain injury, including brain injury, particularly but not limited to, hypoxic-ischemic encephalopathy, preferably in term or preterm neonates, relating to a combination therapy.

Background Art

[0002] Neuroproliferation factors (NGF or hNGF) are neuroactive proteins whose discovery was awarded the Nobel Prize in Medicine (1986, Rita Levi-Montalcini). For decades, their therapeutic potential has been widely studied, particularly in chronic / degenerative diseases. Recently, recombinant human NGF (rhNGF) ophthalmic formulations have been approved by the EMA and FDA for the treatment of neurotrophic keratitis, a rare eye disease. There has been little research on the neuroprotective effects of hNGF in acute lesions of the central nervous system (CNS). Several scientific publications have shown that deficiencies in the production of endogenous neurotrophic factors may contribute to determining the severity of long-term neurological impairment after neonatal hypoxic-ischemic injury (Wang et al., Int J Neurosci 2013; 123: 191-195), and suggest that administering them during the acute phase of the lesion is beneficial (Acta Cir Bras 2017; 32: 270-279, Int J Clin Exp Med 2013; 6: 951-955). In particular, NGF administration limits deficiencies in the development and maturation of complex behaviors assessed in early childhood. Unfortunately, hNGF also has potent hyperalgesic effects (Nat Med 1995; 1: 774-780). (Wild-type) NGF is known to affect target cells by binding to two distinct receptors: (a) receptor tyrosine kinase A (TrkA), which promotes neuronal survival, and (b) the p75 neurotrophin receptor, which is involved in the regulation of cell death (Mesentier-Louro et al., 2017, Int. J. Mol. Sci., vol. 18(98)). Therefore, the polypeptide sequence of wild-type NGF after optic nerve contusion may also have the effect of exacerbating 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 in wild-type human NGF is involved in the binding of NGF to p75, and that mutations in this residue affect binding to p75; see, for example, International Publication No. 2008 / 006893A1.In contrast to wild-type NGF, the polypeptide hNGF of the present invention 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 Sequence ID No. 1, also named CHF6467 (SEQ ID No. 1).

[0003] Hypoxic-ischemic encephalopathy (HIE), or neonatal asphyxia, is a leading cause of severe neurological complications and perinatal mortality. In Western Europe and the United States, its incidence is estimated at 1.5–2 cases per 1,000 live births, with a high prevalence in premature infants (Pediatr Res 2013; 74, Suppl 1, 50-72). Neuronal death following HIE occurs via autophagy mediated by programmed cell death mechanisms, or most likely, through sequential apoptotic-necrotic cell death (Ann Neurol 2011; 69: 743-758), which involves the release of excitotoxic glutamate, activation of NMDA and AMPA receptors, and intracellular Ca2+ 450. 2+ It is caused by a series of events including overload, mitochondrial dysfunction, oxidative stress, caspase activation, and inflammation (Lancet Neurol 2011; 10: 372-382).

[0004] Undoubtedly, brain injury following HI injury is a complex process involving multiple contributing mechanisms and pathways, resulting in both early and delayed injury (Patel, S. et al. (2014). Biochem. Soc. Trans. 42, 564-568). Therefore, there is an urgent need for novel interventions to enhance neuroprotection in addition to therapeutic hypothermia, the current standard treatment (Davidson, JO, et al., (2015). Front. Neurol. 6.). Furthermore, there is evidence that therapeutic hypothermia is more effective in moderate hypoxic-ischemic encephalopathy than in severe hypoxic-ischemic encephalopathy (Sabir, H., et al., (2012). Stroke 43, 3364-3370). There is a growing recognition that one of the main pathogenic factors of neonatal brain injury is inflammation induced by the activation of the central and peripheral immune systems (Hagberg et al., (2015). Nat. Rev. Neurol. 11, 192-208). Immune responses can be triggered within minutes and can persist 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). Neuroproliferation factor (NGF) has been shown to protect certain neurons expressing its signaling receptor, trkA, from a variety of injuries (Holtzman et al., 1995 J. Neurosci. 15). In particular, there is data suggesting that NGF has neuroprotective effects on cells other than nerve cells during brain development (Fodelianaki et al., 2019 Exp. Cell Res. 377).In fact, NGF has been shown to have neuroprotective effects in animal models of neonatal hypoxic-ischemic encephalopathy, suggesting it is a promising substance 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 a standard treatment for improving survival rates and reducing neurodevelopmental sequelae in neonates with moderate to severe hypoxic-ischemic encephalopathy (HIE). Selective head or whole-body cooling initiated within a 6-hour postnatal treatment window, maintaining a core temperature of 33.5°C for approximately 72 hours, improves survival rates and neurological outcomes in neonates with HIE (J Pediatr 2015; 91: S78-S83). While there are relatively few preclinical studies comparing multiple hypothermia treatment temperatures, recent studies in neonatal rat models of HIE suggest that cooling below 33.5°C does not provide further neuroprotection, and therefore, relatively milder cooling should be considered in future clinical trials (Sci Rep 2016; 6: 23430). Hypothermia therapy is not associated with major complications, but is considered beneficial only in infants with a relatively small percentage of HIE (Int J Mol. Sci 2015; 16: 22368-22401). Specifically, therapeutic hypothermia is currently reserved for full-term or near-term newborns (>1800g) and is usually limited to reference centers with specific expertise and equipment.

[0006] International Publication No. 2018087656 relates to a pharmaceutical composition comprising NGF or a molecule having NGF-like activity for use in the treatment of traumatic brain injury, particularly traumatic brain injury or ischemic hypoxic brain injury, including but not limited to hypoxic-ischemic encephalopathy, administered intranasally.

[0007] Hou et al. (Chinese Journal of Primary Medicine and Pharmacy 2017;24(3):338-341) reported that combining head hypothermia with mouse neurogenic factor therapy provides protective effects for the treatment of moderate to severe HIE in neonates. As mentioned above, the use of hypothermia in the treatment of neonatal asphyxia is extensively documented in prior literature (see, e.g., Volpe, 2001; Gunn et al, 2000). However, to date, there has been no teaching or suggestion in the art that hypothermia can be used in combination with the administration of mutant NGF, CHF6467. Nor has there been any suggestion that such a combination therapy could result in such a surprising and unexpected enhancement of the neuroprotective effect obtained.

[0008] Therefore, there is still a need for medications to treat HIE. In particular, medications that can enhance the effects of hypothermia can add extremely high value to current clinical practice. [Overview of the Initiative]

[0009] CHF6467 (hNGFp, Sequence ID No. 1) has neurotrophic activity similar to wild-type human nerve growth factor (NGF), but its pain sensitization activity is 10 times 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 the polypeptide of SEQ ID NO: 1 and a hypothermia method for use in the treatment and / or prevention of traumatic brain injury or ischemic hypoxic brain injury in mammals.

[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 a neonat or newborn or premature neonat, and preferably the neonat or newborn subject is suffering from moderate to severe hypoxic-ischemic encephalopathy.

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

[0015] In a preferred embodiment, the polypeptide is administered repeatedly 1 to 7 times per day, or the polypeptide is administered into 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 0.06 to 0.12 mg / day, and preferably 0.06 mg / day.

[0017] Preferably, the dose of 0.06 mg / day is for a neonat or newborn with a body weight of approximately 3 kg.

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

[0019] The preferred dose is approximately 20 μg / kg to approximately 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 to 1.00 mg / day, preferably each dose of the polypeptide is 0.06 to 0.12 mg / day, and preferably each dose of the polypeptide is 0.06 mg / day.

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

[0022] In a preferred embodiment, the polypeptide is included in a composition containing acetate buffer and / or methionine.

