Nerve growth factor and methods of use thereof for neurodevelopmental disorders associated with neonatal hypoxic-ischemic encephalopathy

Abstract

The present disclosure relates to nerve growth factor (NGF) for use in preventing or treating a neurodevelopmental disorder associated with neonatal hypoxic-ischemic encephalopathy in a subject, wherein the NGF is administered intranasally to the subject.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to European Patent Application No. 24167048.8, filed on 27 March 2024. The contents of that application are incorporated herein by reference in their entirety.

[0003] sequence list

[0004] This application includes a sequence list that has been submitted electronically as an XML file named “36381-0060WO1.XML”. This XML file was created on March 25, 2025, and is 3,053 bytes in size. The contents of this XML file are incorporated herein by reference in their entirety. Technical Field

[0005] This disclosure relates to the prevention and / or treatment of neurodevelopmental disabilities associated with hypoxic-ischemic encephalopathy in newborns.

[0006] This disclosure relates to the prevention and / or treatment of motor impairment associated with hypoxic-ischemic encephalopathy in subjects.

[0007] This disclosure also relates to the prevention and / or treatment of cerebral palsy (CP) associated with hypoxic-ischemic encephalopathy in subjects.

[0008] This disclosure also relates to promoting neurodevelopment in subjects affected by hypoxic-ischemic encephalopathy in newborns. Background Technology

[0009] Hypoxic-ischemic encephalopathy (HIE) is a highly dangerous condition affecting newborns.

[0010] Hypoxic-Ischemic Encephalopathy (HIE) is caused by insufficient blood (ischemia) and oxygen (hypoxia) supply to the fetal or infant brain during or after birth, which determines the formation of brain injury (Finer, NN, et al., Am J Dis Child 1983; 137: 21-5; Lawn, JE, et al., Lancet 2005, 365, 891-900; Ranjan, K., et al., J. Clin. Med. 2023, 12, 6653; Jin S. Hahn Clinical Manifestations of Hypoxic-Ischemic Encephalopathy, Fetal and Neonatal Brain Injury, pp. 247-257, from Part 3 - Diagnosis of the Infant with Brain Injury, published online by Cambridge University Press: December 13, 2017).

[0011] The severity of HIE depends on a variety of factors, such as etiology, degree of hypoxia / ischemia, stage of brain maturation, local cerebral blood flow, and maternal diseases / factors affecting the fetus (Allen, KA et al., Newborn Infant Nurs. Rev. 2011, 11, 125-133).

[0012] HIE affects both preterm and full-term newborns, and it occurs in both developed and developing countries, with a higher incidence in the latter. Several factors are known to contribute to the development of HIE, such as placental abruption, umbilical cord prolapse, and uterine rupture (Ranjan, K. et al., J. Clin. Med. 2023, 12, 6653).

[0013] A high percentage of infants with HIE die within the first two years of their lives, and most of those who survive longer develop a range of neurodevelopmental disorders, such as motor disorders, intellectual disability, epilepsy, cerebral palsy, and learning disabilities (Bruschettini, M. et al., Cochrane Database Syst. Rev. 2020, 8, Cd013202; Allen, KA et al., Newborn Infant Nurs. Rev. 2011, 11, 125-133).

[0014] Based on clinical presentation, HIE was classified into three grades—mild (stage 1), moderate (stage 2), or severe (stage 3)—according to the scoring system developed by Sarnat and Sarnat (Sarnat HB, Sarnat MS. Arch Neurol 1976; 33: 696-705). This classification was primarily based on the following clinical features: the infant's level of consciousness, cranial nerve examination findings, muscle tone, deep tendon reflexes, neonatal reflexes, spontaneous motor activity, and autonomic function (see Jin S. Hahn Clinical Manifestations of Hypoxic-Ischemic Encephalopathy, Fetal and Neonatal Brain Injury, pp. 247-257, from Part 3—Diagnosis of the Infant with Brain Injury, published online by Cambridge University Press: December 13, 2017, Table 16.1).

[0015] One of the most debilitating clinical manifestations experienced by patients with HIE is movement disorder, which is usually caused by muscle tone deregulation. This symptom is not the same in every patient and varies depending on the severity of HIE. In fact, muscle tone is one of the parameters used to stratify patients into the three stages of HIE defined by Sarnath and Sarnath. Specifically, muscle tone is typically normal or hypertonic in patients with mild HIE, hypotonic in patients with moderate HIE, and flaccid in patients with severe HIE (Sarnat HB, Sarnat MS. Arch Neurol 1976; 33: 696-705; Roland EH, Hill A. Semin Pediatr Neurol 1995; 2:57-71; Shankaran S, et al. N Engl J Med 2005; 353: 1574-84; Allen, KA, et al., Newborn Infant Nurs. Rev. 2011, 11, 125-133).

[0016] Furthermore, the severity of motor disorders can vary depending on the type of brain injury caused by hypoxic-ischemic events. Patients with periventricular leukomalacia (PVL) may develop spastic cerebral palsy (CP) presenting as diplegia, quadriplegia, or hemiplegia. Involvement of the basal ganglia (BG) and thalamus leads to extrapyramidal symptoms. Polycystic encephalopathy is associated with quadriplegia (Blair E, Stanley FJ. J Pediatr 1988; 112: 515-19).

[0017] Despite the fact that therapeutic hypothermia has been approved to date and other therapeutic options for treating HIE are under investigation (Ranjan et al., J. Clin. Med. 2023, 12, 6653), the rate of newborns experiencing neurodevelopmental disorders associated with hypoxic-ischemic encephalopathy (HIE) such as cognitive impairment, cerebral palsy, and moderate / severe disability remains unacceptably high (Gluckman PD et al., Lancet. 2005; 365(9460):663-670; Jacobs SE et al., Arch Pediatr Adolesc Med. 2011, 165(8):692-700; Simbruner G. et al., Pediatrics, 2010;126(4):e771-778; Azzopardi DV et al., N Engl J Med. 2009; 361(14):1349-1358). Furthermore, the management of movement disorders associated with this condition remains unsatisfactory, as its heterogeneity among different patients complicates the management process.

[0018] Therefore, there is a need to develop new, effective, durable, and safe treatments for the prevention or treatment of neurodevelopmental disorders (such as motor disorders or cerebral palsy) associated with HIE (e.g., caused by HIE). Summary of the Invention

[0019] As will be described in the experimental section, the inventors have found that when NGF is administered intranasally to subjects who experienced NHIE at birth, it effectively reduces the incidence of major neurodevelopmental disorders, such as cerebral palsy, motor disorders, and intellectual disabilities. In particular, the inventors have also found that when NGF is administered intranasally, it is delivered at particularly high concentrations to damaged brain regions in patients with motor disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE).

[0020] The inventors also unexpectedly discovered that intranasal administration of NGF is effective in preventing or treating movement disorders related to HIE (e.g., affected by or caused by it), regardless of the specific clinical presentation of the movement disorder experienced by the patient. This is due to the efficacy of intranasal NGF in normalizing muscle tone in patients affected by HIE, thereby improving their motor function. Based on the obtained data, intranasal NGF can therefore prevent and / or treat movement disorders related to HIE (e.g., affected by or caused by it) in patients affected by the condition.

[0021] Therefore, one objective of this disclosure is NGF, which is used in subjects to prevent and / or treat neurodevelopmental disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy in newborns.

[0022] Another objective of this disclosure is NGF, which is used for the prevention and / or treatment of movement disorders associated with (e.g., affected by or caused by) HIE.

[0023] Another objective of this disclosure is NGF, which is used in subjects to prevent and / or treat cerebral palsy (CP) associated with (e.g., affected by or caused by) neonatal hypoxic-ischemic encephalopathy.

[0024] Another objective of this disclosure is NGF, which is used to promote neurodevelopment in subjects diagnosed with neonatal hypoxic-ischemic encephalopathy.

[0025] This article discloses and describes methods for preventing or treating neurodevelopmental disorders associated with neonatal hypoxic-ischemic encephalopathy (NHIE) in subjects of need, involving intranasal administration of a therapeutically effective or prophylacticly effective dose of nerve growth factor (NGF). These neurodevelopmental disorders may include motor impairments or neurodevelopmental delays, including conditions such as cerebral palsy. Additionally, impairments may encompass cognitive impairment, behavioral impairment, or sensory impairment. Motor impairments are associated with dystonia, and NGF administration aims to reduce the likelihood or severity of these neurodevelopmental disorders compared to subjects who have not received NGF, particularly those diagnosed with NHIE.

[0026] Subjects at risk of developing such disorders may also benefit from NGF administration, particularly those who have experienced perinatal depression, indicated by factors such as: an Apgar score below 5 at 10 minutes of age, a need for resuscitation (including chest compressions, mechanical ventilation, or CPAP), a pH below 7.00 in the umbilical cord or neonatal blood gases within 60 minutes of birth, or a base deficiency exceeding 15 mmol / L. The need for resuscitation may include chest compressions, mechanical ventilation, or CPAP therapy. This method is particularly suitable for subjects diagnosed with NHIE classified as stage 2 or 3 according to the modified Sarnath staging system, with a gestational age of at least 36 weeks and a birth weight of at least 1800 grams.

[0027] For subjects diagnosed with motor disorders or cerebral palsy associated with NHIE, intranasal administration of NGF is used for therapeutic purposes. It can also be used to prevent cerebral palsy in subjects identified as being at risk due to NHIE. NGF administration is particularly suitable for subjects who have received therapeutic hypothermia within the first six hours after birth. Initial administration of NGF is within one month of birth, preferably within seven days, more preferably within 72 hours, even more preferably within 36 hours, and most preferably within 24 hours. Alternatively, NGF can be first administered between one and 36 months of age, and further preferably between one and 24 months, one and 12 months, one and six months, or one and four months of age, and additionally preferably between three and nine months of age, ideally between three and six months.

[0028] The administration regimen can follow a continuous or intermittent protocol, wherein the continuous protocol has two or more one-week treatment cycles, and the intermittent protocol has treatment cycles interspersed with washout periods. A preferred intermittent protocol comprises at least three cycles, each consisting of seven days of daily NGF administration followed by a 21-day washout period. The total NGF dose administered is from 25 µg / kg to 400 µg / kg, and preferably ranges from 30 µg / kg to 300 µg / kg, 35 µg / kg to 200 µg / kg, 40 µg / kg to 100 µg / kg, 40 µg / kg to 60 µg / kg, and 45 µg / kg to 55 µg / kg, with the most preferred dose being 50 µg / kg. The dose per cycle is from 15 µg / kg to 20 µg / kg, preferably from 16 µg / kg to 17 µg / kg, even more preferably from 16.6 µg / kg to 16.8 µg / kg, with the most preferred dose being 16.7 µg / kg. The daily dose is 2 µg / kg to 3 µg / kg, preferably 2.3 µg / kg to 2.5 µg / kg, and most preferably 2.4 µg / kg. The dose per nostril is 0.3 µg / kg / nostril to 1 µg / kg / nostril, preferably 0.4 µg / kg / nostril to 0.8 µg / kg / nostril, more preferably 0.5 µg / kg / nostril to 0.7 µg / kg / nostril, and most preferably 0.6 µg / kg / nostril. The daily NGF dose is administered twice daily at 12-hour intervals.

[0029] This treatment aims to normalize muscle tone in individuals with NHIE and address motor disorders or dystonia associated with cerebral palsy. Dystonia can include conditions such as hypotonia, bradykinesia, hypertonia, dystonia, ataxia, extrapyramidal symptoms, diplegia, tetraplegia, quadriplegia, hemiplegia, and paraplegia. Hypotonia can be focal or generalized, dystonia can be focal, segmental, or generalized, and hypertonia can include spasticity, rigidity, or paratonia.

[0030] In some cases, NGF is formulated into pharmaceutical compositions for intranasal administration, comprising at least one pharmaceutically acceptable excipient. The NGF concentration in the composition is from 5 µg / ml to 1 mg / ml, preferably from 10 µg / ml to 400 µg / ml, and even more preferably from 15 µg / ml to 200 µg / ml. The composition may consist of NGF, sodium chloride, phosphate buffer, and water. The NGF used is human NGF, preferably recombinant human NGF, and comprises or consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0031] In some cases, methods for treating cerebral palsy associated with hypoxic-ischemic encephalopathy in newborns have also been disclosed, involving the intranasal administration of a therapeutically effective amount of nerve growth factor (NGF) to a recipient. In some cases, the NGF used in this method comprises the sequence identified as SEQ ID NO: 1. This treatment may also reduce the likelihood or severity of cerebral palsy in subjects who have not received NGF, particularly those diagnosed with hypoxic-ischemic encephalopathy in newborns.

[0032] This method is applicable to subjects experiencing motor disorders caused by dystonia, which may include conditions such as hypotonia, bradykinesia, hypertonia, dystonia, ataxia, extrapyramidal symptoms, diplegia, quadriplegia, hemiplegia, and paraplegia. Hypotonia can be focal or generalized, dystonia can be focal, segmental, or generalized, and hypertonia can manifest as spasticity, rigidity, or tonic atony. NGF administration aims to normalize muscle tone in affected subjects to levels comparable to those in healthy individuals of the same age.

[0033] This treatment is indicated for subjects who have experienced perinatal depression, as indicated by the following criteria: for example, an Apgar score of less than 5 at 10 minutes of gestation; resuscitation required at 10 minutes; cord blood pH less than 7.00; or alkali deficiency meeting a specific threshold. Eligible subjects typically have a gestational age of at least 36 weeks and a birth weight of at least 1800 grams.

[0034] NGF administration can begin within the first 24 hours after birth or between one and four months of age. Administration can be continuous (involving two or more weekly treatment cycles) or intermittent (involving treatment cycles alternating with washout periods). The total NGF dose is 25 µg / kg to 400 µg / kg, with a dose of 15 µg / kg to 20 µg / kg per cycle and a daily dose of 2 µg / kg to 3 µg / kg. Additionally, subjects receiving this treatment may also receive therapeutic hypothermia within the first six hours after birth.

[0035] NGF can be formulated into a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient, which may include sodium chloride, phosphate buffer, and water.

[0036] Unless the context clearly indicates otherwise, the singular forms “a” and “the” used in this disclosure and claims include the plural forms.

[0037] It should be understood that wherever the implementation scheme is described in this document using the word "comprising / including", similar implementation schemes described as "consisting of" and / or "substantially consisting of" are also provided.

[0038] The term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" and "B". Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). Attached Figure Description

[0039] Figure 1 The biodistribution of NGF in rat brain regions, as measured by ELISA within 24 hours following intranasal administration, is shown, as described in Example 1. Results are expressed as a percentage of the total amount absorbed in the rat brain within 24 hours following administration.

[0040] Figure 2 The experimental timeline of the study described in Example 2 is shown.

[0041] Figure 3 The retention time in the wire hanging test is shown for sham-operated and hypoxic-ischemic (HI) mice treated with a carrier or rhNGF at 28 days post-birth. N = 10 mice / group. Data are shown as mean and SEM for each group. One-way ANOVA, followed by Tukey comparison test.

[0042] Figure 4 The retention time of sham-operated and HI mice treated with either the loading agent or rhNGF in the rotarod test at day 29 postnatal is shown. N = 10 mice. Data are shown as mean and SEM for each group. One-way ANOVA, followed by Tukey comparison test.

[0043] Figure 5 The number of errors in the tapered beam test in HI and sham-operated mice treated with the load or with rhNGF is shown at 30 days after birth. N = 10 mice / group. Data are shown as mean and SEM for each group. One-way ANOVA, followed by Tukey comparison test.

[0044] Figure 6 The experimental timeline of the study described in Example 3 is shown.

[0045] Figure 7 The rats' weight in grams is shown from baseline (P7, immediately before surgery) to endpoint (early treatment).

[0046] Figure 8 The rats' weight in grams is shown from baseline (P7, immediately before surgery) to P42 (late treatment).

[0047] Figure 9 The neuroscores of sham-operated animals (treated with a carrier or early treatment with hNGF) and NHIE animals (treated with a carrier or early treatment with hNGF) are shown 2 weeks after NHIE induction. Higher scores indicate worse outcomes.

[0048] Figure 10 Neurological scores of sham-operated animals (treated with a carrier or late-stage hNGF) and NHIE animals (treated with a carrier or late-stage hNGF) are shown 2 weeks after NHIE induction. Higher scores indicate worse outcomes.

[0049] Figure 11 Alternating data assessed after NHIE induction (rats treated with a loading agent vs. rats treated with hNGF early) are shown. Data are presented as median and individual values.

[0050] Figure 12 Alternating data assessed after NHIE induction (rats treated with a loading agent vs. rats treated late with hNGF) are shown. Data are presented as median and individual values.