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

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

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

[0026] Preferably, the hypothermia method is maintained for at least about 4 hours, more preferably at least about 6 hours 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 a therapeutically effective amount of CHF6467 to the mammalian mother before and / or during childbirth; and (b) Subjecting mammals to hypothermia after birth. Regarding the treatment of neonatal asphyxia in mammals requiring medical intervention.

[0029] Hypothermia can occur passively by allowing body temperature to drop rather than intentionally maintaining it. Because newborns' body temperature is easily affected, it can quickly become as warm as the surrounding temperature. Alternatively, hypothermia can be actively induced by intentionally lowering the ambient temperature.

[0030] Further aspects of the present invention include: (a) administering a therapeutically effective dose of CHF6467 to a mammal; and (b) Exposing mammals to hypothermia or hypothermic conditions Regarding the treatment of neonatal asphyxia in mammals requiring medical intervention.

[0031] In a preferred embodiment, the mammals are newborns within four weeks of birth. More preferably, the mammals are within two weeks of birth, and even more preferably, within one week of birth.

[0032] Preferably, the mammal is a human.

[0033] A preferred embodiment of the second aspect of the present invention is the same as the above-described embodiment of the first aspect.

[0034] Another aspect of the present invention relates to the treatment of neonatal asphyxia in mammals by administering a therapeutically effective dose of xenon in combination with hypothermia to mammals requiring treatment.

[0035] A further aspect of the present invention relates to the use of CHF6467 in the manufacture of a medicament for the treatment of neonatal asphyxia, wherein the treatment comprises administering CHF6467 to the subject simultaneously, sequentially, or separately in combination with a hypothermia method.

[0036] A further aspect of the present invention relates to the use of a combination of CHF6467 and hypothermia for treating neonatal asphyxia.

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

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

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

[0040] [Figure 1] Effect of CHF6467 on baseline transmission at CA1 hippocampal synapses [Figure 2] CHF6467 restored the reduction induced by OGD (as a preventative measure). Normalized pooled data demonstrate the effects of OGD on excitatory postsynaptic potentials (fEPSP) and the protective effect of CHF6467 perfused at different concentrations (0.03 and 0.1 μg / mL) during OGD. [Figure 3] OGD-induced EPSC reduction is restored by CHF6467 perfusion (therapeutic measure). Panel A shows the time course of normalized EPSC data after treatment with OGD alone (black circles) and OGD + CHF6467 (white circles). The histogram shows the mean normalized EPSC data in the last 10 minutes 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 minutes of OGD. [Figure 5] Diagram of the experimental protocol for a rat model of neonatal hypoxic-ischemic encephalopathy. TTC = 2,3,5-triphenyltetrazolium chloride. [Figure 6] Effects of intranasal administration of CHF6467 (20 and 40 μg / kg) alone and in combination with hypothermia on logarithmically transformed cerebral infarct volume in young rats 72 hours after hypoxic-ischemic injury at 10 days of age. The number of animals is shown in each column. [Figure 7] Hippocampal mRNA levels of a group of rat cytokines and chemokines in simulated-treated, vehicle-treated hypoxic-ischemic encephalopathy (HIE), HIE treated with CHF6467, HIE treated with 20 μg / kg (HIE + CHF 20 μg / ml), HIE treated by 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] Cytokine and chemokine cortical mRNA levels in rat populations treated with sham therapy, 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). Sprays are shown with mean ± standard error (SEM). **p<0.01 and *p<0.05 vs. HIE. [Figure 9] Statistical analysis of the mean cerebral infarction volume after log10 transformation for each treatment group. [Figure 10] Brain penetration of CHF6467 after intranasal administration in control juvenile rats was determined by quantitative analysis of the SSSHPIFHR (SEQ ID NO: 2) peptide. [Figure 11]Brain penetration of CHF6467 after intranasal administration in young rats with hypoxic-ischemic injury. Light-to-weight 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. [Modes for carrying out the invention]

[0041] Detailed description of the invention The neuroprotective effects of CH6467 or the polypeptide SEQ ID NO: 1 were tested using in vitro and in vivo experimental models of hypoxic-ischemic encephalopathy (HIE). In oxygen-glucose deprived (OGD) acute hippocampal slices, 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 abnormal EPSCs (Figure 4). In neonatal rats hypoxicized after permanent occlusion of the left common carotid artery, an established model of neonatal hypoxic-ischemic encephalopathy, intranasal administration of CHF6467 (20 and 40 μg / kg) significantly reduced cerebral infarct volume compared to vehicles treated. Surprisingly, the combination of CHF6467 and hypothermia dramatically reduced cerebral infarct volume by up to 86% compared to vehicles treated at normal temperature. Unexpectedly, the neuroprotective effect of combining CHF6467 with hypothermia was significantly higher than that of monotherapy (CHF6467 or hypothermia) alone, demonstrating a synergistic effect (Figure 6). In particular, the effect of CHF6467 at the preferred in vivo dose (20 μg / kg) was synergistic with hypothermia in providing neuroprotective effects. No deaths, clinical signs, or weight loss were observed with CHF6467 treatment, suggesting high tolerance to CHF6467.

[0042] Therefore, the present invention provides the combined use 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 combined use of CHF6467 and hypothermia, when appropriate dosages are taken into consideration, can significantly reduce mortality and severe neurodevelopmental disorders in infants and neonates with moderate to severe hypoxic-ischemic encephalopathy compared to hypothermia alone, as reported in the experimental section below.

[0043] Furthermore, a group of neuroinflammatory markers in the cortex and hippocampus of pup rats induced with hypoxic-ischemic brain injury were also tested, either with CHF6467 alone or in combination with hypothermia.

[0044] Significant increases in mRNA levels of several cytokines and chemokines in the hippocampus and frontoparietal cortex were measured in hypoxic-ischemic injured rats compared to sham-treated rats. CHF6467 (20 μg / kg, intranasal) significantly counteracted the upmodulation of several neuroinflammatory markers in the hippocampal region of hypoxic-ischemic injured rats, although hypothermia itself was not as effective as CHF6467. In the cortex, CHF6467 (20 μg / kg) itself was effective in counteracting the upmodulation of several neuroinflammatory markers induced by hypoxic-ischemic injury, and this effect was enhanced in some cases by combination with hypothermia.

[0045] Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, and announcements) is incorporated herein by whole reference, whether as described above or below. Nothing in this specification shall be construed as acknowledging that the present invention has no prior rights to any particular teaching, and / or that any particular document other than common general knowledge contains information that is sufficiently clear and complete for a person skilled in the art to implement.

[0046] The expression "and / or," for example "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 the meanings of "and," "or," and both ("and" or "or").

[0047] Unless otherwise indicated, the terms “about,” “ca.” and “substantially” all mean “approximately” or “nearly,” and in the context of numbers or ranges described herein, preferably ±10%, more preferably ±5% of the stated or claimed number 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 there may be additional elements beyond the list introduced by “comprising.” However, it is intended that in certain embodiments of the invention, the term “comprising” encompasses the possibility that no additional elements exist, i.e., “comprising” is understood to mean “consisting of.”

[0049] Unless otherwise specified, relative quantities relating to the present invention are expressed on a weight / weight basis. The expression of a relative quantity of a component characterized by a collective term means that it represents the total quantity of all specific variants or members included in the aforementioned collective term. If a specific component defined by a collective term is present in a specific relative quantity, and that component is further characterized as being a specific variant or member included in its variant, then it means that there are no additional other variants or members included in that variant such that the total relative quantity of the component included in that variant exceeds the specific relative quantity; more preferably, it means that there are no specific variants or members included in that variant at all.