[0051] Figure 13 The primary latency (in seconds) of NHIE rats treated with a carrier or with hNGF (early) in the maze to reach the escape opening is shown. Data are mean ± SEM.

[0052] Figure 14 The initial latency (in seconds) of NHIE rats treated with the carrier or with hNGF (late) in the maze to reach the escape opening is shown. Data are mean ± SEM.

[0053] Figure 15 A and 15B The individual comprehensive health score of each rat is shown, according to early ( Figure 15 A) or late stage ( Figure 15 B) Processing involves partitioning the data.

[0054] Figure 15 C The forest plot shown is the result of contingency analysis performed using the chi-square test. p < 0.05).

[0055] Figure 16 The fractional anisotropy (FA) values ​​of rats treated with a carrier or with hNGF (late stage) are shown, measured by diffusion tensor imaging (DTI). Decreased FA values ​​indicate white matter damage. Detailed Implementation

[0056] The inventors have discovered that intranasal administration of nerve growth factor (NGF) is effective in preventing, improving, or treating neurodevelopmental disorders and their clinical manifestations (e.g., motor disorders and cerebral palsy) associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE). Furthermore, as will be explained below, the inventors have unexpectedly discovered that a regimen in which NGF is first administered intranasally at least one month after the injury (late administration) has a beneficial effect in this pathological condition.

[0057] The terms “treatment” and “prevention” used in this article refer to the eradication / improvement or prevention / delay of the onset of a disease or one or more of its related symptoms.

[0058] Treatment includes therapeutic measures that cure, alleviate, reduce the severity of, and / or halt the progression of a diagnosed pathological condition or symptom. Prevention includes preventive or preventative measures that prevent the target pathological condition or symptom, reduce its likelihood of occurrence, reduce its severity, and / or delay its onset.

[0059] In the context of this application, the following definitions apply.

[0060] As used herein, “mitigation” refers to the slowing of the progression of neurodevelopmental disorders (e.g., mild motor developmental delay, damage, dysfunction, or developmental abnormalities within the nervous system, difficulties with motor, muscle coordination, and motor skills, which may include spasticity, weakness, and poor muscle tone) associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE) in subjects (e.g., human subjects) receiving NGF as described herein, compared to a group diagnosed with HIE who have not received NGF as described herein.

[0061] As used herein, “reduction of severity” means, in subjects (e.g., human subjects) diagnosed with hypoxic-ischemic encephalopathy (HIE) of comparable severity who have not received the NGF described herein, a reduction in the severity of neurodevelopmental disorders (e.g., mild motor developmental delay, damage, dysfunction, or developmental abnormalities within the nervous system, difficulties with motor, muscle coordination, and motor skills, which may include spasticity, weakness, and dystonia) associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE), preferably motor disorders, cerebral palsy, and cognitive impairment.

[0062] As used herein, “prevention” means, in subjects (e.g., human subjects) diagnosed with hypoxic-ischemic encephalopathy (HIE) of comparable severity who have not received the NGF described herein, the prevention of the progression of neurodevelopmental disorders (e.g., mild motor developmental delay, damage, dysfunction, or developmental abnormalities within the nervous system, difficulties with motor, muscle coordination, and motor skills, which may include spasticity, weakness, and dystonia) associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE).

[0063] As used herein, “delayed onset” means, in subjects (e.g., human subjects) diagnosed with hypoxic-ischemic encephalopathy (HIE) of comparable severity who have not received the NGF described herein, a delay in the onset of neurodevelopmental disorders (e.g., mild motor developmental delay, damage, dysfunction, or developmental abnormalities within the nervous system, difficulties with motor, muscle coordination, and motor skills, which may include spasticity, weakness, and dystonia) associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE).

[0064] As used herein, “reduced likelihood of occurrence” means that, compared with a group diagnosed with hypoxic-ischemic encephalopathy (HIE) who have not received the NGF described herein, subjects (e.g., human subjects) diagnosed with HIE who have received the NGF described herein are less likely to develop neurodevelopmental disorders (e.g., those affected by or caused by HIE) associated with HIE. These neurodevelopmental disorders are preferably motor disorders, cerebral palsy, and cognitive impairments.

[0065] Preferably, the NGF reduces the likelihood of the subject having neurodevelopmental disorders (preferably motor disorders, cerebral palsy, and cognitive impairment) associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy in newborns (HIE) by at least 40%, preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, and even more preferably at least 80%.

[0066] As used herein, “reduced severity of occurrence” means that subjects diagnosed with hypoxic-ischemic encephalopathy (HIE) who receive the NGF described herein experience a significantly reduced severity of neurodevelopmental disorders (e.g., mild motor developmental delay, damage, dysfunction, or developmental abnormalities within the nervous system, difficulties with motor, muscle coordination, and motor skills, which may include spasticity, weakness, and dystonia) associated with HIE after NGF treatment compared to a group diagnosed with HIE of comparable severity who do not receive the NGF described herein. These neurodevelopmental disorders are preferably motor disorders, cerebral palsy, and cognitive impairment.

[0067] Therapeutic effective amount refers to the amount of NGF that is effective in treating a disease or condition in a subject (e.g., mammals; e.g., humans).

[0068] The effective preventive dose refers to the amount that, at the required dose and for the required duration, effectively achieves the desired preventive result.

[0069] The first objective of this disclosure relates to nerve growth factor (NGF) for the prevention and / or treatment in subjects of neurodevelopmental disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE) in the newborn, wherein the NGF is administered intranasally to the subjects.

[0070] In some cases, this disclosure relates to NGF for the treatment of neurodevelopmental disorders in subjects that are associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE) in the newborn, wherein the NGF is administered intranasally to the subjects.

[0071] In some cases, this disclosure relates to NGF for alleviating neurodevelopmental disorders in subjects associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE), wherein the NGF is administered intranasally to the subjects.

[0072] In some cases, this disclosure relates to NGF for the purpose of mitigating the severity of neurodevelopmental disorders in subjects associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE), wherein the NGF is administered intranasally to the subjects.

[0073] In some cases, this disclosure relates to NGF for preventing the progression of neurodevelopmental disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE) in subjects, wherein the NGF is administered intranasally to the subjects.

[0074] In some cases, this disclosure relates to NGF for delaying the onset of neurodevelopmental disorders in subjects associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE), wherein the NGF is administered intranasally to the subjects.

[0075] In some cases, this disclosure relates to NGFs used to reduce the likelihood of neurodevelopmental disorders in subjects that are associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE), wherein the NGF is administered intranasally to the subjects.

[0076] In some cases, this disclosure relates to NGFs used to reduce the severity of neurodevelopmental disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE) in subjects, wherein the NGF is administered intranasally to the subjects.

[0077] In some cases, the object is a human object, preferably a newborn or an infant.

[0078] In some cases, the neurodevelopmental disorder is selected from cerebral palsy, cognitive impairment, motor disorder, neurodevelopmental delay, behavioral disorder, and sensory disorder.

[0079] In some cases, the movement disorder is as described below.

[0080] In some cases, the cerebral palsy is as described below.

[0081] In some cases, the subject has been diagnosed with a neurodevelopmental disorder associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn (HIE), and the NGF is used to treat the neurodevelopmental disorder in the subject by intranasal administration.

[0082] The treatment may include one or more of the following: curing, slowing down, reducing the severity of, or halting the progression of neurodevelopmental disorders in the subject.

[0083] In some cases, the subject has been identified as being at risk of developing neurodevelopmental disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE) in the newborn, and the NGF is used to prevent the neurodevelopmental disorders in the subject by intranasal administration.

[0084] The prevention may include one or more of the following: reducing the likelihood of neurodevelopmental disorders occurring in the subject, reducing the severity of their occurrence, or delaying their occurrence.

[0085] In some cases, the subject has been identified as being at risk of developing neurodevelopmental disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn (HIE), and the NGF is used to reduce the likelihood and / or severity of the neurodevelopmental disorder in the subject by intranasal administration.

[0086] In some cases, this disclosure provides a method for preventing and / or treating neurodevelopmental disorders in subjects via intranasal administration of nerve growth factor (NGF) that are associated with, or affected by, neonatal hypoxic-ischemic encephalopathy (HIE). The method involves delivering NGF to the subject to promote neuronal survival, support neurogenesis, and enhance synaptic plasticity in brain regions affected by the neurodevelopmental disorder. NGF is administered via intranasal delivery, which allows for efficient transport across the blood-brain barrier and direct targeting of the central nervous system. This treatment aims to address a range of neurodevelopmental disorders, including cognitive, motor, and sensory impairments, by stimulating the repair of damaged neural circuits and promoting the regeneration of lost or damaged neurons. The method may involve a series of intermittent NGF administration cycles, in which the subject receives treatment over a period of time to maximize neuroprotective and restorative effects while minimizing potential adverse reactions.

[0087] "Neurodevelopmental disorders" refers to a broad category of conditions characterized by impairment of brain function, which can affect cognitive, motor, sensory, or behavioral abilities. In some cases, the neurodevelopmental disorders are selected from cerebral palsy, cognitive impairment, motor disorders, neurodevelopmental delay, behavioral disorders, and sensory disorders.

[0088] These conditions are caused by damage, dysfunction, or developmental abnormalities within the nervous system, leading to long-term or permanent disability. Treatment for neurodevelopmental disorders aims to restore or enhance neurological function, alleviate symptoms, and improve the overall quality of life for affected individuals.

[0089] Another objective of this disclosure is nerve growth factor (NGF) for the prevention and / or treatment in subjects of motor disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE), wherein the NGF is administered intranasally to the subjects.

[0090] In the context of hypoxic-ischemic encephalopathy (HIE) in newborns, "motor impairment" refers to the loss or dysfunction of motor control resulting from brain damage caused by a lack of oxygen and blood flow to the brain during or shortly after birth. In the context of neonatal HIE, motor impairment manifests as difficulties in movement, muscle coordination, and motor skills, which may include spasticity, weakness, dystonia (hypotonia or hypertonia), or bradykinesia. Motor impairment in neonatal HIE can vary in severity, ranging from mild developmental delay to more severe conditions such as cerebral palsy, and can affect an infant's ability to perform basic movements such as crawling, walking, or controlling voluntary muscle movements. The degree and type of motor impairment depend on the extent of brain damage and the timing of the hypoxic-ischemic event during neurodevelopment.

[0091] In some cases, this disclosure relates to NGF for the treatment of motor disorders or one or more symptoms thereof in subjects that are associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE) in the neonatal body, wherein the NGF is administered intranasally to the subjects.

[0092] In some cases, this disclosure relates to NGF for alleviating motor disorders or one or more symptoms thereof associated with (e.g., affected by or caused by) neonatal hypoxic-ischemic encephalopathy (HIE) in a subject, wherein the NGF is administered intranasally to the subject.

[0093] In some cases, this disclosure relates to NGF for the purpose of alleviating the severity of motor impairment or one or more symptoms of neonatal hypoxic-ischemic encephalopathy (HIE) in subjects (e.g., affected by or caused by it), wherein the NGF is administered intranasally to the subjects.

[0094] In some cases, this disclosure relates to NGF for preventing the progression of motor disorders or one or more symptoms associated with (e.g., affected by or caused by) neonatal hypoxic-ischemic encephalopathy (HIE) in subjects, wherein the NGF is administered intranasally to the subjects.

[0095] In some cases, this disclosure relates to NGF for delaying the onset of motor disorders or one or more symptoms of neonatal hypoxic-ischemic encephalopathy (HIE) in subjects, such as those affected by or caused by HIE, wherein the NGF is administered intranasally to the subjects.

[0096] In some cases, this disclosure relates to NGFs used to reduce the likelihood of motor disorders or one or more symptoms of neonatal hypoxic-ischemic encephalopathy (HIE) in subjects, such as those affected by or caused by HIE, wherein the NGF is administered intranasally to the subjects.

[0097] In some cases, this disclosure relates to NGF for reducing the severity of motor impairment or one or more symptoms of neonatal hypoxic-ischemic encephalopathy (HIE) in subjects, such as those affected by or caused by HIE, wherein the NGF is administered intranasally to the subjects.

[0098] In some cases, one or more of the symptoms are selected from difficulty in movement, difficulty in muscle coordination, difficulty in motor skills, weakness, poor muscle tone, and bradykinesia.

[0099] In some cases, the object is a human object. As used herein, “object” refers to any animal (e.g., a mammal) that is to become a recipient of a particular treatment, including but not limited to humans, non-human primates, etc. Generally, when referring to a human object, the terms “object” and “patient” are used interchangeably herein.

[0100] In some cases, the person being referred to is a newborn or an infant.

[0101] In some cases, the movement disorder is caused by dystonia.

[0102] In some cases, NGF is used to normalize muscle tone in subjects with hypoxic-ischemic encephalopathy (HIE). Normalization involves restoring the subject's muscle tone to a level substantially equivalent to that of healthy subjects of the same age.

[0103] In some cases, NGF is used to improve motor impairments associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE) to levels that are compatible with the performance of typical daily activities in age-matched subjects not diagnosed with HIE. In some cases, these daily activities include basic motor skills such as crawling, walking, controlling voluntary muscle movements, sitting upright, feeding, grasping objects, and interacting with the environment.

[0104] In some cases, movement disorders or dystonia are associated with cerebral palsy (e.g., affected by or caused by it).

[0105] Cerebral palsy is a group of conditions affecting movement and muscle tone, typically caused by brain injury or abnormal brain development. Intranasal administration of NGF offers a potential therapeutic intervention for individuals suffering from this debilitating condition.

[0106] In some cases, dystonia is selected from hypotonia, bradykinesia, hypertonia, dystonia, ataxia, extrapyramidal symptoms, diplegia, quadriplegia, quadriplegia, hemiplegia, and paraplegia.

[0107] In some cases, hypotonia can be classified as focal hypotonia or generalized hypotonia.

[0108] Focal hypotonia affects specific muscle groups, while generalized hypotonia affects the entire body. Both types of hypotonia can impair motor function and lead to difficulties in movement, balance, and coordination. NGF administration can help normalize muscle tone and improve overall motor performance in individuals affected by these conditions.

[0109] In some cases, dystonia is selected from focal dystonia, segmental dystonia, and generalized dystonia.

[0110] Focal dystonia affects specific parts of the body, segmental dystonia affects multiple areas of the body, while generalized dystonia involves a wide range of muscles. NGF has the potential to alleviate symptoms of these types of dystonia and restore more functional muscle control.

[0111] In some cases, hypertonia arises from spasticity, rigidity, and tonic atony. Spasticity, rigidity, and tonic atony can lead to muscle stiffness and motor resistance. NGF therapy can help alleviate these symptoms by modulating neural circuits involved in muscle tone, thereby improving motor control and function.

[0112] In some cases, the subject has been diagnosed with a motor disorder associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn (HIE), and the NGF is used to treat the motor disorder in the subject by intranasal application.

[0113] The treatment may include one or more of the following: curing the subject, slowing the movement disorder, reducing its severity, or stopping its progression.

[0114] In some cases, according to this implementation scheme, NGF is applied to the subject less than two years after birth, preferably less than one year after birth, more preferably less than eight months after birth, more preferably less than six months after birth, and even more preferably three to six months after birth.

[0115] In some cases, the subject has been identified as being at risk of developing motor disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE), and the NGF is used to prevent motor disorders in the subject by intranasal administration prior to their occurrence.

[0116] The prevention may include one or more of the following: reducing the likelihood of movement disorders occurring in the subject, reducing the severity of their occurrence, or delaying their occurrence.

[0117] Preferably, according to this embodiment, NGF is applied to the subject from 6 hours to 15 days after birth, more preferably from 24 hours to 7 days after birth.

[0118] This article also discloses a method for preventing and / or treating motor disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy in subjects with this need, the method comprising administering a therapeutically effective amount of nerve growth factor (NGF) intranasally to the subject.

[0119] This method involves delivering NGF to subjects to promote the survival, growth, and repair of motor neurons, thereby enhancing motor function and reducing motor deficits. In some cases, intranasal delivery allows for efficient transport across the blood-brain barrier and targeted action within the central nervous system. By stimulating neural plasticity, synaptic regeneration, and remodeling of motor pathways, NGF administration can improve motor coordination, strength, and overall mobility in subjects with motor dysfunction. This method may involve periodic NGF administration cycles to maximize therapeutic effects, and the treatment is designed to slow the progression of motor impairment, restore motor function, and improve quality of life over time.

[0120] Motor disorders caused by hypoxic-ischemic encephalopathy in newborns can be due to dystonia. This dystonia can affect movement and coordination in affected individuals. By administering NGF, these motor deficits can be addressed and overall motor function improved in subjects. This treatment specifically targets the underlying neural mechanisms leading to motor dysfunction.

[0121] In particular, as described in this method, administration of NGF can help normalize muscle tone in subjects with hypoxic-ischemic encephalopathy in newborns. By targeting the underlying neurological dysfunction causing abnormal muscle tone, NGF can restore more normal motor function, thereby improving the subject's quality of life and ability to perform daily activities.

[0122] Dystonia resulting from hypoxic-ischemic encephalopathy in newborns can be associated with conditions such as cerebral palsy. Cerebral palsy is a group of conditions affecting movement and muscle tone, typically caused by brain injury or abnormal brain development. The treatments outlined in this disclosure offer potential therapeutic interventions for individuals suffering from this debilitating condition.

[0123] Dystonia can manifest in a variety of ways, including hypotonia, bradykinesia, hypertonia, dystonia, ataxia, extrapyramidal symptoms, diplegia, quadriplegia, hemiplegia, and paraplegia. Each of these conditions presents unique challenges in terms of motor control and function, making the use of NGF as a treatment for such conditions highly beneficial. The ability to address multiple forms of dystonia broadens the scope of this approach.

[0124] More specifically, hypotonia (which refers to low muscle tone) can be focal or generalized. Focal hypotonia affects specific muscle groups, while generalized hypotonia affects the entire body. Both types of hypotonia can impair motor function and lead to difficulties in movement, balance, and coordination. NGF administration can help normalize muscle tone and improve overall motor performance in individuals affected by these conditions.

[0125] Similarly, dystonia (a condition characterized by involuntary muscle contractions that cause twisting and abnormal posture) can also be treated with NGF. Dystonia can manifest as focal dystonia affecting a specific part of the body, segmental dystonia affecting multiple areas, or generalized dystonia involving a wide range of muscles. NGF has the potential to alleviate symptoms of these types of dystonia and restore more functional muscle control.

[0126] Hypertonia or elevated muscle tone is another condition that can result from hypoxic-ischemic encephalopathy in newborns. This can include spasticity, rigidity, and tonic defiance, which can lead to muscle stiffness and motor resistance. NGF therapy can help alleviate these symptoms by modulating neural circuits involved in muscle tone, thereby improving motor control and function.

[0127] Another objective of this disclosure is nerve growth factor (NGF) for the prevention and / or treatment in subjects of cerebral palsy (CP) or its clinical manifestations associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE), wherein the NGF is administered intranasally to the subjects.

[0128] In some cases, this disclosure relates to NGF for the treatment of cerebral palsy (CP) or its clinical manifestations associated with (e.g., affected by or caused by) neonatal hypoxic-ischemic encephalopathy (HIE) in subjects, wherein the NGF is administered intranasally to the subjects.

[0129] In some cases, this disclosure relates to NGF for the purpose of alleviating cerebral palsy (CP) or its clinical manifestations in subjects that are associated with (e.g., affected by or caused by) neonatal hypoxic-ischemic encephalopathy (HIE), wherein the NGF is administered intranasally to the subjects.

[0130] In some cases, this disclosure relates to NGF for the purpose of reducing the severity of cerebral palsy (CP) or its clinical manifestations associated with (e.g., affected by or caused by) neonatal hypoxic-ischemic encephalopathy (HIE) in subjects, wherein the NGF is administered intranasally to the subjects.

[0131] In some cases, this disclosure relates to NGF for preventing the progression of cerebral palsy (CP) or its clinical manifestations associated with (e.g., affected by or caused by) neonatal hypoxic-ischemic encephalopathy (HIE) in subjects, wherein the NGF is administered intranasally to the subjects.

[0132] In some cases, this disclosure relates to NGF for delaying the onset of cerebral palsy (CP) or its clinical manifestations associated with (e.g., affected by or caused by) neonatal hypoxic-ischemic encephalopathy (HIE) in subjects, wherein the NGF is administered intranasally to the subjects.

[0133] In some cases, this disclosure relates to NGFs used to reduce the likelihood of cerebral palsy (CP) or its clinical manifestations associated with (e.g., affected by or caused by) neonatal hypoxic-ischemic encephalopathy (HIE) in subjects, wherein the NGF is administered intranasally to the subjects.

[0134] In some cases, this disclosure relates to NGF for reducing the severity of cerebral palsy (CP) or its clinical manifestations associated with (e.g., affected by or caused by) neonatal hypoxic-ischemic encephalopathy (HIE) in subjects, wherein the NGF is administered intranasally to the subjects.

[0135] In some cases, the object is a human object, preferably a newborn or an infant.

[0136] In some cases, the clinical manifestations are selected from movement disorders and cognitive impairments.

[0137] In some cases, the movement barrier is as described above.

[0138] In some cases, the subject has been diagnosed with cerebral palsy associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn, and the NGF is used to treat the cerebral palsy in the subject by intranasal application.

[0139] The treatment may include one or more of the following: curing, slowing, reducing the severity, or stopping the progression of cerebral palsy in the subject.

[0140] In some cases, the subject has been identified as being at risk of developing cerebral palsy associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn, and the NGF is used to prevent the cerebral palsy in the subject by intranasal administration.

[0141] The prevention may include one or more of the following: reducing the likelihood of cerebral palsy occurring in the subject, reducing the likelihood of it reaching a severe level, or delaying its onset.

[0142] In some cases, this disclosure provides a method for preventing and / or treating cerebral palsy (CP) in subjects via intranasal administration of nerve growth factor (NGF) (e.g., SEQ ID NO: 1 or any NGF composition described herein). The method involves administering NGF to a subject diagnosed with CP to promote neuronal survival, enhance synaptic plasticity, and restore neurovascular function in the affected brain regions. Intranasal administration of NGF ensures efficient delivery to the central nervous system while minimizing systemic exposure. The treatment is designed to target motor and cognitive impairments commonly associated with CP by promoting the repair of damaged neural pathways, enhancing neurotrophic support for surviving neurons, and stimulating the growth of new synaptic connections. NGF administration is performed over a specified duration, and preferred treatment regimens involve intermittent administration cycles to optimize therapeutic effects while minimizing potential side effects. This method offers a promising approach to improving both motor function and quality of life in individuals with CP by enhancing brain plasticity and promoting long-term neurorepair.

[0143] Another object of the present invention is nerve growth factor (NGF) for promoting neural development in subjects diagnosed with hypoxic-ischemic encephalopathy (HIE), wherein the NGF is administered intranasally to the subjects.

[0144] In some cases, promoting neurodevelopment includes enhancing neuronal survival, synapse formation, and / or the establishment of neural networks necessary for normal cognitive, motor, and sensory functions.

[0145] In some cases, this disclosure provides a method for promoting neurodevelopment in subjects diagnosed with hypoxic-ischemic encephalopathy (NHIE) by intranasal administration of nerve growth factor (NGF). The method involves delivering NGF to the subject to stimulate the growth, differentiation, and maturation of neurons, thereby supporting healthy brain development. NGF is administered via intranasal delivery, which allows for efficient transport across the blood-brain barrier and direct action on the central nervous system. This treatment is particularly beneficial during critical periods of brain development, during which NGF enhances neuronal survival, synapse formation, and the establishment of neural networks essential for normal cognitive, motor, and sensory functions. The method is designed to address neurodevelopmental disorders caused by NHIE by promoting neurotrophic support for developing neurons and stimulating brain plasticity. Administration regimens may include cyclical NGF treatment cycles to optimize its neuroprotective and neuroregenerative effects, ultimately supporting healthier neurodevelopment and improving long-term neurological outcomes.

[0146] Neurodevelopment refers to the maturation and organization of the nervous system (including the brain and spinal cord) throughout the prenatal, neonatal, and early postnatal stages of life. This process encompasses the growth, differentiation, migration, and maturation of neurons, as well as the formation of synaptic connections and neural networks necessary for cognitive, motor, sensory, and emotional functions. Neurodevelopment is a highly dynamic and sensitive process that occurs during critical developmental windows and is influenced by genetic, environmental, and physiological factors. Disorders of neurodevelopment can lead to a variety of neurological and neuropsychiatric disorders, including developmental delays, intellectual disabilities, autism spectrum disorders, and other neurodevelopmental disorders. Normal progression of neurodevelopment is crucial for the establishment of functional brain circuits and for the overall health and function of an individual's nervous system.

[0147] In some cases, hypoxic-ischemic encephalopathy (HIE) of the newborn is selected from mild hypoxic-ischemic encephalopathy (HIE) (stage 1), moderate hypoxic-ischemic encephalopathy (HIE) (stage 2) and severe hypoxic-ischemic encephalopathy (HIE) (stage 3) as determined by the staging system developed by Sarnat and Sarnat (Sarnat HB, Sarnat MS. Arch Neurol 1976; 33: 696-705).

[0148] In some cases, hypoxic-ischemic encephalopathy (HIE) in newborns is either moderate or severe.

[0149] In some cases, the object is a newborn or an infant.

[0150] In some cases, the subject was born at a gestational age of at least 36 weeks.

[0151] In some cases, the subject weighed at least 1800 grams at birth.

[0152] In some cases, the subject has at least one of the following signs of perinatal suppression:

[0153] - An Apgar score of less than 5 at 10 minutes of age.

[0154] - Resuscitation is required within 10 minutes (e.g., chest compressions, mechanical ventilation, or CPAP).

[0155] - The pH of the umbilical cord blood gas or the neonatal blood gas obtained before 60 minutes of age is less than 7.00.

[0156] - An alkali deficiency of ≥12 mmol / L, ≥15 mmol / L, or ≥16 mmol / L in umbilical cord (arterial or venous) air or infant air (arterial or venous) obtained before 60 minutes of age.

[0157] In some cases, the subject received therapeutic hypothermia treatment that began within 6 hours of birth.

[0158] In some cases, the NGF applied according to this disclosure is applied within one month after birth (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days), preferably within seven days after birth, more preferably within 72 hours after birth (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours). (hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours), preferably within 36 hours after birth, and even more preferably within 24 hours after birth for the first application to the subject.

[0159] In some cases, the NGF applied according to this disclosure is applied to the subject one month or later after birth, preferably one month to 36 months after birth, more preferably one month to 24 months after birth, even more preferably one month to 12 months after birth, and even more preferably one month to six months after birth, and most preferably one month to four months after birth.

[0160] In some cases, the NGF applied according to this disclosure is first applied to the subject three to nine months after birth, preferably three to six months after birth.

[0161] In some cases, the NGF used according to this disclosure is administered to the subject daily or every two / three days throughout the treatment period.

[0162] In some cases, the treatment period is between 7 and 90 days. In other cases, the treatment period is between 15 and 60 days.

[0163] In some cases, the NGF applied according to this disclosure is applied to the target one to three times daily.

[0164] In some cases, the administration regimen is a continuous administration regimen, in which NGF is administered according to the same regimen during the treatment period.

[0165] Alternatively, several treatment cycles can be performed.

[0166] In some cases, the administration regimen is an intermittent administration regimen, which has two or more treatment cycles that alternate with the washout period.

[0167] In some cases, the NGF applied according to this disclosure is administered to the subject for at least three cycles, wherein each cycle consists of seven days of daily intranasal administration of NGF followed by a 21-day washout period (i.e., a period during which no NGF is administered to the subject).

[0168] The effective amount of NGF used in each application, the duration of treatment, and the number of daily applications are selected by the technician based on the characteristics of the patient, the severity of the movement disorder or other condition to be prevented or treated, and the assessment tests performed during treatment.

[0169] In some cases, the total dose of NGF applied to the subject according to the present disclosure is from 25 µg / kg to 400 µg / kg, preferably from 30 µg / kg to 300 µg / kg, more preferably from 35 µg / kg to 200 µg / kg, more preferably from 40 µg / kg to 100 µg / kg, more preferably from 40 µg / kg to 60 µg / kg, more preferably from 45 µg / kg to 55 µg / kg, and even more preferably from 50 µg / kg.

[0170] In some cases, the dose of NGF applied to the subject according to the present disclosure per cycle is from 15 µg / kg to 20 µg / kg, more preferably from 16 µg / kg to 17 µg / kg, even more preferably from 16.6 µg / kg to 16.8 µg / kg, and most preferably from 16.7 µg / kg.

[0171] In some cases, the daily dose of NGF applied to the subject according to the present disclosure is from 2 µg / kg to 3 µg / kg, preferably from 2.3 µg / kg to 2.5 µg / kg, and more preferably 2.4 µg / kg.

[0172] In some cases, the dose of NGF applied to the subject according to the present disclosure is from 0.3 µg / kg / nostril to 1 µg / kg / nostril, more preferably from 0.4 µg / kg / nostril to 0.8 µg / kg / nostril, more preferably from 0.5 µg / kg / nostril to 0.7 µg / kg / nostril, and most preferably 0.6 µg / kg / nostril.

[0173] In some cases, the daily dose of NGF used according to this disclosure is administered to the subject twice daily at 12-hour intervals.

[0174] In some cases, the amount of NGF administered intranasally is about 5 µg to about 1 mg. In some cases, the amount of NGF administered intranasally is about 10 µg to about 400 µg. In some cases, the amount of NGF administered intranasally is about 15 µg to about 200 µg. In some cases, the amount of NGF administered intranasally is about 20 µg.

[0175] In some cases, subjects are treated under hypothermic conditions prior to NGF treatment.

[0176] This disclosure provides a method for treating newborns diagnosed with hypoxic-ischemic encephalopathy (NHIE) under hypothermic conditions. The method involves administering controlled therapeutic hypothermia to the newborn, wherein the newborn's body temperature is maintained below the normal range, typically 32°C to 34°C, for a defined duration, typically 72 hours. This therapeutic hypothermia is initiated as soon as possible after diagnosis of NHIE, typically within the first six hours of life, to reduce brain damage caused by oxygen deprivation and improve neurological outcomes. The newborn is carefully monitored throughout the treatment, with continuous assessment of core body temperature, neurological function, and vital signs to ensure the hypothermic condition is maintained safely without causing further harm. During this period, the newborn may be placed in a specialized cooling device or incubator where the temperature is regulated to provide optimal conditions for neuroprotection. Once the cooling phase is complete, gradual rewarming is performed to restore the newborn to normal body temperature. This method is effective in reducing the degree of brain damage, enhancing recovery, and improving long-term neurological function in infants with NHIE.

[0177] In another embodiment, this disclosure provides a late-term therapeutic administration of nerve growth factor (NGF) to a subject to treat or prevent brain diseases. In this method, NGF is administered starting one month after birth, with a defined therapeutic window extending up to a maximum of 36 months after birth. Preferably, administration occurs between one and 24 months after birth, more preferably between one and 12 months, even more preferably between one and six months, and most preferably between one and four months. Administration of NGF during this period aims to support ongoing neuronal maturation, synaptic connections, and neural plasticity, which remain crucial after the neonatal period. By delivering NGF within these timeframes, this treatment can mitigate progressive neuronal damage, enhance cognitive and motor function, and improve long-term neurological outcomes.

[0178] In another embodiment, this disclosure describes the administration of nerve growth factor (NGF) within a defined postnatal window of three to nine months after birth, with a preferred time range of three to six months after birth.

[0179] In some cases, the NGF is human NGF.

[0180] In some cases, the human NGF has the following amino acid sequence as SEQ ID NO: 1:

[0181] Alternatively, the human NGF has the following amino acid sequence as SEQ ID NO: 2:

[0182] Alternatively, the human NGF may be a mixture of NGFs having the sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

[0183] The amino acid sequence of human NGF in SEQ ID NO: 2 differs from that of NGF in SEQ ID NO: 1 only in the presence of two additional amino acids at the C-terminus. Both forms of NGF are present in human cells and are therefore considered to be wild-type human NGF.

[0184] Therefore, when “human NGF” or “wild-type human NGF” is mentioned in this application, it means the human NGF of SEQ ID NO: 1 or SEQ ID NO: 2.

[0185] In some cases, the NGF is generated using recombinant DNA technology. In some cases, the NGF is human recombinant NGF (rhNGF). Methods for generating rhNGF are known to those skilled in the art, such as those described in WO0022119A1 and WO2013092776A1.

[0186] In some cases, the purity of the NGF is greater than 70%. In other cases, the purity of the NGF is greater than 80%, greater than 90%, greater than 95%, greater than 98%, or greater than 99%. The purity of NGF can be determined by conventional methods known to those skilled in the art, such as by HPLC analysis. Essentially pure means a substance that is at least 70% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0187] Another object of this disclosure is a pharmaceutical composition for intranasal administration comprising NGF as described above and at least one pharmaceutically acceptable excipient, said pharmaceutical composition for the prevention and / or treatment in subjects of motor disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn (HIE), said pharmaceutical composition being administered intranasally to said subjects.

[0188] In some cases, the movement barrier is as described above.