[0050] Unless otherwise specified in the context, the term "neuronal 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 “NGF mutein,” or “its mutein” 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 further detail herein. The polypeptide of Sequence ID No. 1 is NGF mutein. It refers to any species, preferably a mammalian species, but human NGF mutein is always preferred. Preferably, NGF mutein has 80–99.5% sequence identity with NGF, particularly human NGF, and more preferably, mutein has 90–99% sequence identity with NGF, particularly human NGF.

[0052] As used herein, the term "hypothermia" refers to subjecting a specific subject (e.g., a neonatal subject) to hypothermic conditions by passive or active techniques, for example, by lowering their body temperature, preferably by 3 to 5°C. Preferred hypothermia temperatures are 32°C to 36°C, preferably 32°C to 35°C, and preferably 32°C or 33°C.

[0053] As used herein, the term “simultaneously” is used to mean that CHF6467 is administered at the same time as the hypothermia method, while the term “in combination” is used to mean that CHF6467 is administered “sequentially” thereafter, if not simultaneously, within a time frame in which both CHF6467 and the hypothermia method exhibit therapeutic effects, i.e., within a time frame in which both are available to act therapeutically within the same time frame. Therefore, when a neonatal subject is exposed to hypothermic conditions, “sequential” administration may occur within 5 minutes, 10 minutes, or several hours before or after the hypothermia method, provided that the circulating half-life or target tissue residence time of CHF6467 is such that CHF6467 is present in a therapeutically effective dose.

[0054] The terms “patient” and “subject” are used interchangeably herein, and are used in particular in reference to patients / subjects characterized by the eye disorders described herein.

[0055] In the context of this invention, the term "prevent" is understood in a broad sense, including not only the initiation of prevention of brain injury but also the prevention of the progression of brain injury. In particular, in the context of injuries including brain injury and / or brain impairment, the term "prevent" also includes preventing the further progression of brain injury.

[0056] In the context of this invention, the term "treatment" is understood in a broad sense and is not limited to that, but includes improvement of the symptoms of the disorder.

[0057] According to the present invention, "effective amount" is an amount or dose that achieves the desired reaction or desired effect, either as a single dose or in combination with further doses.

[0058] The term "pharmaceutically acceptable" generally indicates that a particular substance can be administered to a subject, sometimes and preferably in combination with a drug, without causing an adverse effect that would be unacceptable to the drug at the dosage in which it is used.

[0059] The terms “pharmaceutically acceptable carrier” and “pharmaceutically acceptable excipient” are used to indicate one or more solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarders, etc., that are physiologically compatible and suitable for administration to the subjects described herein, or that do not interfere with such administration. Such pharmaceutically 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. In particular for liquid pharmaceutical compositions, it may be preferable to include an isotonic agent in the composition, such as sugar, polyalcohols such as mannitol or sorbitol, or sodium chloride.

[0060] A pharmaceutically acceptable carrier may further contain auxiliary substances such as wetting agents or emulsifiers, preservatives or buffers that enhance the shelf life or efficacy of the drug. A pharmaceutically acceptable carrier is typically included in the composition according to the present invention.

[0061] The term “pharmaceutically active agent” refers to an agent that can be administered to a subject if it is beneficial to improve the symptoms of a disease or disorder. Furthermore, a “pharmaceutically active agent” may have a positive or beneficial effect on the subject’s condition or disease state when administered to the subject in a therapeutically effective dose. Preferably, a pharmaceutically active agent has therapeutic properties and may be administered to improve, alleviate, reduce, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. A pharmaceutically active agent may have preventive properties and may 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 pharmaceutically active ingredient for the treatment of cystic fibrosis, as claimed. In another example, a pharmaceutically active protein may be used to treat cells or individuals that do not normally express a protein, or do not express it at a desired level, or do not express a protein accurately. For example, a pharmaceutically active protein may compensate for mutations or a lack of sufficiently high expression by supplying the desired protein. The term "pharmaceutically active peptide or protein" includes the entire protein or polypeptide, and may also refer to its pharmaceutically active fragment. It also includes analogues of pharmaceutically active peptides or proteins.

[0062] As used herein, the terms “subject” and “patient” refer to mammals. For example, mammals in the context of the present invention include humans, non-human primates, domestic animals (but not limited to dogs, cats, sheep, cattle, goats, pigs, horses, etc.), laboratory animals (but not limited to mice, rats, rabbits, etc.), and captured animals such as zoo animals. As used herein, the terms “subject” and “patient” particularly include humans. A subject (human or animal) has two pairs of chromosomes; that is, the subject is diploid. The term “patient” means a subject that has, is at risk of having, has, or is expected to have a certain condition, and a subject that can be subjected to treatment, such as the administration of a drug. A patient’s condition can be chronic and / or acute. Accordingly, “patient” may be described as a subject that is subjected to treatment and / or requires treatment.

[0063] The term "treatment" is understood in a broad sense to mean the treatment of an object with the goal of preventing or treating a condition in that object. In a preferred embodiment, treatment specifically includes the administration of a drug to the object.

[0064] The agents according to the present invention, also referred to herein as “sequence number 1 polypeptide,” will be described in more detail. The terms “sequence number 1 polypeptide” and similar terms refer herein to a polypeptide comprising the amino acid sequence defined in sequence number 1 and / or an agent having equivalent biological activity. Accordingly, 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 sequence number 1 polypeptide that contains a binding site enabling it to exert substantially the same biological activity as the full-length sequence number 1 polypeptide, or a gene encoding such a polypeptide. The term “substantially identical biological activity” means 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 sequence number 1 polypeptide in the assays described in Examples 1 to 3. An example of a biologically equivalent analog of a polypeptide could be a fusion protein containing at least a portion of the amino acid sequence of the polypeptide of SEQ ID NO: 1, but it could also be a homologous analog of the polypeptide. Furthermore, a complete synthetic molecule that mimics the specific biological activity of the polypeptide of SEQ ID NO: 1 is considered to constitute a "biologically equivalent analog."

[0065] More preferably, the term “polypeptide of SEQ ID NO: 1” and similar terms refer herein to a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 1; such a drug may optionally be a fusion protein comprising, in particular, the amino acid sequence defined in SEQ ID NO: 1. Preferably, the term “polypeptide of SEQ ID NO: 1” and similar terms refer herein to a polypeptide consisting of the amino acid sequence defined in SEQ ID NO: 1; in this embodiment, the drug consists of a polypeptide comprising 118 amino acid residues in the order defined in SEQ ID NO: 1. In this embodiment and other embodiments, the polypeptide may optionally have one, two, or three cysteine ​​bonds such that cysteine ​​(Cys,C) residues are covalently bonded to each other to form intramolecular disulfide crosslinks. The cysteine ​​bonds are preferably equivalent to those in wild-type human NGF.

[0066] The polypeptides of the present invention may optionally be characterized by further post-translational modifications. Such post-translational modifications may optionally include glycosylation and / or phosphorylation. However, preferably, the polypeptides of the present invention are not glycosylated and / or phosphorylated. Indeed, the test examples herein demonstrate beneficial effects and beneficial-to-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), and considering that the polypeptides used therein were typically obtained by cytoplasmic recombination expression in bacteria that do not result in glycosylation and / or phosphorylation, it is reasonable to consider that the beneficial effects of the present invention are independent of such types of post-translational modifications.

[0067] Typically, the polypeptides according to the present invention are non-natural polypeptides that are not spontaneously produced by the subjects to whom the polypeptide is administered.

[0068] 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 (e.g., des-nona variants) and proteases (e.g., trypsin). When the polypeptide according to the present invention is essentially free of host cell proteins, degradation products (e.g., des-nona variants) and proteases (e.g., trypsin), it may also be referred to as a “pure polypeptide”. Preferably, the polypeptide according to the present invention is administered as a pure polypeptide. More preferably, the pure polypeptide comprising 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, and more preferably 99.9% or more, based on the total protein in the composition. Most preferably, the pure polypeptide according to the present invention has a purity that conforms to Good Manufacturing Practices (GMP).