[0189] Another object of this disclosure is a pharmaceutical composition for intranasal administration comprising NGF as described above and at least one pharmaceutically acceptable excipient, said pharmaceutical composition for the prevention and / or treatment in subjects of cerebral palsy (CP) associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy (HIE), said pharmaceutical composition being administered intranasally to said subjects.

[0190] In some cases, the cerebral palsy (CP) is as described above.

[0191] Another object of this disclosure is a pharmaceutical composition for intranasal administration comprising the NGF as described above and at least one pharmaceutically acceptable excipient, said pharmaceutical composition for the prevention and / or treatment in subjects of neurodevelopmental disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn (HIE), said pharmaceutical composition being administered intranasally to said subjects.

[0192] In some cases, the neurodevelopmental disorder is as described above.

[0193] Another object of this disclosure is a pharmaceutical composition for intranasal administration comprising NGF as described above and at least one pharmaceutically acceptable excipient, said pharmaceutical composition for promoting neural development in subjects affected by hypoxic-ischemic encephalopathy (HIE), said pharmaceutical composition being administered intranasally to said subjects.

[0194] In some cases, the neural development is as described above.

[0195] In some cases, the pharmaceutical composition used according to this disclosure is a liquid intranasal composition.

[0196] In some cases, the pharmaceutical composition used according to this disclosure comprises an effective amount of the NGF as described above and at least one pharmaceutically acceptable excipient suitable for intranasal use. In some cases, the pharmaceutically acceptable excipient is selected from solvents, thickeners, mucosal adhesives, buffers, antioxidants, surfactants, preservatives, and penetration enhancers.

[0197] In some cases, the concentration of NGF in the liquid intranasal composition used according to this disclosure is from about 5 µg / ml to about 1 mg / ml. In some cases, the concentration of NGF in the liquid intranasal composition used according to this disclosure is from about 10 µg / ml to about 400 µg / ml. In some cases, the concentration of NGF in the liquid intranasal composition used according to this disclosure is from about 15 µg / ml to about 200 µg / ml. In some cases, the concentration of NGF in the liquid intranasal composition used according to this disclosure is about 20 µg / ml.

[0198] In some cases, the solvent is water.

[0199] In some cases, the mucosal adhesive is glycerin. In some cases, the concentration of the mucosal adhesive (e.g., glycerin) is from 0.05% w / v to 0.2% w / v. In some cases, the concentration of the mucosal adhesive (e.g., glycerin) is 0.1% w / v.

[0200] In some cases, the antioxidant is methionine. In some cases, the concentration of the antioxidant (e.g., methionine) is from about 0.005 mg / ml to about 0.02 mg / ml. In some cases, the antioxidant (e.g., methionine) is about 0.01 mg / ml.

[0201] In some cases, the surfactant is Kolliphor P188. In some cases, the concentration of the surfactant (e.g., Kolliphor P188) is from about 0.05% w / v to about 0.2% w / v. In some cases, the surfactant (e.g., Kolliphor P188) is about 0.1% w / v.

[0202] In some cases, the buffer is a phosphate buffer.

[0203] In some cases, the penetration enhancer is n-dodecyl-β-D-maltose. In some cases, the concentration of the penetration enhancer (e.g., n-dodecyl-β-D-maltose) is from about 0.1% w / v to about 1% w / v. In some cases, the penetration enhancer (e.g., n-dodecyl-β-D-maltose) is about 0.5% w / v.

[0204] In some cases, the liquid intranasal composition used according to this disclosure comprises, or is composed of, NGF, sodium chloride, phosphate buffer and water as described above.

[0205] In some cases, the liquid intranasal composition used according to this disclosure comprises or consists of the following: NGF as described above, sodium chloride, phosphate buffer, Kolliphor P188, L-methionine, and water.

[0206] In some cases, the liquid intranasal composition used according to this disclosure comprises or consists of the following: NGF as described above, sodium chloride, phosphate buffer, Kolliphor P188, L-methionine, glycerol, n-dodecyl-β-D-maltodextrin, and water.

[0207] In some cases, the liquid intranasal composition used according to this disclosure comprises or consists of the following: NGF, NaH2PO4 H2O, NaCl, Kolliphor P188, β-L-methionine. In some cases, the liquid nasal composition contains dodecyl-β-D-maltose. In some cases, the liquid nasal composition contains glycerol. In some cases, the liquid nasal composition contains water.

[0208] In some cases, the liquid intranasal composition used according to this disclosure comprises or is composed of the following components:

[0209] - The concentration of NGF as described above is:

[0210] Approximately 5 µg / ml to approximately 1 mg / ml

[0211] Approximately 10 µg / ml to approximately 400 µg / ml, or

[0212] From approximately 15 µg / ml to approximately 200 µg / ml,

[0213] - NaH2PO4 H2O, its concentration is:

[0214] Approximately 5 to approximately 8 mg / ml, or

[0215] Approximately 6.9 mg / mL,

[0216] - NaCl, its concentration is:

[0217] Approximately 5 to approximately 6.5 mg / ml, or

[0218] Approximately 5.84 mg / mL

[0219] - Kolliphor P188, its concentration is:

[0220] Approximately 0.05% w / v to approximately 0.2% w / v, or

[0221] Approximately 0.1% w / v,

[0222] -L-methionine, its concentration is:

[0223] Approximately 0.05 mg / ml to approximately 0.2 mg / ml, or

[0224] Approximately 0.1 mg / ml,

[0225] - Optionally, n-dodecyl-β-D-maltodextrin, at a concentration of:

[0226] Approximately 0.1% w / v to approximately 1% w / v, or

[0227] Approximately 0.5% w / v,

[0228] and / or

[0229] - Glycerin, with the following concentration:

[0230] Approximately 0.05% w / v to approximately 0.2% w / v, or

[0231] Approximately 0.1% w / v, and

[0232] - water.

[0233] The pharmaceutical compositions used according to this disclosure can be properly formulated using suitable methods known in the art or by methods disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton, Pa.

[0234] In some cases, the NGF used in the methods of this disclosure is in the form of a pharmaceutical composition as described above.

[0235] This disclosure will be further described in the following embodiments, which do not limit the scope of this disclosure as set forth in the claims.

[0236] Example

[0237] Example 1

[0238] The biodistribution of NGF in rat brain regions following a single intranasal administration was evaluated.

[0239] The formulation containing 1.2 mg / mL rhNGF (i.e., containing SEQ ID NO: 1) was administered intranasally to rats once.

[0240] Rats were sacrificed at different time points corresponding to 2 hours, 4 hours, 8 hours and 24 hours after treatment.

[0241] Samples were collected from the parietal cortex, hypothalamus, thalamus, striatum, hippocampus, brainstem, frontal cortex, and medial septum for quantification of rhNGF by ELISA and calculation of cumulative uptake (2 to 24 hours).

[0242] Prior to analysis, brain samples were homogenized on ice using an ultraturax device and centrifuged to recover the supernatant. A commercial ELISA kit (RayBiotech, catalog ELH-BNGF) was used according to the supplier's instructions. The calibration curve range for NGF determination was set from 20.5 to 5000 pg / mL.

[0243] like Figure 1 As shown, NGF uptake is evident in all brain tissues, but the protein is particularly concentrated in areas involved in motor activity, especially in the hypothalamus and thalamus.

[0244] Example 2

[0245] The effects of NGF on motor disorders associated with hypoxic-ischemic encephalopathy in newborns were evaluated in a representative mouse model of the disease.

[0246] Ipsilateral ischemic injury was induced based on the Rice-Vannucci model with minor modifications (Rice et al., Ann. Neurol., 9 (1981), pp. 131-141; RC Vannucci et al., J. Cerebr. Blood Flow Metabol., 24 (2004), pp. 1090-1097; SJ Vannucci and H. Hagberg, J. Exp. Biol., 207 (2004), pp. 3149-3154; Xu et al., Mol. Neurobiol., 53 (2016), pp. 5962-5970).

[0247] Specifically, C57BL / 6 mice (day 7 postnatal) were anesthetized with 1.5% sevoflurane and 98.5% O2 (oxygen concentrator, model LFY-I-5). Throughout the procedure, the animals' body temperature was maintained at 37 ± 0.5°C using a heating pad. Under a surgical stereomicroscope, a midline skin incision (0.5 cm) was made in the neck, exposing the right common carotid artery (CCA), which was double-ligated with 6-0 sutures to isolate the upstream and downstream areas of the area to be cut. The CCA was cut between the two ligatures using specific micro-clippers. The incision was flushed with 1% lidocaine and sutured with 6.0 polypropylene sutures. The animals were returned to their mothers and monitored continuously during the 1-hour recovery period. Next, the young mice were subjected to hypoxia (60 minutes) induced by placing them in a hypoxic chamber circulated with a balanced gaseous mixture (8% O2 and 92% N2), the composition of which was monitored using an oxygen monitor. The hypoxic chamber was placed in a water bath heated to 37°C. At the end of the procedure, the young mice were returned to their mothers. The animals were continuously monitored for 30 minutes, then every 30 minutes for 2 hours, and then daily until euthanasia.

[0248] athletic performance test

[0249] The following tests were conducted to evaluate the animal’s motor performance: suspension line test, swivel bar test, and beam walking test.

[0250] - Suspension line test

[0251] The suspension wire test examines forelimb motor strength in mice (Crawley, JN What's Wrong with MyMouse: Behavioral Phenotyping of Transgenic and Knockout Mice. 2nd ed., Wiley-Interscience, 2007). In this test, mice are trained to suspend themselves from a 2 mm diameter wire using only their forelimbs (SansBio, China). The wire is held 40 cm above a soft pillow by two posts (Dirnagl, U. 2010, Royl, G. 2009, Brain Res. 1265, 148-157). Mice were trained for two days, from day 21 (P21) to day 28, with three trials per day. The time until the mouse falls (holding time) was recorded, and the average of the three trials was further analyzed.

[0252] - Crossbeam walking test

[0253] Sensorimotor coordination was tested using a balanced beam (45 cm long; 30% tilt). From day 21 after birth (P21) to P28, each mouse was tested three times on each beam, two days a week. Latency for crossing the beam was scored and averaged. The test was terminated if the mouse failed to cross the beam within the specified time, and the maximum time (180 seconds) was measured (Anzilotti et al., Cell Death Dis., 2015 Dec 3;6(12):e2004).

[0254] - Spinning rod test

[0255] From day 21 (P21) to P28, each mouse underwent three trials on each rotundus, two days a week. Motor coordination and balance were assessed using a five-station mouse rotundus apparatus (Ugo Basile; Milan, Italy). At each station, the rod was 6 cm long and 3 cm in diameter. Mice were trained to maintain balance at increasing speeds (up to a constant speed of 14 rpm) for three consecutive trials. The testing phase consisted of a rotundus trial performed weekly. In this phase, the rotation speed was increased from 4 rpm to 14 rpm over 60 seconds. The maximum 60-second latency was assigned to mice that did not fall at all (Giampa et al., PLoS One, 2010 Oct 15;5(10):e13417).

[0256] Drug administration

[0257] Starting 7 hours after HI induction, mice were treated daily with either a carrier or rhNGF (50 ug / Kg) (i.e., containing SEQ ID NO: 1) administered intranasally for 3 weeks. Intranasal administration was performed as follows.

[0258] Using your dominant hand, load 10 µl of the compound or carrier into a P-20 micropipette. Place the tip of the filled pipette at a 45-degree angle near the mouse's left nostril. Position the droplet close enough to both the left and right nostrils so that the mouse can aspirate it. Immediately after the mouse aspirates the droplet, expel any remaining compound from the pipette tip to form another droplet, which the mouse aspirates through the same nostril approximately 2 to 3 seconds later. The mouse should remain in this position for 15 seconds after administration.

[0259] Research Design

[0260] Behavioral assessments were conducted 28 to 30 days after birth.

[0261] The animals were divided into the following groups:

[0262] - Sham surgery / loador (n = 10)

[0263] - Sham surgery + rhNGF (50 µg / Kg) (n = 10)

[0264] - HI + carrier (n = 10)

[0265] - HI + rhNGF (50 µg / Kg) (n = 10)

[0266] like Figure 2 As shown, intranasal intermittent delivery of rh-NGF (50 ug / kg, administered for 21 days) was initiated 7 hours after HI (when secondary energy exhaustion begins (Tetorou K et al., FrontSynaptic Neurosci. 2021 Aug 24; 13:709301)) and continued for the following 3 weeks.

[0267] Data were analyzed using GraphPad Prism version 8.04 (GraphPad Software). Data from the behavioral experiment are presented as mean ± SEM. Differences between groups were analyzed using one-way ANOVA followed by Tukey post-hoc tests, with treatment (drug or carrier) used as a factor in the analysis.

[0268] result

[0269] In the suspension test, mice were trained to suspend their bodies from a wire using only their forelimbs. Univariate ANOVA analysis showed that the high-intensity state (HI) significantly affected the duration of suspension in mice (F(3, 36) = 7.16, P = 0.0007). Figure 3 At 28 days after birth, the hold time of HI mice (25.9 ± 2.27 seconds) was significantly shorter than that of sham-operated mice (40.5 ± 2.68 seconds), indicating a decrease in grip strength in HI mice (p = 0.0012). Figure 3 Interestingly, intranasal administration of rhNGF daily for 3 weeks, starting 7 hours after HI, showed a significant rescue of muscle strength. In fact, HI / rhNGF mice showed a retention time of 36.5 ± 2.21 seconds compared to HI mice treated with the load (p = 0.0024). Figure 3 Conversely, the same treatment did not affect the retention time in sham-operated mice (39.6 ± 2.79 seconds; p = 0.99). Figure 3 ).

[0270] The rotarod test is commonly used to measure motor coordination and balance in mice (Crawley, JN, What's Wrong with My Mouse: Behavioral Phenotyping of Transgenic and Knockout Mice. 2nd ed., Wiley-Interscience, 2007). In the rotarod test, mice trained 29 days after birth to suspend themselves on a wire using only their forelimbs were able to walk continuously on the rotarod and rarely fell off after continuous training. Univariate ANOVA analysis showed that the HI state had a significant effect on the holding time of the mice (F(3, 36) = 7.71, P = 0.0004). In fact, quantitative measurements showed that the holding time of the sham-operated mice was 989.7 ± 41.47 seconds, compared to a significantly shorter holding time in the HI group (567.3 ± 51.03 seconds, p = 0.014). Figure 4 As observed in the suspension test, intranasal administration of rhNGF daily for 3 weeks, starting 7 hours after HI, significantly increased the retention time of HI mice on the wheel. In fact, HI / rhNGF mice showed a retention time of 889.8 ± 27.79 seconds compared to HI mice treated with the load (p = 0.011). Figure 4 Conversely, the same treatment did not affect the sham-operated mice's retention time on the wheel (865 ± 119.26 seconds; p = 0.63). Figure 4 ).

[0271] The cone-beam test is commonly used to evaluate hind leg motor dysfunction in mice (Schaar, K. L, et al., ExpTransl Stroke Med. 2010 Jul 19;2(1):13). Sham-operated mice used a central plate and crossed the beam without many errors (mistakes) on the edges (2.9 ± 0.43). Figure 5 However, HI mice showed a consistent increase in foot mishaps at 30 days after birth (7 ± 0.47, p < 0.0001). Figure 5 Univariate ANOVA analysis showed that HI status had a significant effect on the number of errors in HI mice (F (3, 36) = 20.68, P < 0.0001). Similar to other motor tests, intranasal administration of rhNGF daily for 3 weeks starting 7 hours after HI normalized the number of errors in HI mice compared with mice treated with a carrier (5.2 ± 0.49, p = 0.04). Figure 5 Conversely, the same treatment did not affect the number of errors in sham-operated mice (3.5 ± 0.42; p = 0.76). Figure 5 ).

[0272] The publicly available data comprehensively demonstrate that intranasal administration of NGF can restore muscle strength, motor coordination, and balance in a mouse model of motor disorders associated with HIE. Therefore, these data support the efficacy of intranasal NGF in the prevention and / or treatment of motor disorders associated with hypoxic-ischemic encephalopathy in newborns.

[0273] Example 3

[0274] Intranasal administration of recombinant human nerve growth factor (rhNGF) in a preclinical model of hypoxic-ischemic encephalopathy in newborns.

[0275] The following study aimed to evaluate the therapeutic efficacy of recombinant human nerve growth factor (rhNGF, cenegermin-bkbj; i.e., containing SEQ ID NO: 1) in a rodent model of neonatal hypoxic-ischemic injury (NHIE). The effects of two rhNGF treatment regimens (early or late) were evaluated. Animals treated with the NHIE model were longitudinally monitored using a series of behavioral tests and neuroimaging techniques.