[0069] The polypeptide comprising Sequence ID No. 1 differs from the amino acid sequence of human neuron growth factor (NGF, also referred to as wild-type human NGF or wild-type NGF) in two locations. The differences 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, have a remarkable effect in treating or preventing brain injury, particularly traumatic brain injury or ischemic hypoxic brain injury, including but not limited to hypoxic-ischemic encephalopathy, and have no side effects.

[0070] Among several variants of human NGF (hNGF variants), the variant human NGF P61SR100E is considered the most promising for the treatment of brain injury. However, specific uses of the treatment and / or prevention according to the present invention are not shown in the literature, and the literature also does not show that each polypeptide is available in a purity sufficient to enable its medical use in humans. Therefore, the polypeptides for use according to the present invention described and provided herein offer several unexpected advantages over wild-type human NGF. Administration of the agents according to the present invention causes a reduction in cerebral infarct volume (see, for example, Example 2), and said administration is painless.

[0071] The polypeptide of Sequence ID No. 1 does not exist in nature and may also be referred to as a non-natural polypeptide. The drug according to the present invention is not wild-type NGF, and in particular is not wild-type human NGF.

[0072] Preferably, the non-natural polypeptide according to the present invention is provided in high purity. Optionally, the polypeptide may be soluble in an aqueous medium.

[0073] Preferably, the treatment and / or prevention does not result in side effects or adverse effects in subjects receiving or having received the polypeptide. Preferably, the administration of the polypeptide of the present invention does not cause any undesirable effects in mammalian subjects.

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

[0075] It is important to note that the absence of pain not only results in a more comfortable (or less unpleasant) treatment than the administration of a painful reference compound (such as wild-type NGF), but also has at least a causal relationship to the success of treating or preventing ischemic hypoxic brain injury, including, but not limited to, traumatic brain injury or hypoxic-ischemic encephalopathy itself. Given that the polypeptide according to the present invention is preferably administered intranasally, the absence of pain allows subjects to accept the administration of the polypeptide without adverse reactions such as rubbing, washing, or otherwise removing the polypeptide to avoid pain, and as a result, the polypeptide exerts therapeutically beneficial effects, such as treating or preventing ischemic brain injury, including, but not limited to, hypoxic-ischemic encephalopathy. Therefore, the absence of pain associated with the polypeptide of the present invention is suitable for overcoming consumer caregiver hesitation and regulatory concerns. In other words, the absence of pain is associated with a significantly increased benefit-risk ratio compared to pain-inducing drugs.

[0076] In particular, preferably, the treatment and / or prevention does not result in hyperalgesia in mammalian subjects. In one embodiment, subjects administered with the polypeptide of the present invention do not have mechanical allodynia. More specifically, mechanical allodynia is not induced in subjects administered with the polypeptide, and as a result, subjects administered with the polypeptide do not have mechanical allodynia.

[0077] Typically, the drug of the present invention is well tolerated by the target population. In particular, preferably, administration of the polypeptide according to the present invention does not involve the formation of anti-drug antibodies that neutralize the drug in the target population. Indeed, since the amino acid sequence of the polypeptide according to the present invention differs from that of wild-type human NGF by only two amino acid positions, it is reasonable to assume that immunological tolerance in humans is particularly advantageous, and it is reasonable to assume that administration of the polypeptide according to the present invention does not involve the formation of anti-drug antibodies in humans.

[0078] Preferably, the administration according to the present invention has a positive effect on one or more of the following: inflammation, innervation, and angiogenesis.

[0079] The pathological conditions according to the present invention are generally characterized by mechanical and / or pathological destruction (e.g., degeneration) of intercellular junctions of nerve cells, and the death of nerve cells, glial cells, and occasionally vascular tissue in the site affected by such destruction. Non-limiting examples of pathological conditions or states according to the present invention include traumatic events involving the central nervous system, such as brain injury or traumatic brain injury (TBI) or conditions / states associated therewith, or conditions / states directly arising therefrom, such as hypoxic / ischemic brain injury (HIBI) or hypoxic-ischemic encephalopathy (HIE) in neonatal patients, cerebral palsy in children, or other conditions / states caused by mechanical trauma or hypoxic / ischemic encephalopathy.

[0080] This also includes brain injuries caused by various types of cerebral ischemia, such as hypoxic / ischemic encephalopathy (primary or secondary injury), birth injuries, cerebral palsy in children, ruptured cerebral aneurysms, arteriovenous malformations, and cerebral venous sinus thrombosis.

[0081] According to the present invention, pathological conditions caused by changes in the blood vessels / circulatory system include, for example, traumatic injuries; hemorrhagic injuries; atherosclerotic injuries; cardiogenic injuries; and hypoxic-ischemic syndromes, including hypertensive injuries.

[0082] According to the present invention, the target is a pathological condition caused by inflammatory injury due to infectious causes such as meningitis / encephalitis, regardless of whether the cause is fungi, bacteria, viruses, prions, or other pathogens.

[0083] At the pediatric level, such conditions include, for example, fetal and / or neonatal hypoxia / asphyxia; severe head trauma; acute cerebral hemorrhage; poisoning (carbon monoxide, cyanide); cerebral venous sinus thrombosis; coagulation factor deficiencies; or genetic disorders.

[0084] According to the present invention, the polypeptide of SEQ ID NO: 1 can be administered to subjects requiring such administration. Subjects requiring such administration may be subjects having the disorders described herein, subjects at risk of having such disorders, or subjects otherwise suffering from such disorders. The drug is administered to the subject in a therapeutically effective dose. The therapeutically effective dose may be determined by a physician in light of the disclosures herein.

[0085] In particular, the polypeptide according to the present invention is administered to mammalian subjects. The subjects may also be referred to as "patients." Most preferably, the mammalian subject is human.

[0086] The subjects may be newborns, defined as children from birth up to 12 weeks of age, while neonates are defined as children from birth up to 4 weeks of age. Treatment of newborns and neonates is considered in this invention. A premature infant is a child born before the end of pregnancy, and a premature infant is a child born more than 3 weeks before the end of pregnancy.

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

[0088] In particular, administration to the brain can be carried out by utilizing the unique anatomical relationships and functions of the nasal cavity. It has long been recognized that therapeutic substances (including biological agents) applied to the cribriform plate region of the upper anterior nasal cavity can reach the central nervous system via perforations that allow the olfactory nerve to pass from the skull through the nasal cavity. Furthermore, for drugs taken up by nerve tissue and transported along axonal projections, there are auxiliary pathways via the olfactory nerve itself and its related pathways, as well as via the nasal branch of the trigeminal nerve, which allow therapeutically appropriate concentrations of the drug to reach many areas of the brain.

[0089] In the present invention, intranasal administration is preferably carried out by a device suitable for local administration that acts to absorb the drug via the nasal mucosa, and preferably by a particulate formulation having a diameter of 10 to 50 micrometers.

[0090] Examples of devices suitable for intranasal administration of the compositions of the present invention consist of MAD (mucosal atomizer device) type devices or commercially available devices that operate in a similar manner. By attaching the MAD to a syringe, the entire drug can be atomized, ensuring optimal absorption in the nasal mucosa and minimizing diffusion into the external environment and lung structure. Due to its structure, the MAD is an easy-to-use and highly effective device.

[0091] Intranasal administration of the polypeptide of the present invention using a MAD device is an effective and readily available method that allows access to this active ingredient at the CNS level.