[0276] To replicate the consequences of perinatal ischemic injury, investigate its neurological outcomes, and evaluate the potential role of pharmacological treatment, the chosen animal model involved subjecting 7-day-old rats to unilateral common carotid artery ligation followed by 3 hours of exposure to hypoxia (a neonatal hypoxic-ischemic encephalopathy model, NHIE, as described in Carloni et al., Neurobiol Dis. 2006 Jan;21(1):119-26). From a neurodevelopmental perspective, this stage in rats is considered equivalent to that in full-term human newborns.

[0277] method

[0278] animal

[0279] All animal procedures were performed according to protocols approved by the Italian Ministry of Health, authorization number n° 1014 / 2024-PR. Pups of the Sprague-Dawley strain of the brown rat (Rattus Norvegicus) were used in the experiments discussed below. Pregnant dams were received at approximately 15 days of gestation. Special care was provided to the dams before and after parturition, and they were housed in breeding facilities authorized by the Ministry of Health. Pups were evenly distributed among the dams, if possible. Veterinarians monitored both dams and pups for any signs of distress.

[0280] Neonatal hypoxic-ischemic encephalopathy (NHIE) model

[0281] On day 7 after birth (P7), animals were anesthetized and prepared for surgery to detach the right carotid artery. Young mice underwent ligation of the right common carotid artery, followed by 3 hours of hypoxia (92% nitrogen and 8% oxygen) in a ventilated cage. Throughout the ligation procedure, animals were maintained under deep general anesthesia, with their body temperature kept at physiological levels using a feedback-controlled heating pad. Anesthesia was induced with 3% isoflurane in an O2 / N2O (30:70) ratio and maintained with 1% to 1.5% isoflurane in an O2 / N2O (30:70) ratio. The vascular surgery for carotid artery ligation lasted 10 minutes. Young mice were then transferred to a hypoxic chamber for 3 hours without further manipulation. Sham-operated mice received carotid artery exposure without ligation or hypoxia. At the end of the procedure, animals were assigned to their specific experimental groups (loader, early or late hNGF treatment). A schematic diagram of the experimental timeline is shown in [link to diagram]. Figure 6 As shown in the image.

[0282] Early treatment (early)

[0283] The animals were treated immediately after the hypoxia. The drug (cinegmentin-bkbj) was administered intranasally at a dose of 100 µg / kg, divided evenly into three rounds, 90 minutes apart. After a three-day rest period following the cessation of treatment, the procedure was repeated twice.

[0284] Late-stage treatment (late stage)

[0285] The animals in this group received the same treatment as described above, but starting 7 days after NHIE / sham surgery.

[0286] Sensorimotor deficits were assessed using neurological scoring.

[0287] Sensorimotor deficits were assessed using a neurological scoring system, as described in Valente A., et al., J Cereb Blood Flow Metab. 2023 Jul;43(7):1077-1088. Scores ranged from 0 (no deficit) to 56 (worst neurological outcome) and included both generalized and focal deficits. Generalized deficits were described using a score of 0 to 28 to assess the rat's overall health. This score provided information about the rat's physical appearance, namely: fur (0 to 2), ears (0 to 2), eyes (0 to 4), posture (0 to 4), spontaneous activity (0 to 4), and the presence of seizures (0 to 12). Focal deficits were described using a scale of 0 to 28 and were evaluated by the following observations: body symmetry (0 to 4), gait (0 to 4), ability to climb 45° inclined surfaces (0 to 4), circling behavior (0 to 4), forelimb symmetry (0 to 4), compulsive circling (0 to 4), and whisker response (0 to 4).

[0288] Cognitive deficit

[0289] T-Maze: On day 28, hippocampal-dependent spatial memory was measured in rats using a standard two-arm T-maze apparatus (50 × 40 × 10 cm per arm). The animals were tested by researchers unaware of their identities. Animals were placed in the starting arm of the T-maze and allowed to freely choose to enter one of the two arms in each of seven trials. Successful alternation was defined as alternating arm entry, while unsuccessful alternation occurred when the rat returned to the most recently explored arm. The total number of arm entries and the order of entry were recorded for each rat, and the percentage of correct choices was calculated as: (Number of correct alternations / Maximum number of alternations) × 100. Rats with intact spatial memory will remember previously visited arms and will preferentially enter new, unexplored arms (alternation rate ≥ 60%).

[0290] Barnes Maze: This test assesses spatial learning and memory and is conducted on day 28. The maze consists of circular platforms with multiple openings around its perimeter, only one of which leads to an escape tunnel or a safe area. The test begins with an adaptation trial (day 1), during which each rat is placed under a beaker in the center of an open area for 30 seconds, then guided to the escape tunnel within 10 to 15 seconds, and given 2 minutes to spontaneously enter the escape tunnel. The rat is allowed to remain in the escape tunnel for 1 minute before being returned to its cage. In the learning phase (days 2–4), the rat is placed in the center of the area for 10 seconds, and then allowed to explore the area for at least 2 minutes to find the escape tunnel (initial latency) and enter it (secondary latency). The testing phase (day 5) consists of the following: the escape tunnel is removed from the maze to assess the initial latency; the escape opening is sealed (“dummy escape opening”) to prevent the rat from falling out; and the rat’s ability to find the escape tunnel within 2 minutes is recorded.

[0291] Magnetic resonance imaging (MRI)

[0292] Rats anesthetized with isoflurane (1% to 1.5%, in 30% O2, 70% N2O) were placed in a magnet, and both body temperature and respiration were monitored throughout the experiment, allowing for adjustment of the optimal level of anesthesia as needed. MRI acquisition was then initiated. At the end of the experiment, the animals were removed from the mat and returned to their cages, where they were monitored until they awoke.

[0293] Brain imaging was performed on a 7T small-aperture animal scanner (BioSpec®; Bruker, Ettlingen, Germany) running ParaVision 6.01 and equipped with orthogonal 1H CryoProbe™ (Bruker, Ettlingen, Germany) surface coils as transmitters and receivers. Diffusion tension imaging (DTI) was obtained to quantify white matter (WM) damage.

[0294] DTI. Acquired echo-plane imaging sequence (TR / TE = 7000 / 32 ms, resolution 125 × 125 µm) 2 FOV 1.5 × 1.5 cm 2 The acquisition matrix was 120 × 120, and the slice thickness was 0.3 mm. An 800 mm band was applied along 19 isotropic directions. 2The diffusion coding b-factor was calculated at a rate of / second, with two B0 unweighted images acquired for each repetition. The diffusion tensor was computed using FSL software. First, a group-averaged full tensor template was created using a population-based DTI map construction algorithm, which employs a tensor-based registration procedure embedded in the DTI-TK software library. Then, the average template was resampled to an in-plane resolution of 100 × 100 µm. 2 The slices were 0.2 mm thick and skeletonized. Anisotropy fraction (FA) images of all objects were normalized relative to the mean template using a differential homeomorphic transformation. This transformation was applied to all DTI measures (MD, RD, AD), which were warped to the mean skeleton for region of interest (ROI) based analysis. The diffusion tensor was computed using FSL software. First, a group-averaged full tensor template was created using a population-based DTI map construction algorithm, employing a tensor-based registration procedure embedded in the DTI-TK software. The mean template was then resampled to an in-plane resolution of 100. 100 lm² and slice thickness 0.2 mm. Then, high-resolution FA maps based on population templates were generated and skeletonized.

[0295] The target area analyzed is the entire corpus callosum, which extends from +2 mm to -3 mm relative to the anterior fontanelle in the anteroposterior direction.

[0296] Execution

[0297] Animals were deeply anesthetized prior to sacrifice. Cerebrospinal fluid (CSF) and blood were collected from the cisterna magna and vena cava, respectively. The CSF was immediately frozen on dry ice. Blood was collected into dedicated tubes washed with EDTA and centrifuged at 2000 rcf for 20 minutes at 4°C to obtain plasma, which was stored in an ultra-cryogenic device at -80°C until use. Fresh tissues were perfused and fixed or rapidly frozen for histological or biochemical analysis.

[0298] result

[0299] Growth curves. Regardless of the treatment regimen, rats receiving hNGF showed a steeper growth curve than rats receiving the load. Figure 7 and 8 This indicates that rats treated with hNGF (both early and late stages) tended to reach essentially physiological body weight at the endpoint compared to rats treated with the carrier. Furthermore, although rats treated with hNGF (early stage) did not reach the same body weight as sham-operated animals ( Figure 7However, rats treated with hNGF (late stage) reached and exceeded the body weight of sham-operated animals at P42. Figure 8 Weight loss indicates brain injury (Llovera et al., Sci Transl Med. 2015 Aug 5;7(299):299ra121). Therefore, these results suggest that rats treated with NGF had less brain injury than those treated with the carrier and their overall health was restored to a level comparable to sham-operated animals.

[0300] Neurological scoring. Sensorimotor deficits were analyzed at week 2 following NHIE. The NHIE model induced significant sensorimotor deficits. Compared to the carrier, administration of hNGF (early) improved behavior in rats at week 2 following NHIE. Figure 9 For NHIE rats receiving hNGF (late stage), the trend toward improvement in sensorimotor deficits was significant. Figure 10 Therefore, when compared with NHIE rats, both treatments showed beneficial effects on sensorimotor deficits.

[0301] T-maze. The ability to alternate between the two arms of the T-maze was assessed at week 3 following NHIE induction. NHIE induced a slight deficiency in the rats' ability to alternate maze arms. No effect was observed after hNGF (early). Figure 11 Conversely, data associated with NHIE rats receiving hNGF (late) showed a conserved alternation capacity compared to rats receiving the load. Figure 12 ).

[0302] Barnes Maze. This test (performed 28 days after NHIE induction) evaluated learning and memory tasks. After three days of training, all rats were able to learn the location of the escape hole in the maze. Figure 13 and Figure 14 No effect was observed after hNGF (early stage). Figure 13 It is noteworthy that NHIE rats receiving hNGF (late stage) were able to rapidly locate the escape pore from day 1. Figure 14 ).

[0303] Comprehensive health score. The data above represent different aspects of rat health, including weight gain and sensorimotor and cognitive deficits. A comprehensive score was created based on the distribution of each animal across the quartiles at different readouts (body weight at P42, neurological score at week 2 (w2), T-maze at week 3 (w3), and Barnes maze at week 4 (w4)). Quartiles were defined within the NHIE / veh group. Data falling into Q4 (best performance) received a score of 4, and data falling into other quartiles were correspondingly scored 1 - worst performance. Figure 15 (A to 15B). For contingency analysis, 2.75 is considered the cutoff value indicating a favorable outcome. Figure 15 C). NHIE rats had worse outcomes than sham-operated rats, while rats receiving hNGF (late) showed the best scores among NHIE rats. This suggests that late administration of hNGF has a surprisingly higher efficacy in restoring health status compared to early administration in NHIE rats.

[0304] MRI. The effect on white matter damage was assessed 5 weeks post-injury using diffusion tensor imaging (DTI) MRI analysis. Figure 16 As shown, NHIE rats receiving hNGF (late) exhibited an increased anisotropy fraction compared to rats receiving the carrier, indicating the protective effect of hNGF on white matter.

[0305] In summary, the results discussed above indicate that hNGF administration in a rat model of NHIE has beneficial effects on both the typical sensorimotor and cognitive deficits of the disease. Surprisingly, later administration of hNGF showed superior efficacy against cognitive deficits compared to earlier administration, which could be used to determine the therapeutic window in humans to maximize the effects of hNGF on motor and neurodevelopmental disorders caused by NHIE.

[0306] Example 4

[0307] This study aims to evaluate the efficacy, safety, pharmacokinetics, and immunogenicity of a pharmaceutical composition containing nerve growth factor (NGF) for the treatment of neonatal hypoxic-ischemic encephalopathy (NHIE) in infants. The study population will include infants diagnosed with NHIE based on the modified Sarnath staging. Eligible participants may have experienced standard therapeutic hypothermia within six hours of birth.

[0308] This study will consist of a control group receiving standard care (therapeutic hypothermia only) and an experimental group receiving different doses of a drug composition containing NGF, administered intranasally. The drug composition contains recombinant human NGF (rhNGF) in a sterile solution or suspension and is designed for safe and effective neonatal administration.

[0309] Inclusion criteria for this study required informed consent from parents or legal guardians, demonstrating a full understanding of the informed consent form (ICF). This included understanding the nature and purpose of the study, potential risks and side effects, and the ability to cooperate with the investigators while adhering to all study requirements. Both male and female participants were eligible. The study included infants born at gestational age greater than 36 weeks and with a corrected age of 1 to 3 months. Participants must have experienced perinatal suppression, indicated by at least one of the following: an Apgar score less than 5 at 10 minutes; requiring resuscitation at 10 minutes (e.g., chest compressions, mechanical ventilation, or CPAP); having cord blood gas or neonatal blood gas with a pH below 7.00 (obtained within 60 minutes of birth); or a base deficiency of 15 mmol / L or higher in a neonatal blood gas sample (arterial or venous) collected from the cord or within the first 60 minutes after birth. Additionally, participants must have a diagnosis of neonatal hypoxic-ischemic encephalopathy (NHIE) classified as stage 2 or 3 according to the modified Sarnath staging system. Finally, all participants must have received standard therapeutic hypothermia treatment, which begins within the first six hours after birth.

[0310] Exclusion criteria for this study included birth weight less than 1800 grams and microcephaly (defined as head circumference three standard deviations or more below the gestational age mean). Patients in a critical condition, those with motor disorders identified by the investigator as being caused by reasons other than those studied, and individuals with congenital malformations that posed an unacceptable risk to safety or interfered with the study evaluation were also excluded. Additionally, participants identified by newborn genetic screening as having a positive result for neurodevelopmental disorder (NDD) and / or neuromuscular disorder (NMD) through chromosomal microarray analysis, whole-exome sequencing, or genome sequencing were ineligible. The study also excluded individuals taking certain concomitant medications (to be determined), individuals who had recently undergone surgery or planned to undergo surgery during the study period, and patients who had participated in, were currently participating in, or planned to participate in another interventional study within the specified period. Finally, individuals who could not comply with the study procedures were excluded.

[0311] The primary objective of this study was to evaluate the efficacy of intranasal cinegrin in reducing the incidence of major neurodevelopmental disorders by age two in infants diagnosed at birth with hypoxic-ischemic encephalopathy (NHIE). Secondary objectives included assessing its efficacy in reducing the incidence of cerebral palsy and other neurodevelopmental disorders, as well as evaluating its safety and tolerability throughout the study treatment. Additionally, the study aimed to determine the systemic pharmacokinetic profile of intranasal cinegrin after administration and to evaluate its immunogenicity in infants diagnosed with NHIE. As an exploratory objective, the study will also evaluate biomarkers associated with target binding.

[0312] The primary endpoint of this study was the assessment of neurodevelopmental outcomes using the Bayley Scales of Infant and Toddler Development-4 (BATD-4), which evaluates cognitive, linguistic, motor, socio-emotional, and adaptive behaviors. Secondary endpoints included standardized neurological examinations classified according to the Gross Motor Function Classification System (GMFCS), brain MRI results, and assessments using EEG, visual evoked potentials (VEP), motor evoked potentials (MEP), and brainstem auditory evoked potentials (BAEP). Other secondary endpoints included autism assessment, quality of life (QoL) questionnaires, and monitoring the incidence of serious and non-serious adverse events throughout the study. The study also measured changes in serum anti-drug antibody (ADA) levels from baseline to 30 days after initiation of intranasal cinegrimin treatment. Exploratory endpoints will focus on neuroinflammatory biomarkers, including IL-1β, IL-6, IL-8, TNF-α, IL-10, IFN-γ, and MIP-1α, as well as neurodegenerative markers such as Tau, neurofilament light (NfL), and glial fibrillary acidic protein (GFAP). Additionally, this study will evaluate the neuroendocrine profile by measuring levels of vasopressin (ADH), cortisol, adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), T3, and T4.

[0313] The assessment criteria for normal, mild, moderate and severe forms of NHIE are shown in Table 1.

[0314] Table 1.

[0315] Evaluation criteria Normal (N) Mild Moderate (Mod) Severe (S) Level of consciousness Alertness / Appropriate Awareness Hyperalert Drowsiness catatonia or coma Spontaneous activities normal Normal or increased reduce none posture normal Normal or mild distal flexion Distal flexion, full extension brain removal tension normal Normal or increased height of trunk and limbs Hypotonia (focal or generalized) relaxation Sucking reflex normal Normal or incomplete weak Missing Moro reflex powerful Strong, low threshold Incomplete Missing Autonomous System Pupils are equal in size and respond to light; heart rate and respiration are normal. Pupils are equal in size and respond to light; heart rate and respiration are normal. Pupil constriction; bradycardia or periodic / irregular breathing Pupil deviation / dilation / unresponsiveness; variable heart rate or apnea Seizures none none Common, focal or multifocal Uncommon (excluding brain removal)

[0316] Assess the tension in both the limbs and the trunk / neck—hypotonia in either limb or trunk is considered a criterion.

[0317] Implementation Plan

[0318] Implementation Scheme 1 (E1). Nerve growth factor (NGF) for the prevention and / or treatment in subjects of neurodevelopmental disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy in newborns, wherein the NGF is administered intranasally to the subjects.

[0319] E2. The NGF used according to E1, wherein the NGF is used to cure neurodevelopmental disorders in the subject that are associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy in newborns.

[0320] E3. The NGF used according to E1 or E2, wherein the NGF is used to alleviate neurodevelopmental disorders in the subject associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy in the newborn.

[0321] E4. An NGF used according to any one of E1 to E3, wherein the NGF is used to reduce the severity of neurodevelopmental disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy in the subject.

[0322] E5. An NGF according to any one of E1 to E4, wherein the NGF is used to prevent the progression of neurodevelopmental disorders associated with (e.g., affected by or caused by) neonatal hypoxic-ischemic encephalopathy in the subject.

[0323] E6. An NGF according to any one of E1 to E5, wherein the NGF is used to delay the onset of neurodevelopmental disorders associated with (e.g., affected by or caused by) neonatal hypoxic-ischemic encephalopathy in the subject.

[0324] E7. The NGF according to any one of E1 to E6, wherein the NGF is used to improve neurodevelopmental disorders in the subject that are associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn to a level that is compatible with the performance of typical daily activities in subjects of the same age who are not affected by hypoxic-ischemic encephalopathy of the newborn.

[0325] E8. The NGF applied according to E7, wherein the daily activities include basic motor skills such as crawling, walking or controlling voluntary muscle movements, sitting upright, eating, grasping objects, and interacting with the surrounding environment.

[0326] E9. The NGF used according to any one of E1 to E8, wherein the neurodevelopmental disorder is a motor disorder or one or more of its symptoms.

[0327] E10. The NGF applied according to E9, wherein one or more of the symptoms are selected from difficulty in movement, difficulty in muscle coordination, difficulty in motor skills, weakness, poor muscle tone and bradykinesia.

[0328] E11. The NGF applied according to E9 or E10, wherein the movement disorder is caused by dystonia.

[0329] E12. The NGF used in any of E9 to E11, wherein the NGF is used to normalize the muscle tone of the object.

[0330] E13. The NGF applied according to E12, wherein normalizing the muscle tone of the subject includes restoring the muscle tone of the subject to a level substantially equivalent to that of a subject of the same age who has not suffered from hypoxic-ischemic encephalopathy in the newborn.

[0331] E14. The NGF according to any one of E9 to E13, wherein the motor disorder or the dystonia is associated with (e.g., affected by or caused by) cerebral palsy.

[0332] E15. The NGF used according to any one of E11 to E14, wherein the dystonia is selected from hypotonia, bradykinesia, hypertonia, dystonia, ataxia, extrapyramidal symptoms, diplegia, quadriplegia, quadriplegia, hemiplegia, and paraplegia.

[0333] E16. The NGF applied according to E15, wherein the hypotonia is selected from focal hypotonia and generalized hypotonia.

[0334] E17. The NGF applied according to E15 or E16, wherein the dystonia is selected from focal dystonia, segmental dystonia and generalized dystonia.

[0335] E18. The NGF used according to any one of E15 to E17, wherein the hypertonia is selected from spasticity, rigidity, and tonic defiance.

[0336] E19. An NGF according to any one of E9 to E18, wherein the subject has been diagnosed with a motor disorder associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn (HIE), and the NGF is used to treat the motor disorder in the subject by intranasal application.

[0337] E20. An NGF according to any one of E9 to E18, wherein the subject has been identified as being at risk of developing a motor disorder associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn (HIE), and the NGF is used to prevent the motor disorder in the subject by intranasal administration prior to the occurrence of the motor disorder.

[0338] E21. The NGF used according to any one of E1 to E8, wherein the neurodevelopmental disorder is cerebral palsy or a clinical manifestation thereof.

[0339] E22. The NGF used according to E21, wherein the clinical manifestations are selected from movement disorders or one or more of their symptoms and cognitive impairment.

[0340] E23. The NGF applied according to E22, wherein one or more symptoms of the movement disorder are selected from difficulty in movement, difficulty in muscle coordination, difficulty in motor skills, weakness, poor muscle tone and bradykinesia.

[0341] E24. The NGF applied according to E22 or E23, wherein the movement disorder is caused by dystonia.

[0342] E25. The NGF used in any of E22 to E24, wherein the NGF is used to normalize the muscle tone of the object.

[0343] E26. The NGF applied according to E25, wherein normalizing the muscle tone of the subject includes restoring the muscle tone of the subject to a level substantially equivalent to that of a subject of the same age who has not suffered from hypoxic-ischemic encephalopathy of the newborn.

[0344] E27. The NGF used according to any one of E24 to E26, wherein the dystonia is selected from hypotonia, bradykinesia, hypertonia, dystonia, ataxia, extrapyramidal symptoms, diplegia, quadriplegia, hemiplegia, and paraplegia.

[0345] E28. The NGF applied according to E27, wherein the hypotonia is selected from focal hypotonia and generalized hypotonia.

[0346] E29. The NGF applied according to E27 or E28, wherein the dystonia is selected from focal dystonia, segmental dystonia and generalized dystonia.

[0347] E30. The NGF used according to any one of E27 to E29, wherein the hypertonia is selected from spasticity, rigidity, and tonic defiance.

[0348] E31. An NGF according to any one of E21 to E30, wherein the subject has been diagnosed with cerebral palsy associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn, and the NGF is used to treat the cerebral palsy in the subject by intranasal application.

[0349] E32. An NGF according to any one of E21 to E30, wherein the subject has been identified as being at risk of developing cerebral palsy associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn, and the NGF is used to prevent said cerebral palsy in the subject by intranasal administration.

[0350] E33. The NGF used according to any one of E1 to E8, wherein the neurodevelopmental disorder is a cognitive disorder.

[0351] E34. The NGF used according to any one of E1 to E8, wherein the neurodevelopmental disorder is a behavioral disorder.

[0352] E35. The NGF used according to any one of E1 to E8, wherein the neurodevelopmental disorder is a sensory disorder.

[0353] E36. NGF, which is used to promote neural development in subjects diagnosed with neonatal hypoxic-ischemic encephalopathy, wherein the NGF is administered intranasally to the subjects.

[0354] E37. The NGF applied according to E36, wherein promoting neural development includes enhancing neuronal survival, synapse formation, and / or the establishment of neural networks necessary for normal cognitive, motor, and sensory functions.

[0355] E38. The NGF used according to any one of E1 to E37, wherein the neonatal hypoxic-ischemic encephalopathy (HIE) is selected from mild neonatal hypoxic-ischemic encephalopathy (HIE) (stage 1), moderate neonatal hypoxic-ischemic encephalopathy (HIE) (stage 2), and severe neonatal hypoxic-ischemic encephalopathy (HIE) (stage 3) as determined by the staging system developed by Sarnat and Sarnat (Sarnat HB, Sarnat MS. Arch Neurol 1976; 33: 696-705).

[0356] E39. The NGF according to any one of E1 to E38, wherein the gestational age at birth of the subject is at least 36 weeks.

[0357] E40. The NGF according to any one of E1 to E39, wherein the subject weighs at birth at a weight of at least 1800 grams.

[0358] E41. The NGF applied according to any one of E1 to E40, wherein the subject has at least one of the following signs of perinatal suppression:

[0359] An Apgar score of less than 5 at 10 minutes of age

[0360] Resuscitation (e.g., chest compressions, mechanical ventilation, or CPAP) is required within 10 minutes of birth.

[0361] The pH of the umbilical cord blood gas or the neonatal blood gas obtained before 60 minutes of age is less than 7.00.

[0362] An alkali deficiency of ≥15 mmol / L, ≥12 mmol / L, or ≥16 mmol / L in umbilical cord (arterial or venous) air or infant air (arterial or venous) obtained before 60 minutes of age.

[0363] E42. The NGF used according to any one of E1 to E41, wherein the subject has received therapeutic hypothermia treatment that began no earlier than 6 hours after birth.

[0364] E43. The NGF applied according to any one of E1 to E42, wherein the NGF is first applied to the subject within one month after birth (e.g., 1 to 30 days), preferably within seven days after birth, more preferably within 72 hours after birth, more preferably within 36 hours after birth, and even more preferably within 24 hours after birth.

[0365] E44. The NGF applied according to any one of E1 to E42, wherein the NGF is first applied to the subject one month or later after birth, preferably one month to 36 months after birth, and more preferably one month to four months after birth.

[0366] E45. The NGF applied according to any one of E1 to E42, wherein the NGF is first applied to the subject three to nine months after birth, preferably three to six months after birth.

[0367] E46. The NGF used according to any one of E1 to E45, wherein the NGF is administered to the subject in an intermittent administration regimen having two or more treatment cycles alternating with a washout period.

[0368] E47. The NGF applied according to E46, wherein the NGF is administered to the subject for at least three cycles, wherein each cycle comprises seven days of daily intranasal administration of NGF, followed by 21 days of washout.

[0369] E48. The NGF used according to any one of E1 to E47, wherein the total dose of NGF applied to the subject is from 25 µg / kg to 400 µg / kg, preferably from 30 µg / kg to 300 µg / kg, more preferably from 35 µg / kg to 200 µg / kg, more preferably from 40 µg / kg to 100 µg / kg, more preferably from 40 µg / kg to 60 µg / kg, more preferably from 45 µg / kg to 55 µg / kg, and even more preferably from 50 µg / kg.

[0370] E49. The NGF used according to any one of E46 to E49, wherein the dose of NGF administered to the subject per cycle is from 15 µg / kg to 20 µg / kg, most preferably 16.7 µg / kg.

[0371] E50. The NGF used according to any one of E1 to E49, wherein the daily dose of NGF applied to the subject is from 2 µg / kg to 3 µg / kg, more preferably 2.4 µg / kg.

[0372] E51. The NGF used according to any one of E1 to E50, wherein the dose of NGF applied to the subject per nostril is from 0.3 µg / kg / nostril to 1 µg / kg / nostril, most preferably 0.6 µg / kg / nostril.

[0373] E52. The NGF used according to any one of E1 to E51, wherein the daily dose of the NGF is administered to the subject twice daily at 12-hour intervals.

[0374] E53. The NGF used in any of E1 to E52, wherein the NGF is a human NGF.

[0375] E54. The NGF used according to any one of E1 to E53, wherein the human NGF has the amino acid sequence of SEQ ID NO: 1.

[0376] E55. The NGF used according to any one of E1 to E53, wherein the human NGF has the amino acid sequence of SEQ ID NO: 2.

[0377] E56. The NGF used according to any one of E1 to E53, wherein the human NGF is a mixture of NGFs having the sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

[0378] E57. The NGF used according to any one of E1 to E56, wherein the NGF is generated by recombinant DNA technology.

[0379] E58. The NGF used according to any one of E1 to E57, wherein the NGF is a human recombinant NGF (rhNGF).

[0380] E59. The NGF used according to E58, wherein the NGF is a human recombinant NGF (rhNGF) produced by the method described in WO0022119A1 or WO2013092776A1.

[0381] E60. NGF used according to any one of E1 to E59, wherein the purity of the NGF is greater than 70%.

[0382] E61. The NGF used according to any one of E1 to E60, wherein the purity of the NGF is greater than 80%, greater than 90%, greater than 95%, greater than 98%, or greater than 99%.

[0383] E62. NGF used according to E60 or E61, wherein the purity of the NGF is determined by HPLC.

[0384] E63. A pharmaceutical composition for intranasal administration comprising NGF as described in any one of E1 to E62 and at least one pharmaceutically acceptable excipient, said pharmaceutical composition for the prevention and / or treatment in a subject of neurodevelopmental disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn, said pharmaceutical composition being administered intranasally to said subject.

[0385] E64. A pharmaceutical composition according to the application described in E63, wherein the pharmaceutical composition is used to cure, in the subject, a neurodevelopmental disorder associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy in the newborn.

[0386] E65. A pharmaceutical composition used according to E63 or E64, wherein the pharmaceutical composition is used in the subject to alleviate neurodevelopmental disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy in the newborn.

[0387] E66. A pharmaceutical composition for use according to any one of E63 to E65, wherein the pharmaceutical composition is used in the subject to reduce the severity of neurodevelopmental disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy in the newborn.

[0388] E67. A pharmaceutical composition for use according to any one of E63 to E66, wherein the pharmaceutical composition is used in the subject to prevent the progression of neurodevelopmental disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy in the newborn.

[0389] E68. A pharmaceutical composition for use according to any one of E63 to E67, wherein the pharmaceutical composition is used in the subject to delay the onset of neurodevelopmental disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy in the newborn.

[0390] E69. A pharmaceutical composition for use according to any one of E63 to E68, wherein the pharmaceutical composition is used to improve neurodevelopmental disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy in the subject to a level that is compatible with the performance of typical daily activities in subjects of the same age not affected by hypoxic-ischemic encephalopathy.

[0391] E70. A pharmaceutical composition according to E69, wherein the daily activities include basic movements such as crawling, walking or controlling voluntary muscle movements, sitting upright, eating, grasping objects, and interacting with the surrounding environment.

[0392] E71. A pharmaceutical composition for use according to any one of E63 to E70, wherein the neurodevelopmental disorder is a motor disorder or one or more of its symptoms.

[0393] E72. The pharmaceutical composition used according to E71, wherein one or more of the symptoms are selected from difficulty in movement, difficulty in muscle coordination, difficulty in motor skills, weakness, poor muscle tone and bradykinesia.

[0394] E73. A pharmaceutical composition used according to E71 or E72, wherein the movement disorder is caused by dystonia.

[0395] E74. A pharmaceutical composition for use according to any one of E71 to E73, wherein the pharmaceutical composition is used to normalize the muscle tone of the subject.

[0396] E75. The pharmaceutical composition used according to E74, wherein normalizing the muscle tone of the subject comprises restoring the muscle tone of the subject to a level substantially equivalent to that of a subject of the same age who has not suffered from hypoxic-ischemic encephalopathy of the newborn.

[0397] E76. A pharmaceutical composition for use according to any one of E71 to E75, wherein the motor disorder or the dystonia is associated with (e.g., affected by or caused by) cerebral palsy.

[0398] E77. A pharmaceutical composition for use according to any one of E73 to E76, wherein the dystonia is selected from hypotonia, bradykinesia, hypertonia, dystonia, ataxia, extrapyramidal symptoms, diplegia, quadriplegia, hemiplegia, and paraplegia.

[0399] E78. The pharmaceutical composition used according to E77, wherein the hypotonia is selected from focal hypotonia and generalized hypotonia.

[0400] E79. A pharmaceutical composition used according to E77 or E78, wherein the dystonia is selected from focal dystonia, segmental dystonia and generalized dystonia.

[0401] E80. A pharmaceutical composition for use according to any one of E77 to E79, wherein the hypertonia is selected from spasticity, rigidity, and tonic defiance.

[0402] E81. A pharmaceutical composition for use according to any one of E71 to E80, wherein the subject has been diagnosed with a motor disorder associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn (HIE), and the pharmaceutical composition is used to treat the motor disorder in the subject by intranasal administration.

[0403] E82. A pharmaceutical composition for use according to any one of E71 to E80, wherein the subject has been identified as being at risk of developing a motor disorder associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn (HIE), and the pharmaceutical composition is used to prevent the motor disorder in the subject by intranasal administration prior to the occurrence of the motor disorder.

[0404] E83. A pharmaceutical composition for use according to any one of E63 to E70, wherein the neurodevelopmental disorder is cerebral palsy or a clinical manifestation thereof.

[0405] E84. The pharmaceutical composition used according to E83, wherein the clinical manifestation is selected from movement disorders or one or more of their symptoms and cognitive impairment.

[0406] E85. The pharmaceutical composition used according to E84, wherein one or more symptoms of the movement disorder are selected from difficulty in movement, difficulty in muscle coordination, difficulty in motor skills, weakness, poor muscle tone and bradykinesia.

[0407] E86. A pharmaceutical composition used according to E84 or E85, wherein the movement disorder is caused by dystonia.

[0408] E87. A pharmaceutical composition for use according to any one of E84 to E86, wherein the pharmaceutical composition is used to normalize the muscle tone of the subject.