[0092] Accordingly, devices filled with the compositions described herein may be used to administer the polypeptides of the present invention to the nasal mucosa and olfactory epithelium for the purpose of delivery to the brain parenchyma via the olfactory pathway and / or nerve endings. Accordingly, devices combined with drugs may be used in clinical practice for the purpose of inducing neuronal regeneration in the brain, for example, to treat the consequences of hypoxic-ischemic brain injury or disease, and to treat conditions or states that are currently not pharmacologically treatable. The compositions described herein may be administered once or more times daily, with a total dose ranging from 0.01 to 1.00 mg / day per patient.

[0093] Generally, the polypeptide according to the present invention is administered repeatedly or as a single dose.

[0094] In one embodiment, the polypeptide is administered as a single dose. In this embodiment, the polypeptide is administered as a single dose, and administration is not continued 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 after an appropriate rest period is also envisioned to enable the treatment of chronically progressive or relapsing conditions.

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

[0097] Preferably, polypeptides are administered after brain injury. Alternatively, polypeptides may be used prophylactically in situations where brain injury is anticipated or possible (such as neurosurgical intervention or the onset of 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 embodiments administered within one day after the diagnosis of brain injury or injury. However, polypeptides also have neuroprotective effects when administered several days after the onset of brain injury. Preferably, polypeptides are administered at least three days after the induction of brain injury.

[0099] The agents and compositions described herein are administered in effective amounts. In the case of treating a particular disorder, the desired response is preferably related to inhibiting the course of the disease. This includes delaying the progression of the disease, and preferably inhibiting or regressing its progression. The desired response to the treatment of a disease or condition may also include delaying or preventing the onset of the disease or condition. In some embodiments, the desired response includes completely curing the symptoms of the disorder, both locally and / or systemically.

[0100] The effective dose of the drugs or compositions described herein depends on the condition or disorder being treated, the severity of the disorder, individual parameters of the person receiving the drug, such as age, physiological condition, any associated conditions, body size and weight, duration of treatment, type of associated treatment (if any), specific route of administration, and other parameters. Therefore, the dosage of the drugs described herein will vary depending on these parameters. If the patient's response to the initial dose is insufficient, a higher dose (or an effective higher dose achieved by another, more localized route of administration) may be used.

[0101] According to the present invention, an appropriate and therapeutically effective dose 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, may be determined based on an appropriate and therapeutically effective dose 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 pharmacological responses and evaluating the potential toxicity of the treatment agents. In some embodiments, the dose administered to the subjects is the dose described in Example 2 or Example 3.

[0103] Preferably, the polypeptide dose is determined at or before the start of treatment. In one embodiment, the dose is adjusted for subsequent doses as the condition being treated progresses. In another embodiment, the dose is not adjusted for subsequent doses, and as a result, subsequent doses correspond to the initial dose.

[0104] Polypeptides are active both intranasally and systemically. For intranasal administration, preferred doses per subject / each dose are 0.10–1.00 mg / day, preferably 0.06–0.12 mg / day, of polypeptide per nostril, more preferably about 0.06 mg / day per nostril. These doses may be administered separately to one nostril or both nostrils, depending on the recipient's preference, tolerance, and access to the nostrils. Most preferably, these indicated doses are specifically for administration to humans.

[0105] The following describes compositions comprising the polypeptides of the present invention. Such compositions are also part of the present invention in the specific context of 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. Accordingly, the present invention relates to the medical use of such compositions.

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

[0107] In one embodiment, the polypeptide is contained in an aqueous medium, which is intended for administration to a mammalian subject.

[0108] A specific aqueous composition for use according to the present 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, and pH 5.5. It is dissolved in 0.9% saline and can be delivered intranasally, preferably in doses of 20 μg / kg or 40 μg / kg (total volume 4 μL, 2 μL / nostril), alternately into each nostril, preferably with an administration interval of 1 to 5 minutes, preferably 2 minutes. Methods for preparing such compositions are known in the art.

[0109] In some embodiments, the polypeptide of Sequence ID No. 1 described herein is contained in a composition, for example, a liquid composition, comprising one or more further carriers and / or one or more excipients. As used herein, “carrier” means a natural or synthetic organic or inorganic component that is combined with an active ingredient to enable, enhance, or, for example, simplify the application of the active ingredient. As used herein, “excipient” is intended to refer to all substances that may be present in the pharmaceutical composition of the present invention and that are not the active ingredient itself.

[0110] Preferably, the composition of the present invention contains 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 contained in an aqueous medium, and the aqueous medium is administered to a mammalian subject. The aqueous medium is, for example, an aqueous solution.

[0111] Therefore, the polypeptide of SEQ ID NO: 1 described herein may be present in a composition, for example, a pharmaceutical composition. The composition described herein preferably comprises the polypeptide of SEQ ID NO: 1 as a sterile and preferably pharmaceutically active peptide or protein, and may optionally contain further agents described herein or not. The composition may be in any state, such as liquid, frozen, or lyophilized.

[0112] The compositions described herein may include salts, buffers, preservatives, carriers, diluents, and / or excipients, all of which are preferably pharmaceutically acceptable. The term “pharmaceutically acceptable” means a substance that is non-toxic and / or does not interact with the action of the active ingredient of the pharmaceutical composition.

[0113] A suitable buffering material used in the present invention includes 20 mM acetate buffer.

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

[0115] Suitable preservatives for use in compositions according to the present invention include those known in the art, and, in particular, but are not limited to, benzyl alcohol, benzalkonium and its salts, m-cresol, phenol, chlorobutanol, parabens, and thimerosal. These and other preservatives may be included in compositions according to the present invention as appropriate.

[0116] Accordingly, the present invention provides the polypeptide of SEQ ID NO: 1 for therapeutic use, i.e., for use in methods of treating the body of a human or animal by therapy. Therapy may include prevention and / or treatment of a condition. In terms of potential therapeutic use, the polypeptide may also be referred to as a pharmaceutically active protein or peptide.

[0117] Depending on the circumstances, administration according to the present invention may be accompanied by the administration of at least another therapeutic agent, such as an agent intended to counteract inflammation, edema / swelling, increased intracranial pressure, infection, seizures, pain, or psychological sequelae. The other therapeutic agent may be part of a composition comprising the polypeptide according to the present invention, or it may be administered separately to the subject at the same or different sites, via the same or different routes of administration.

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

[0119] The drug may be administered once, twice, three, four, five, six, seven or more times a day, depending on the patient's needs, with each dose being 0.01 to 1.00 mg / day of CHF6467 as described above.

[0120] In one embodiment, for example, the composition may be administered two to four times a day, for example three times a day, at a dose of 0.02 to 0.04 mg of CHF6467 per dose.

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

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

[0123] Such treatment cycles can last, for example, from one day to two weeks.

[0124] For example, the pharmaceutical composition may be provided in the form of a pre-adjusted number of fractional amounts (e.g., any number from 10 to 20), each containing, in a non-limiting example, 1 ml of a diluted physiological solution or any other suitable carrier containing 0.5 to 2.0 mg of CHF6467. Such fractional amounts may be contained in vials or, in particularly user-friendly embodiments, also in pre-dosed syringes suitable for connection to a MAD device. Embodiments of fractional amounts are provided to enable administration of CHF6467 at doses of 0.01 to 1.00 mg / day. By possible embodiments, the composition of the present invention may contain, for example, 1 ml of fractional amount containing 1 mg of CHF6467, and this type of formulation is particularly suitable for children weighing about 10 kg. Alternatively, the composition may contain 1 ml of fractional amount of a suitable carrier containing 2 mg of CHF6467 for children weighing, for example, about 20 kg. With the knowledge of dosages advised herein, it is readily apparent to those skilled in the art that appropriate fractional amounts of a pharmaceutical composition can be prepared based on body weight ranges.