[0409] E88. The pharmaceutical composition used according to E87, wherein normalizing the muscle tone of the subject comprises restoring the muscle tone of the subject to a level substantially equivalent to that of a subject of the same age who has not suffered from hypoxic-ischemic encephalopathy of the newborn.

[0410] E89. A pharmaceutical composition for use according to any one of E85 to E88, wherein the dystonia is selected from hypotonia, bradykinesia, hypertonia, dystonia, ataxia, extrapyramidal symptoms, diplegia, quadriplegia, hemiplegia, and paraplegia.

[0411] E90. The pharmaceutical composition used according to E89, wherein the hypotonia is selected from focal hypotonia and generalized hypotonia.

[0412] E91. A pharmaceutical composition used according to E89 or E90, wherein the dystonia is selected from focal dystonia, segmental dystonia and generalized dystonia.

[0413] E92. A pharmaceutical composition for use according to any one of E89 to E91, wherein the hypertonia is selected from spasticity, rigidity, and tonic defiance.

[0414] E93. A pharmaceutical composition according to any one of E83 to E92, wherein the subject has been diagnosed with cerebral palsy associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn, and the pharmaceutical composition is used to treat the cerebral palsy in the subject by intranasal administration.

[0415] E94. A pharmaceutical composition for use according to any one of E83 to E92, wherein the subject has been identified as being at risk of developing cerebral palsy associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn, and the pharmaceutical composition is used to prevent said cerebral palsy in the subject by intranasal administration.

[0416] E95. A pharmaceutical composition for use according to any one of E63 to E70, wherein the neurodevelopmental disorder is a behavioral disorder or a cognitive disorder.

[0417] E96. A pharmaceutical composition for use according to any one of E63 to E70, wherein the neurodevelopmental disorder is a sensory disorder.

[0418] E97. A pharmaceutical composition for intranasal administration comprising NGF as described in any one of E1 to E62 and at least one pharmaceutically acceptable excipient, said pharmaceutical composition for promoting neurodevelopment in a subject diagnosed with hypoxic-ischemic encephalopathy of the newborn, said pharmaceutical composition being administered intranasally to said subject.

[0419] E98. A pharmaceutical composition used according to E97, wherein promoting neurodevelopment includes enhancing neuronal survival, synapse formation, and / or the establishment of neural networks necessary for normal cognitive, motor, and sensory functions.

[0420] E99. A pharmaceutical composition for use according to any one of E63 to E98, wherein the neonatal hypoxic-ischemic encephalopathy (HIE) is selected from mild neonatal hypoxic-ischemic encephalopathy (HIE) (stage 1), moderate neonatal hypoxic-ischemic encephalopathy (HIE) (stage 2), and severe neonatal hypoxic-ischemic encephalopathy (HIE) (stage 3) as determined according to the staging system developed by Sarnat and Sarnat (Sarnat HB, Sarnat MS. ArchNeurol 1976; 33: 696-705).

[0421] E100. A pharmaceutical composition for use according to any one of E63 to E99, wherein the subject is born at a gestational age of at least 36 weeks.

[0422] E101. A pharmaceutical composition for use according to any one of E63 to E100, wherein the subject has a birth weight of at least 1800 grams.

[0423] E102. A pharmaceutical composition according to any one of E63 to E101, wherein the subject has at least one of the following signs of perinatal suppression:

[0424] - An Apgar score of less than 5 at 10 minutes of age.

[0425] - Resuscitation is required within 10 minutes (e.g., chest compressions, mechanical ventilation, or CPAP).

[0426] - The pH of the umbilical cord blood gas or the neonatal blood gas obtained before 60 minutes of age is less than 7.00.

[0427] - An alkali deficiency of ≥12 mmol / L, ≥15 mmol / L, or ≥16 mmol / L in umbilical cord (arterial or venous) air or in infants less than 60 minutes old.

[0428] E103. A pharmaceutical composition according to any one of E63 to E102, wherein the subject has received therapeutic hypothermia treatment initiated before 6 hours after birth.

[0429] E104. A pharmaceutical composition for use according to any one of E63 to E103, wherein the pharmaceutical composition is applied within one month after birth (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days), preferably within seven days after birth, more preferably within 72 hours after birth (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, or 23 hours). (Hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours), more preferably within 36 hours after birth, and even more preferably within 24 hours after birth, for the first application to the subject.

[0430] E105. A pharmaceutical composition for use according to any one of E63 to E103, wherein the pharmaceutical composition is first administered to the subject one month or later after birth, preferably one month to 36 months after birth, more preferably one month to 24 months after birth, even more preferably one month to 12 months after birth, and most preferably one month to six months after birth.

[0431] E106. A pharmaceutical composition according to any one of E63 to E103, wherein the pharmaceutical composition is first administered to the subject three to nine months after birth, preferably three to six months after birth.

[0432] E107. A pharmaceutical composition according to any one of E63 to E106, wherein the pharmaceutical composition is administered to the subject in an intermittent administration regimen having two or more treatment cycles alternating with a washout period.

[0433] E108. The pharmaceutical composition used according to E107, wherein the pharmaceutical composition is administered to the subject for at least three cycles, wherein each cycle comprises seven days of daily intranasal administration of NGF, followed by a 21-day washout period (i.e., a period during which no NGF is administered to the subject).

[0434] E109. A pharmaceutical composition for use according to any one of E63 to E108, wherein the total dose of NGF administered to the subject is from 25 µg / kg to 400 µg / kg, preferably from 30 µg / kg to 300 µg / kg, more preferably from 35 µg / kg to 200 µg / kg, more preferably from 40 µg / kg to 100 µg / kg, more preferably from 40 µg / kg to 60 µg / kg, more preferably from 45 µg / kg to 55 µg / kg, and even more preferably from 50 µg / kg.

[0435] E110. A pharmaceutical composition for the application according to any one of E107 to E109, wherein the dose of NGF administered to the subject per cycle is from 15 µg / kg to 20 µg / kg, more preferably from 16 µg / kg to 17 µg / kg, even more preferably from 16.6 µg / kg to 16.8 µg / kg, and most preferably 16.7 µg / kg.

[0436] E111. A pharmaceutical composition for use according to any one of E63 to E110, wherein the daily dose of NGF administered to the subject is 2 µg / kg to 3 µg / kg, preferably 2.3 µg / kg to 2.5 µg / kg, more preferably 2.4 µg / kg.

[0437] E112. A pharmaceutical composition for use according to any one of E63 to E111, wherein the dose of NGF administered to the subject per nostril is from 0.3 µg / kg / nostril to 1 µg / kg / nostril, more preferably from 0.4 µg / kg / nostril to 0.8 µg / kg / nostril, more preferably from 0.5 µg / kg / nostril to 0.7 µg / kg / nostril, and most preferably 0.6 µg / kg / nostril.

[0438] E113. A pharmaceutical composition according to any one of E63 to E112, wherein a daily dose of NGF according to the present disclosure is administered to the subject twice daily at 12-hour intervals.

[0439] E114. A pharmaceutical composition for use according to any one of E63 to E113, wherein the NGF is human NGF.

[0440] E115. A pharmaceutical composition for use according to any one of E63 to E114, wherein the human NGF has the amino acid sequence of SEQ ID NO: 1:

[0441]

[0442] E116. A pharmaceutical composition for use according to any one of E63 to E114, wherein the human NGF has the amino acid sequence of SEQ ID NO: 2:

[0443]

[0444] E117. A pharmaceutical composition for use according to any one of E63 to E114, wherein the human NGF is a mixture of NGFs having the sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

[0445] E118. A pharmaceutical composition for use according to any one of E63 to E117, wherein the NGF is produced by recombinant DNA technology.

[0446] E119. A pharmaceutical composition for use according to any one of E63 to E118, wherein the NGF is recombinant human NGF (rhNGF).

[0447] E120. A pharmaceutical composition for use according to E119, wherein the NGF is a recombinant human NGF (rhNGF) produced by the method described in WO0022119A1 or WO2013092776A1.

[0448] E121. A pharmaceutical composition for use according to any one of E63 to E120, wherein the purity of NGF is greater than 70%.

[0449] E122. A pharmaceutical composition for use according to any one of E63 to E121, wherein the purity of NGF is greater than 80%, greater than 90%, greater than 95%, greater than 98%, or greater than 99%.

[0450] E123. A pharmaceutical composition used according to E121 or E122, wherein the purity of NGF is determined by HPLC.

[0451] E124. A pharmaceutical composition for use according to any one of E63 to E123, wherein the pharmaceutical composition is a liquid intranasal composition.

[0452] E125. A pharmaceutical composition for any of the applications described in E63 to E124, wherein the pharmaceutical composition comprises an effective amount of the NGF and at least one pharmaceutically acceptable excipient suitable for intranasal use.

[0453] E126. A pharmaceutical composition for any of the applications described in E63 to E125, wherein the pharmaceutically acceptable excipient is selected from solvents, thickeners, mucosal adhesives, buffers, antioxidants, surfactants, preservatives, and penetration enhancers.

[0454] E127. A pharmaceutical composition for use according to any one of E124 to E126, wherein the concentration of the NGF in the liquid intranasal composition for use according to this disclosure is from about 5 µg / ml to about 1 mg / ml, preferably from about 10 µg / ml to about 400 µg / ml, more preferably from about 15 µg / ml to about 200 µg / ml, and even more preferably from about 20 µg / ml.

[0455] E128. A pharmaceutical composition used according to E126 or E127, wherein the solvent is water.

[0456] E129. A pharmaceutical composition for use according to any one of E126 to E128, wherein the mucosal adhesive is glycerin.

[0457] E130. A pharmaceutical composition for any of the applications described in E126 to E129, wherein the concentration of the mucosal adhesive (e.g., glycerin) is from 0.05% w / v to 0.2% w / v, preferably 0.1% w / v.

[0458] E131. A pharmaceutical composition according to E126, wherein the antioxidant is methionine.

[0459] E132. A pharmaceutical composition according to E126 or E131, wherein the concentration of said antioxidant (e.g., methionine) is from about 0.005 mg / ml to about 0.02 mg / ml, preferably about 0.01 mg / ml.

[0460] E133. A pharmaceutical composition for use according to any one of E126 to E132, wherein the surfactant is Kolliphor P188.

[0461] E134. A pharmaceutical composition for any of the applications described in E126 to E133, wherein the concentration of the surfactant (e.g., Kolliphor P188) is from about 0.05% w / v to about 0.2% w / v, preferably about 0.1% w / v.

[0462] E135. A pharmaceutical composition for use according to any one of E126 to E134, wherein the buffer is a phosphate buffer.

[0463] E136. A pharmaceutical composition for use according to any one of E126 to E135, wherein the penetration enhancer is n-dodecyl-β-D-maltodextrin.

[0464] E137. A pharmaceutical composition for any of the applications described in E126 to E136, wherein the concentration of the penetration enhancer (e.g., n-dodecyl-β-D-maltodextrin) is from about 0.1% w / v to about 1% w / v, preferably about 0.5% w / v.

[0465] E138. A pharmaceutical composition for any of the applications described in E124 to E137, wherein the liquid intranasal composition comprises, or consists of, NGF, sodium chloride, phosphate buffer, and water.

[0466] E139. A pharmaceutical composition for any of the applications described in E124 to E138, wherein the liquid intranasal composition comprises or consists of the following: the NGF, sodium chloride, phosphate buffer, Kolliphor P188, L-methionine, and water.

[0467] E140. A pharmaceutical composition for any of the applications described in E124 to E139, wherein the liquid intranasal composition for the application described in this disclosure comprises or consists of the following: NGF, sodium chloride, phosphate buffer, Kolliphor P188, L-methionine, glycerol, n-dodecyl-β-D-maltodextrin, and water.

[0468] E141. A pharmaceutical composition for use according to any one of E124 to E140, wherein the liquid intranasal composition for use according to this disclosure comprises or consists of: NGF, NaH2PO4. H2O, NaCl, Kolliphor P188, -L-methionine.

[0469] E142. A pharmaceutical composition for any of the applications described in E124 to E141, wherein the liquid intranasal composition for the application according to this disclosure comprises or is composed of the following components:

[0470] - The concentration of NGF as described above is:

[0471] Approximately 5 µg / ml to approximately 1 mg / ml

[0472] Approximately 10 µg / ml to approximately 400 µg / ml, or

[0473] From approximately 15 µg / ml to approximately 200 µg / ml,

[0474] - NaH2PO4 H2O, its concentration is:

[0475] Approximately 5 to approximately 8 mg / ml, or

[0476] Approximately 6.9 mg / mL,

[0477] - NaCl, its concentration is:

[0478] Approximately 5 to approximately 6.5 mg / ml, or

[0479] Approximately 5.84 mg / mL

[0480] - Kolliphor P188, its concentration is:

[0481] Approximately 0.05% w / v to approximately 0.2% w / v, or

[0482] Approximately 0.1% w / v,

[0483] -L-methionine, its concentration is:

[0484] Approximately 0.05 mg / ml to approximately 0.2 mg / ml, or

[0485] Approximately 0.1 mg / ml,

[0486] - Optionally, n-dodecyl-β-D-maltodextrin, at a concentration of:

[0487] Approximately 0.1% w / v to approximately 1% w / v, or

[0488] Approximately 0.5% w / v,

[0489] and / or

[0490] - Glycerin, with the following concentration:

[0491] Approximately 0.05% w / v to approximately 0.2% w / v, or

[0492] Approximately 0.1% w / v, and

[0493] - water.

[0494] E143. An NGF application according to any one of E1 to E62 or a pharmaceutical composition according to any one of E63 to E142, wherein the subject is a human subject.

[0495] E144. An NGF or pharmaceutical composition used according to E143, wherein the subject is a newborn or an infant.

[0496] E145. Nerve growth factor (NGF) for the prevention and / or treatment of motor disorders associated with hypoxic-ischemic encephalopathy in subjects, wherein the NGF is administered intranasally to the subjects.

[0497] E146. The NGF applied according to E145, wherein the movement disorder is caused by dystonia.

[0498] E147. The NGF used according to E145 or E146, wherein the NGF is used to normalize muscle tone in subjects suffering from hypoxic-ischemic encephalopathy in newborns.

[0499] E148. The NGF according to any one of E145 to E147, wherein the motor disorder or the dystonia is associated with cerebral palsy.

[0500] E149. The NGF used according to any one of E145 to E148, wherein the dystonia is selected from hypotonia, bradykinesia, hypertonia, dystonia, ataxia, extrapyramidal symptoms, diplegia, quadriplegia, quadriplegia, hemiplegia, and paraplegia.

[0501] E150. The NGF used according to E149, wherein the hypotonia is selected from focal hypotonia and generalized hypotonia.

[0502] E151. The NGF used according to E149, wherein the dystonia is selected from focal dystonia, segmental dystonia and generalized dystonia.

[0503] E152. The NGF used according to E149, wherein the hypertonia is selected from spasticity, rigidity, and tonic defiance.

[0504] E153. The NGF used according to any one of E145 to E152, wherein the NGF is administered once or three times daily for a treatment period of 7 to 90 days, preferably 15 to 60 days.

[0505] E154. The NGF used according to any one of E145 to E153, wherein the amount of NGF applied each time is 5 µg to 1 mg, more preferably 10 µg to 400 µg, even more preferably 15 µg to 200 µg, even more preferably 20 µg.

[0506] E155. A pharmaceutical composition for intranasal administration comprising NGF and at least one pharmaceutically acceptable excipient, said pharmaceutical composition for the prevention and / or treatment in subjects of motor disorders associated with hypoxic-ischemic encephalopathy (HIE), said pharmaceutical composition being administered intranasally to said subjects.

[0507] E156. A pharmaceutical composition according to E155, wherein the NGF is present in the composition at a concentration of 5 µg / ml to 1 mg / ml, more preferably 10 µg / ml to 400 µg / ml, and even more preferably 15 µg / ml to 200 µg / ml.

[0508] E157. A pharmaceutical composition according to E155 or E156, comprising NGF, sodium chloride, phosphate buffer and water, preferably consisting of NGF, sodium chloride, phosphate buffer and water.

[0509] E158. A pharmaceutical composition for use according to any one of E155 to E157, wherein the NGF is human NGF, more preferably recombinant human NGF.

[0510] E159. A pharmaceutical composition for use according to any one of E155 to E158, wherein the human NGF has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0511] E160. The NGF used according to E1, E7 to E18, E20 to E30, E32 to E62, wherein the NGF is used to reduce the likelihood and / or severity of the neurodevelopmental disorder in the subject by intranasal application.

[0512] E161. The NGF used according to E145 to E159, wherein the NGF is used to reduce the likelihood and / or severity of the movement disorder in the subject by intranasal application.