[0125] The possibility of administering drugs intranasally to bypass the blood-brain barrier and deliver them to the brain was proposed more than 10 years ago and is currently used for administering several types of sedatives and anesthetics. Intranasal administration allows for dose reduction and limitation, and therefore reduces the potential for side effects.

[0126] In further embodiments, the one or more divided doses may be pre-adjusted for administration to children or adults and further divided into single doses per body weight range containing an appropriate amount of CHF6467.

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

[0128] As described above, in one embodiment, the one or more devices for administering the composition may be MAD (mucosal atomizer device) type devices.

[0129] As a non-limiting example, CHF6467 may be in the form of a kit. The kit of the present invention may be provided in a number of pre-adjusted fractions (e.g., any number from 10 to 20), each containing, as a non-limiting example, 1 ml of a diluted physiological solution containing 0.01 to 1.00 mg of CHF6467, and for pre-filled syringes, it is possible to administer 0.01 to 1.00 mg / subject or 3 to 333 μg / kg of CHF6467. Such fractions may be already contained in sterile syringes that are already equipped with a MAD device for intranasal administration of polypeptides. In a possible embodiment, the kit of the present invention may contain, for example, 10 syringes containing 0.01 to 1 mg of CHF6467 for a neonatal patient weighing approximately 3 kg. With the knowledge of doses reported herein, it is readily possible for those skilled in the art to adapt appropriate pharmaceutical kits for different weight ranges. In this manner, it is possible to protect the intranasal administration of an appropriate amount of CHF6467 that can stimulate NGF receptors (TrkA and p75) present in most of the brain's serotonergic and cholinergic regions. Furthermore, this type of kit allows for the intranasal administration of CHF6467 at home by the parents of neonates (given 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 with neurological defects caused by major head trauma (including patients of pediatric or neonatal age), but also in all children affected by brain injury 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 aneurysms, arteriovenous malformations, and cerebral venous sinus thrombosis).

[0131] In another preferred embodiment of the present invention, neonates are subjected to hypothermia before treatment with CHF6467.

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

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

[0134] In a preferred embodiment, CHF6467 is administered simultaneously with hypothermia, and in a preferred embodiment of the present invention, CHF6467 is administered in a therapeutically effective dose.

[0135] In another preferred embodiment, CHF6467 is administered in a dose below a therapeutically effective dose.

[0136] In other words, CHF6467, if administered without hypothermia, is given in insufficient amounts to produce the desired therapeutic effect.

[0137] More preferably, the combined use of CHF6467 and hypothermia has a synergistic effect; in other words, the combination is synergistic.

[0138] In one particularly preferred embodiment, CHF6467 is administered before the hypoxic / ischemic injury itself, or otherwise before a definitive diagnosis of the suspected hypoxic / ischemic injury. Thus, in one preferred embodiment, CHF6467 is administered to the neonatal infant via the mother, for example, by administration to the mother before or during labor.

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

[0140] In another preferred embodiment, CHF6467 is administered to a neonat or newborn immediately after birth for clinical reasons, such as the suspected possibility of clinically significant hypoxic / ischemic injury, but before a definitive diagnosis has been made. This situation is facilitated by the fact that CHF6467 can be administered rapidly and easily without interfering with other important clinical activities or interventions. [Examples]

[0141] Example 1: Acute hippocampal slices in oxygen-glucose deficient (OGD) method C57Bl6 / J mice (30-50 days old) were decapitated and sacrificed, their 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 at room temperature for 1 hour. A single slice was placed on a nylon mesh, fully immersed in a recording chamber, and perfused with aCSF with continuous oxygen supply (3 mL / min, approximately 30°C). Extracellular electric field recording was performed using bipolar electrodes placed on Schaefer collateral fibers, and excitatory postsynaptic field potentials (fEPSPs) were induced using glass microelectrodes filled with aCSF placed in the CA1 region of the radiation layer. Using whole-cell patch-clamp recording, the inventors evaluated the neuroprotective effects of CHF6467 on single neuron parameters altered by OGD. Specifically, the inventors measured spontaneous excitatory postsynaptic potentials (sEPSCs) and membrane potentials (Vm). The inventors also evaluated the time window of the neuroprotective effect 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 gas-purging with 95% N2-5% CO2. After OGD, slices were reperfused with glucose- and oxygen-containing CSF. Hypothermia was achieved by perfusing slices with glucose-containing, oxygenated (95% O2-5% CO2) aCSF (+4°C).

[0142] result The inventors tested the neuroprotective effects of CHF6467 alone or in combination with hypothermia therapy in acute hippocampal slices of mice exposed to OGD.

[0143] First, the effect of CHF6467 on basal neurotransmission in the hippocampus was tested. The inventors recorded fEPSCs for 20 minutes under control conditions, and then added CHF6467 to the bath solution at various concentrations (0.001 to 0.1 μg / mL) for 15 minutes. 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 restore synaptic function upon return to normal oxygen conditions depends on the duration of oxygen-induced gastric drainage (OGD). Under our test conditions, applying 10 minutes of OGD induced an irreversible decrease in fEPSP (data not shown). Applying CHF6467 to the bath during OGD restored the fEPSP decrease observed under control conditions. Slices treated with CHF6467 during OGD showed complete recovery of fEPSP amplitude (Figure 2).

[0145] To investigate the therapeutic window in an in vitro model, a whole-cell patch-clamp test was performed to evaluate the neuroprotective effect of CHF6467 on a single neuron. The inventors recorded induced excitatory postsynaptic potentials (EPSCs) that were irreversibly reduced 15 minutes after oral gastrointestinal dilation (OGD). The inventors observed a neuroprotective effect when CHF6467 was applied after OGD (Figure 3A), while there was no change in basal neurotransmission when CHF6467 was applied under normal conditions (Figure 3B).

[0146] To investigate the potential synergistic effect between CHF6467, currently the only approved treatment for HIE, and hypothermia, the inventors conducted a study applying CHF6467 and hypothermia after oral gastrointestinal disease (OGD). Under the inventors' test conditions, hypothermia was able to partially restore the reduction in EPSCs induced by OGD (Figure 4). By co-applying CHF6467 during hypothermia, the reduction in EPSCs was completely restored (Figure 4).

[0147] In vitro and in vivo doses and administration protocols for CHF6467 were selected in dose-response studies to support potential clinical applications. Maximum attenuation of neuronal damage in vitro was observed with 0.03 μM CHF6467. Surprisingly, the neuroprotective effect of CHF6467 in vitro was dramatically enhanced by 4°C hypothermia, leading to near-complete recovery of electrophysiological damage to neurons.

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

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

[0150] Example 2: A rat model of neonatal hypoxic-ischemic encephalopathy. method Animals. The experiments and methods of animal use followed the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1996) of the National Institutes of Health. The experiment protocol was approved by the Animal Experimentation Committee of the Faculty 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: Following the Rice-Vannucci model (Ann Neurol 1981; 9: 131-141), surgical intervention was performed on 7-day-old Wistar rats (Harlan, MI, Italy). Briefly, the pups were anesthetized with isoflurane gas in oxygen:nitric oxide (1:1) (induction, 4%; maintenance, 1%). A midline incision was made, and the left common carotid artery was ligated using 4-0 silk. Postoperatively, all pups were returned to their mothers for 1.5 hours for recovery and nursing. Subsequently, the pups were placed in a surrounded, breathable plexiglass chamber (W20, D20, H17) partially submerged in a water bath and exposed to heated, humidified gas (8% oxygen, 92% nitrogen) for 120 minutes. After injury, the pup mice were returned to their mothers for 1 hour, and then placed in a chamber at normal body temperature (37°C) or hypothermia (32°C) for 4 hours. CHF6467 was administered intranasally at a dose of 20 μg / kg, which had been shown to be well-tolerated in previous experiments, immediately after hypoxia and 24 hours later. Subsequently, all pup rats were maintained with their mothers and sacrificed by decapitation under isoflurane anesthesia after 72 hours. To measure infarct area and volume, 1 mm thick coronal sections were cut with a blade, incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37°C for 20 minutes, and then fixed in 4% paraformaldehyde for 24 hours. Infarct area was measured using a computer image analysis system (Image-Pro Plus 3.0, Silver Spring, Maryland, USA). The infarct volume was measured as previously described (Ann Neurol 1981; 9: 131-141).

[0152] CHF6467 intranasal preparation and administration. After the end of the hypoxic state, the rats were placed in a supine position. CHF6467 (batch number NGF183704-TR1, concentration 2 mg / mL, 20 mM acetate buffer, 20 mM methionine, pH 5.5) was dissolved in 0.9% saline and administered intranasally to each nostril alternately at a dose of 20 μg / kg (total volume 4 μL, 2 μL / nostril) with an administration interval of 2 minutes.

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

[0154] result The inventors investigated the neuroprotective effects 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 test protocol is shown in Figure 5. In this model, exposure to hypoxia (8% O2 and 92% nitrogen, 2 hours) after left carotid artery occlusion resulted in ipsilateral cerebral infarction in approximately 80% of rats administered via vehicle, as previously described (Landucci et al., Neurosci Lett. 2018; 668:103-107).

[0155] Observations of fresh cerebral hemispheres on the ipsilateral side after 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), the inventors found that in the HIE group, the volume was 70.4 ± 7.8 mm. 3 Significant cerebral infarction was observed (Figure 6).

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

[0157] Hypothermia (32°C, 4 hours initially, followed by 1 hour after the end of the hypoxic state) significantly reduced the severity of the condition compared to animals treated with vehicle therapy at normal body temperature (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 neuroprotective effect of the combination of hypothermia and CHF6467 surpassed the combined neuroprotective effect of any single agent.

[0158] The mean log-converted percentage reduction in cerebral infarction volume in the vehicle + normal body temperature group after various treatments was as follows: - Normothermia+CHF6467 20μg / kg:-13.6% - Normothermia+CHF6467 40μg / kg:-24.3% - Hypothermia method + vehicle: -13.4% - Hypothermia + CHF6467 20μg / kg: -47.9%.

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

[0160] Analysis of interaction effects Statistical analysis was performed using SAS version 9.4 (SAS Institute Inc., Cayley, North Carolina, USA). Statistical analysis of the interaction effect between CHF6467 and hypothermia was based on log10 transformed data using an ANOVA model assuming separate variances for each treatment group. Fixed effects of hypothermia (with / without), CHF6467 (with / without), and their interaction were included in the model. Data from the group treated with normal body temperature + CHF6467 40 μg / kg were excluded because they did not contribute to the evaluation of the interaction effect (this study did 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 Figure 9, which shows the mean log10-converted stroke volume for each treatment group. For an additive effect (no interaction) between CHF6467 20 μg / kg and hypothermia, parallel lines are predicted. The absence of parallelism suggests an interaction between the effects of CHF6467 20 μg / kg and hypothermia. The neuroprotective effect of combining CHF6467 20 μg / kg and hypothermia was greater than the sum of the individual effects of CHF6467 and hypothermia.

[0162] Table 1 shows the effects 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: Effects of CHF6467 alone or in combination with hypothermia on body weight [Table 1] TIFF0007902193000003.tif111161

[0163] Furthermore, CHF6467 may be used as a neuroprotective agent in combination with hypothermia in neonates with HIE.

[0164] Example 3: Effect of CHF6467 on the expression of neuroinflammatory markers in a young rat model of hypoxic-ischemic brain injury. method Animal Testing Procedures. All procedures and conditions, including those involving animal testing, have been reviewed and approved by the local ethics committee and approved by the Italian Ministry of Health (Approval No. 671 / 2019-PR).

[0165] The experimental procedure was carried out as previously described (1986, Rita Levi-Montalcini). Briefly, seven-day-old Wistar rat pups were anesthetized with isoflurane gas (induction, 4-5%; maintenance, 1%). The midline was incised, and the left common carotid artery was ligated with 4-0 silk. After surgery, all pups were returned to their mothers for one hour for recovery and nursing. Subsequently, the pups were placed in a surrounded, breathable plexiglass chamber (W20, D20, H17) partially submerged in a water bath and exposed to heated, humidified gas (8% oxygen, 92% nitrogen) for 120 minutes. After injury, the pups were returned to their mothers for one hour, and then placed in a chamber at normal body temperature (37°C) or hypothermia (32°C) for four hours. CHF6467 was administered intranasally at a dose of 20 μg / kg immediately after and 24 hours after hypoxia. Subsequently, all the offspring rats were kept together with their mothers and sacrificed by decapitation.

[0166] CHF6467 intranasal preparation and administration. After the end of the hypoxic state, rats were placed in a supine position. CHF6467 (batch number NGF183704-TR1, concentration 2 mg / mL, 20 mM acetate buffer, 20 mM methionine, pH 5.5) was dissolved in 0.9% saline and administered intranasally to each nostril alternately at doses of 20 μg / kg or 40 μg / kg (total volume 4 μL, 2 μL / nostril) with a 2-minute administration interval.

[0167] RNA extraction from hippocampal and cortical brain regions. Phenol / guanidine lysis of samples: For both hippocampal and cortical samples, 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 adding chloroform, the homogenate was centrifuged to separate the aqueous and organic phases. The upper aqueous layer was extracted using a QiaCube Connect automated system (Qiagen). A protocol including a DNase step was selected to remove gDNA. RNA samples were eluted in RNase-free water and stored at -80°C until the reverse transcription step.

[0168] RNA sample enrichment was determined and prepared using a Nanodrop (ThermoFisher) analyzer to transcribe 1000 ng of RNA in a single RT reaction for each sample. Further ezDNase treatment was performed before the RT reaction to remove gDNA. TM The enzymatic process was carried out. The RT reaction was performed according to the temperature cycling conditions provided by the manufacturer.

[0169] A group 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] During the experiment, the following genes were targeted (Thermofisher catalog numbers are 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 plates; PCR reactions were performed according to the temperature cycling conditions indicated by the manufacturer. Results were calculated using the 2-ΔΔCt method based on the amplification of housekeeping genes.

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

[0173] result Significant increases in mRNA levels of a group of cytokines and chemokines in the hippocampus and frontoparietal cortex were measured in hypoxic-ischemic injured rats compared to sham-treated 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 hypoxic-ischemic injured rats, 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 upmodulation of several neuroinflammatory markers induced by hypoxic-ischemic injury (however, only cxcl2 reached statistical significance), and this effect was enhanced in combination with hypothermia for several neuroinflammatory markers (ccl7, ccl2, IL-6, TNF-α, cxcl2) (Figure 8).

[0175] Furthermore, the combined use of CHF6467 with hypothermia enhanced the effects of monotherapy on ccl2, ccl7, TNF-α, and IL-6. CHF6467 may exert a major modulatory effect on the immunoinflammatory response induced by hypoxic-ischemia in the brains of immature rodents, and this effect does not overlap when combined with hypothermia.

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

[0177] Procedures for test animals All procedures and conditions, including those involving test animals, were reviewed and approved by the local ethics committee and approved by the Italian Ministry of Health (Approval No. 671 / 2019-PR). As previously described (Landucci et al., 2018, Neurosci. Lett. 668. doi:10.1016 / j.neulet.2018.01.023.), 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 using 4-0 silk. Postoperatively, all pups were returned to their mothers for 1 hour of recovery and nursing. Subsequently, the rat pups were placed in a ventilated, enclosed plexiglass chamber (W20, D20, H17) partially submerged in a water bath and exposed to heated, humidified gas (8% oxygen, 92% nitrogen) for 120 minutes. After injury, the pups were returned to their mothers for 1 hour, and then placed in a chamber at normal body temperature (37°C) or hypothermia (32°C) for 4 hours. CHF6467 was administered intranasally at a dose of 40 μg / kg immediately after the end of the hypoxic state. Furthermore, 40 μg / kg intranasal administration was given to the group of animals that had not suffered hypoxic-ischemic injury.

[0178] CHF6467 intranasal preparation and administration After the end of the hypoxic state, the rats were placed in a supine position. CHF6467 (concentration 2 mg / mL, 20 mM acetate buffer, 20 mM methionine, pH 5.5) was dissolved in 0.9% saline and administered intranasally to each nostril alternately at doses of 20 μg / kg or 40 μg / kg (total volume 4 μL, 2 μL / nostril) with a 2-minute interval between doses.

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

[0180] Protein digestion Brain homogenate is reduced (5 mM DTT, 95°C for 5 minutes) and alkylated (15 mM IAA, 24°C in the dark for 45 minutes). The surfactant is removed from the sample buffer using a Pierce spin column. Then, 20% ACN is added to the digestion buffer, followed by trypsin at a ratio of 1:20 (w / w), incubated at 37°C for 5 hours, and then trypsin is added again at a ratio of 1:20 (w / w), incubated overnight at 37°C. 1.5 ng / g of AQUA peptide is added dropwise to all samples, and the reaction is quenched with 0.1% TFA.

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

[0182] CHF6467 detection and quantification The selected SEC fraction was collected, dried in SpeedVac, redissolved in 3% ACN and 0.1% TFA, and then subjected to Orbitrap Fusion. TM Lumos TM Pibrid TM UltiMate with Mass Spectrometer TMNanoLC-MS analysis was performed using a 3000 RSLC nanosystem. The nanoLC was operated in pre-enrichment mode, and separation was performed using an EASY-Spray PepMap RSLC C18 column, 50 cm × 75 μm. The mobile phase gradient was 5–40% solution B for 100 minutes, with solution B being 80% ACN and 0.1% FA. Mass spectrometry consisted of a full-scan, time-specified PRM to detect the CHF6467 peptide of interest and its corresponding heavily labeled peptide. A transient calibration curve was created to quantify the level of CHF6467 signaling in brain matrix. Eight concentration ranges between 0.09–12 ng / g were first tested for tissue analysis from control animals, and then eight concentration ranges between 0.06–13.5 ng / g were tested before analysis of HIE animals.

[0183] result Sequence analysis of naturally occurring mature human and rat NGF isoforms and CHF6467 variants allowed the inventors to identify long homology regions and several unique trypsin peptides (Table 2). Considering the animal model of this study, the inventors selected two peptides that are proteotypes of the CHF6467 variants: SSSHPIFHR (m / z 534.265) and QAAWEFIR (m / z 510.759). The inventors also monitored the contribution of a peptide potentially reporting both exogenous and endogenous isoforms: QYFFETK (m / z 481.727, SEQ ID NO: 4). Finally, the inventors identified a rat-selective peptide, ALTTDDK (m / z 382.188, SEQ ID NO: 5), which specifically reports a potential endogenous response.

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

[0185] The inventors followed up on this study by confirming the degree of brain penetration of CHF6467 administered intranasally to young rats with hypoxic-ischemic injury (Landucci et al., 2018 Neurosci. Lett. 668. doi:10.1016 / j.neulet.2018.01.023.). The cerebral cortex and hippocampus were analyzed from six animals, and ipsilateral and contralateral tissues were compared. CHF6467 was detected in all analyzed samples except for three ipsilateral hippocampi lost during sample preparation due to tube rupture, and high reproducibility was observed between sample replicas 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 tested quantification range (0.06–13.5 ng / g), suggesting a level of less than 60 pg / g within this group of animals with hypoxic-ischemic injury.

[0186] This study demonstrated that CHF6467, after intranasal administration, actually enters the brain, reaching the hippocampus and cerebral cortex, and may exert neuroprotective and anti-neuroinflammatory effects.

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

Claims

1. A pharmaceutical product comprising the polypeptide of Sequence ID No. 1 for use in the treatment and / or prevention of traumatic brain injury or ischemic hypoxic brain injury in mammals, to be used in combination with hypothermia.

2. The pharmaceutical product according to claim 1, wherein the polypeptide is administered at a dose of 20 μg / kg.

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

4. A pharmaceutical product according to any one of claims 1 to 3, wherein the target mammal is human.

5. The pharmaceutical product according to claim 4, wherein the target mammal is a neonat or a newborn or a premature newborn.

6. The pharmaceutical product according to claim 5, wherein the neonat or newborn subject is affected by moderate to severe hypoxic-ischemic encephalopathy.

7. A pharmaceutical product according to any one of claims 1 to 6, which is administered intranasally or repeatedly over a period of 1 to 30 days.

8. The pharmaceutical product according to any one of claims 1 to 6, which is administered intranasally or repeatedly over a period of 2 to 14 days.

9. The pharmaceutical product according to any one of claims 1 to 8, which is administered repeatedly one to seven times a day, or administered into one or both nostrils.

10. A pharmaceutical product according to any one of claims 1 to 9, wherein the polypeptide is administered in a dose of 0.01 to 1.00 mg / day.

11. The pharmaceutical product according to claim 10, wherein the polypeptide is administered in a dose of 0.06 to 0.12 mg / day.

12. The pharmaceutical product according to claim 10, wherein the polypeptide is administered at a dose of 0.06 mg / day.

13. A pharmaceutical product according to any one of claims 1 to 12, which is administered repeatedly, with each dose of polypeptide being 0.01 to 1.00 mg / day.

14. The pharmaceutical product according to claim 13, wherein the dose of each polypeptide is 0.06 to 0.12 mg / day.

15. The pharmaceutical product according to claim 13, wherein the dose of each polypeptide is 0.06 mg / day.

16. A pharmaceutical product according to any one of claims 1 to 15, which is administered within approximately 1 to 24 hours after brain injury.

17. A pharmaceutical product according to any one of claims 1 to 16, further comprising an acetate buffer and / or methionine.

18. A pharmaceutical product according to any one of claims 1 to 17, which is administered simultaneously with a hypothermia procedure and / or within 24 hours after a hypothermia procedure.

19. The pharmaceutical product according to any one of claims 1 to 18, wherein the hypothermia method is initiated within 1 to 10 hours after a hypoxic-ischemic (HI) injury.

20. The pharmaceutical product according to claim 19, wherein the hypothermia method is initiated within 2 to 8 hours after a hypoxic-ischemic (HI) injury.

21. The pharmaceutical product according to claim 19, wherein the hypothermia method is initiated within 4 to 6 hours after a hypoxic-ischemic (HI) injury.

22. A pharmaceutical product according to any one of claims 1 to 21, wherein the hypothermia method is maintained for approximately 1 to approximately 72 hours after hypoxic-ischemic (HI) injury.

23. A pharmaceutical product according to any one of claims 1 to 22, wherein the body temperature of a mammal is maintained at a temperature of approximately 32°C to approximately 36°C.

24. The pharmaceutical product according to claim 23, wherein the body temperature of a mammal is maintained at a temperature of approximately 32°C to approximately 35°C.

25. The pharmaceutical product according to any one of claims 1 to 24, 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 approximately 32°C to approximately 35°C.

26. The pharmaceutical product according to any one of claims 1 to 25, 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 approximately 32°C to approximately 35°C.

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