[0513] E162. A pharmaceutical composition for use according to any one of E63, E70 to 80, E82 to 92, E92 to 96, E99 to 144, wherein the pharmaceutical composition is used to reduce the likelihood and / or severity of the neurodevelopmental disorder in the subject by intranasal administration.

[0514] E163. The NGF applied according to any one of E1 to E45, E48 to E62, E143 to E144, wherein the NGF is applied to the object in a continuous application scheme.

[0515] E164. A pharmaceutical composition for use according to any one of E63 to E106, E109 to E144, wherein the pharmaceutical composition is administered to the subject in a continuous administration regimen.

[0516] E165. An NGF or pharmaceutical composition according to any one of E1 to E164, wherein the NGF or pharmaceutical composition is administered intranasally to the subject.

Claims

1. Nerve growth factor (NGF) for the prevention and / or treatment in subjects of neurodevelopmental disorders associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy in newborns, wherein the NGF is administered intranasally to the subjects.

2. The NGF used according to claim 1, wherein the neurodevelopmental disorder includes motor disorder or neurodevelopmental delay.

3. The NGF of claim 1 or 2, wherein the neurodevelopmental disorder includes cerebral palsy.

4. The NGF of any one of claims 1 to 3, wherein the neurodevelopmental disorder comprises one or more of cognitive disorders, behavioral disorders, or sensory disorders.

5. The NGF according to any one of claims 1 to 4, wherein the movement disorder is caused by dystonia.

6. The NGF applied according to any one of claims 1 to 5, wherein the application reduces the likelihood of neurodevelopmental disorders compared to subjects and / or subjects who have not received the NGF, optionally wherein the subjects who have not received the NGF have been diagnosed with hypoxic-ischemic encephalopathy of the newborn.

7. The NGF applied according to any one of claims 1 to 6, wherein the application reduces the severity of neurodevelopmental disorders compared to subjects who have not received the NGF, optionally wherein the subjects who have not received the NGF have been diagnosed with hypoxic-ischemic encephalopathy of the newborn.

8. The NGF of any one of claims 1 to 7, wherein the subject has been identified as being at risk of developing neurodevelopmental disorders associated with hypoxic-ischemic encephalopathy in newborns.

9. The NGF of any one of claims 1 to 8, wherein the object has undergone perinatal suppression, indicated by at least one of the following: - An Apgar score of less than 5 at 10 minutes of age. - Resuscitation is required within 10 minutes (e.g., chest compressions, mechanical ventilation, or CPAP). - The pH of the umbilical cord blood gas or the neonatal blood gas obtained at a time when the baby is less than 60 minutes old is less than 7.00, and / or - An alkali deficiency of ≥15 mmol / L, ≥12 mmol / L, or ≥16 mmol / L in umbilical cord (arterial or venous) air or infant air obtained before 60 minutes of age.

10. The NGF of claim 9, wherein the need for resuscitation includes chest compressions, mechanical ventilation, and / or continuous positive airway pressure therapy.

11. The NGF of any one of claims 1 to 10, wherein the subject is diagnosed with neonatal hypoxic-ischemic encephalopathy (NHIE) classified as stage 2 or 3 according to the modified Sarnath staging system.

12. The NGF of any one of claims 1 to 11, wherein the gestational age of the subject at birth is at least 36 weeks, and / or wherein the weight of the subject at birth is at least 1800 grams.

13. The NGF of any one of claims 1 to 12, wherein the subject is at risk of developing a movement disorder, and the NGF is used to treat the movement disorder in the subject by intranasal application.

14. The NGF of any one of claims 1 to 13, wherein the subject has been diagnosed with a motor disorder associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn, and the NGF is used to treat the motor disorder in the subject by intranasal application.

15. The NGF of any one of claims 1 to 14, wherein the subject has been diagnosed with cerebral palsy associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn, and the NGF is used to treat the cerebral palsy in the subject by intranasal application.

16. The NGF of any one of claims 1 to 15, wherein the subject has been identified as being at risk of developing cerebral palsy associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn, and the NGF is used to prevent the cerebral palsy in the subject by intranasal application.

17. The NGF of any one of claims 1 to 15, wherein the subject has been diagnosed with hypoxic-ischemic encephalopathy of the newborn.

18. The NGF of any one of claims 1 to 17, wherein the subject has received therapeutic hypothermia treatment initiated within the first six hours after birth.

19. The NGF used in any one of claims 1 to 18, wherein the NGF is first applied to the subject within one month after birth, preferably within seven days after birth, more preferably within 72 hours after birth, more preferably within 36 hours after birth, or more preferably within 24 hours after birth.

20. The NGF of any one of claims 1 to 19, wherein the NGF is first applied to the subject within 24 hours after birth.

21. The NGF of any one of claims 1 to 18, wherein the NGF is first applied to the subject one to 36 months after birth, more preferably one to 24 months after birth, more preferably one to 12 months after birth, even more preferably one to six months after birth, and most preferably one to four months after birth.

22. The NGF of any one of claims 1 to 21, wherein the NGF is first applied to the subject three to nine months after birth, preferably three to six months after birth.

23. The NGF of any one of claims 1 to 22, wherein the NGF is administered to the subject in a continuous administration regimen having two or more treatment cycles, wherein each of the two or more treatment cycles is one week long.

24. The NGF of any one of claims 1 to 22, wherein the NGF is administered to the subject in an intermittent administration regimen having two or more treatment cycles, wherein each of the two or more treatment cycles is one week long and alternates with a washout period.

25. The NGF used according to any one of claims 1 to 22 or 24, wherein the NGF is administered to the subject in an intermittent administration regimen having two or more treatment cycles alternating with a washout period.

26. The NGF of claim 25, wherein the NGF is applied to the subject for at least three cycles, wherein each cycle comprises seven days of daily intranasal application of the NGF.

27. The NGF of any one of claims 1 to 22 or 24 to 26, wherein the NGF is administered to the subject for at least three cycles, wherein each cycle comprises seven days of daily intranasal administration of the NGF, followed by a 21-day washout period (i.e., a period during which the NGF is not administered to the subject).

28. The NGF used in any one of claims 1 to 27, wherein the total dose of the NGF applied to the subject is from 25 µg / kg to 400 µg / kg, preferably from 30 µg / kg to 300 µg / kg, more preferably from 35 µg / kg to 200 µg / kg, more preferably from 40 µg / kg to 100 µg / kg, more preferably from 40 µg / kg to 60 µg / kg, more preferably from 45 µg / kg to 55 µg / kg, and even more preferably from 50 µg / kg.

29. The NGF used in any one of claims 1 to 28, wherein the dose of the NGF applied to the subject per cycle is from 15 µg / kg to 20 µg / kg, more preferably from 16 µg / kg to 17 µg / kg, even more preferably from 16.6 µg / kg to 16.8 µg / kg, and most preferably from 16.7 µg / kg.

30. The NGF used according to any one of claims 1 to 29, wherein the daily dose of the NGF applied to the subject is from 2 µg / kg to 3 µg / kg, preferably from 2.3 µg / kg to 2.5 µg / kg, more preferably 2.4 µg / kg.

31. The NGF used according to any one of claims 1 to 30, wherein the dose of the NGF applied to the subject per nostril is from 0.3 µg / kg / nostril to 1 µg / kg / nostril, more preferably from 0.4 µg / kg / nostril to 0.8 µg / kg / nostril, more preferably from 0.5 µg / kg / nostril to 0.7 µg / kg / nostril, and most preferably 0.6 µg / kg / nostril.

32. The NGF of any one of claims 1 to 31, wherein the daily dose of the NGF of the application according to this disclosure is administered to the subject twice daily at 12-hour intervals.

33. The NGF used in any one of claims 1 to 32, wherein the NGF normalizes muscle tone in a subject suffering from hypoxic-ischemic encephalopathy of the newborn.

34. The NGF of any one of claims 1 to 33, wherein the motor disorder or the dystonia is associated with cerebral palsy.

35. The NGF used according to any one of claims 1 to 34, wherein the dystonia is selected from hypotonia, bradykinesia, hypertonia, dystonia, ataxia, extrapyramidal symptoms, diplegia, quadriplegia, quadriplegia, hemiplegia, and paraplegia.

36. The NGF of claim 35, wherein the hypotonia is selected from focal hypotonia and generalized hypotonia.

37. The NGF of claim 35, wherein the dystonia is selected from focal dystonia, segmental dystonia and generalized dystonia.

38. The NGF of claim 35, wherein the excessive muscle tone is selected from spasticity, rigidity, and tonic defiance.

39. The NGF of any one of claims 1 to 38, wherein the NGF is formulated into a pharmaceutical composition for intranasal administration, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable excipient.

40. The NGF of claim 39, wherein the NGF is present in the pharmaceutical composition at a concentration of 5 µg / ml to 1 mg / ml, more preferably 10 µg / ml to 400 µg / ml, and even more preferably 15 µg / ml to 200 µg / ml.

41. The NGF used according to claim 39 or 40, wherein the pharmaceutical composition comprises, or is composed of, NGF, sodium chloride, phosphate buffer and water.

42. The NGF used in any one of claims 1 to 41, wherein the NGF is a human NGF, more preferably a recombinant human NGF.

43. The NGF of any one of claims 1 to 42, wherein the human NGF comprises or consists of the amino acid sequence of SEQ ID NO:

1.

44. The NGF of any one of claims 1 to 42, wherein the human NGF comprises or consists of the amino acid sequence of SEQ ID NO:

2.

45. A method for preventing and / or treating neurodevelopmental disorders associated with hypoxic-ischemic encephalopathy in subjects in need, the method comprising administering a therapeutically effective or preventively effective amount of nerve growth factor (NGF) intranasally to the subject.

46. ​​The method of claim 45, wherein the neurodevelopmental disorder comprises motor disorder or neurodevelopmental delay.

47. The method of claim 45 or 46, wherein the neurodevelopmental disorder comprises cerebral palsy.

48. The method of any one of claims 45 to 47, wherein the neurodevelopmental disorder comprises one or more of cognitive disorders, behavioral disorders, or sensory disorders.

49. The method of any one of claims 45 to 48, wherein the movement disorder is caused by dystonia.

50. The method of any one of claims 45 to 49, wherein the application reduces the likelihood of neurodevelopmental disorders compared to subjects and / or subjects who have not received the NGF, optionally wherein the subjects who have not received the NGF have been diagnosed with hypoxic-ischemic encephalopathy of the newborn.

51. The method of any one of claims 45 to 50, wherein the administration reduces the severity of neurodevelopmental disorders compared to subjects who have not received the NGF, optionally wherein the subjects who have not received the NGF have been diagnosed with hypoxic-ischemic encephalopathy of the newborn.

52. The method of any one of claims 45 to 51, wherein the subject has been identified as being at risk of developing neurodevelopmental disorders associated with hypoxic-ischemic encephalopathy in newborns.

53. The method of any one of claims 45 to 52, wherein the object has undergone perinatal suppression, indicated by at least one of the following: - An Apgar score of less than 5 at 10 minutes of age. - Resuscitation is required within 10 minutes (e.g., chest compressions, mechanical ventilation, or CPAP). - The pH of the umbilical cord blood gas or the neonatal blood gas obtained at a time when the baby is less than 60 minutes old is less than 7.00, and / or - An alkali deficiency of ≥15 mmol / L, ≥12 mmol / L, or ≥16 mmol / L in umbilical cord (arterial or venous) air or infant air obtained before 60 minutes of age.

54. The method of claim 53, wherein the need for resuscitation includes chest compressions, mechanical ventilation, and / or continuous positive airway pressure therapy.

55. The method of any one of claims 45 to 54, wherein the subject is diagnosed with neonatal hypoxic-ischemic encephalopathy (NHIE) classified as stage 2 or 3 according to the modified Sarnath staging system.

56. The method of any one of claims 45 to 55, wherein the gestational age of the subject at birth is at least 36 weeks, and / or the weight of the subject at birth is at least 1800 grams.

57. The method of any one of claims 45 to 56, wherein the subject is at risk of developing a movement disorder, and the NGF is used to treat the movement disorder in the subject by intranasal application.

58. The method of any one of claims 45 to 57, wherein the subject has been diagnosed with a motor disorder associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn, and the NGF is used to treat the motor disorder in the subject by intranasal application.

59. The method of any one of claims 45 to 58, wherein the subject has been diagnosed with cerebral palsy associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn, and the NGF is used to treat the cerebral palsy in the subject by intranasal application.

60. The method of any one of claims 45 to 59, wherein the subject has been identified as being at risk of developing cerebral palsy associated with (e.g., affected by or caused by) hypoxic-ischemic encephalopathy of the newborn, and the NGF is used to prevent the cerebral palsy in the subject by intranasal administration.

61. The method of any one of claims 45 to 60, wherein the subject has been diagnosed with hypoxic-ischemic encephalopathy of the newborn.

62. The method of any one of claims 45 to 61, wherein the subject has received therapeutic hypothermia treatment initiated within the first six hours after birth.

63. The method of any one of claims 45 to 62, wherein the NGF is first applied to the subject within one month after birth, preferably within seven days after birth, more preferably within 72 hours after birth, more preferably within 36 hours after birth, or more preferably within 24 hours after birth.

64. The method of any one of claims 45 to 63, wherein the NGF is first applied to the subject within 24 hours after birth.

65. The method of any one of claims 45 to 64, wherein the NGF is first applied to the subject one to 36 months after birth, more preferably one to 24 months after birth, even more preferably one to 12 months after birth, and even more preferably one to six months after birth, and most preferably one to four months after birth.

66. The method of any one of claims 45 to 65, wherein the NGF is first applied to the subject three to nine months after birth, preferably three to six months after birth.

67. The method of any one of claims 45 to 66, wherein the NGF is applied to the subject in a continuous administration regimen having two or more treatment cycles, wherein each of the two or more treatment cycles is one week long.

68. The method of any one of claims 45 to 67, wherein the NGF is administered to the subject in an intermittent administration regimen having two or more treatment cycles, wherein each of the two or more treatment cycles is one week long and alternates with a washout period.

69. The method of any one of claims 45 to 66 or 68, wherein the NGF is administered to the subject in an intermittent administration regimen having two or more treatment cycles alternating with a washout period.

70. The method of claim 69, wherein the NGF is applied to the subject for at least three cycles, wherein each cycle comprises seven days of daily intranasal application of the NGF.

71. The method of any one of claims 45 to 66 or 68 to 70, wherein the NGF is applied to the subject for at least three cycles, wherein each cycle comprises seven days of daily intranasal application of the NGF, followed by a 21-day washout period (i.e., a period during which the NGF is not applied to the subject).

72. The method of any one of claims 45 to 71, wherein the total dose of the NGF applied to the subject is from 25 µg / kg to 400 µg / kg, preferably from 30 µg / kg to 300 µg / kg, more preferably from 35 µg / kg to 200 µg / kg, more preferably from 40 µg / kg to 100 µg / kg, more preferably from 40 µg / kg to 60 µg / kg, more preferably from 45 µg / kg to 55 µg / kg, and even more preferably from 50 µg / kg.

73. The method of any one of claims 45 to 72, wherein the dose of the NGF applied to the subject per cycle is from 15 µg / kg to 20 µg / kg, more preferably from 16 µg / kg to 17 µg / kg, even more preferably from 16.6 µg / kg to 16.8 µg / kg, and most preferably from 16.7 µg / kg.

74. The method of any one of claims 45 to 73, wherein the daily dose of the NGF applied to the subject is 2 µg / kg to 3 µg / kg, preferably 2.3 µg / kg to 2.5 µg / kg, more preferably 2.4 µg / kg.

75. The method of any one of claims 45 to 74, wherein the dose of the NGF applied to the subject per nostril is from 0.3 µg / kg / nostril to 1 µg / kg / nostril, more preferably from 0.4 µg / kg / nostril to 0.8 µg / kg / nostril, more preferably from 0.5 µg / kg / nostril to 0.7 µg / kg / nostril, and most preferably 0.6 µg / kg / nostril.

76. The method of any one of claims 45 to 75, wherein the daily dose of NGF applied according to this disclosure is administered to the subject twice daily at 12-hour intervals.

77. The method of any one of claims 45 to 76, wherein the NGF normalizes muscle tone in a subject suffering from hypoxic-ischemic encephalopathy of the newborn.

78. The method of any one of claims 45 to 77, wherein the motor disorder or the dystonia is associated with cerebral palsy.

79. The method of any one of claims 45 to 78, wherein the dystonia is selected from hypotonia, bradykinesia, hypertonia, dystonia, ataxia, extrapyramidal symptoms, diplegia, quadriplegia, quadriplegia, hemiplegia, and paraplegia.

80. The method of claim 79, wherein the hypotonia is selected from focal hypotonia and generalized hypotonia.

81. The method of claim 79, wherein the dystonia is selected from focal dystonia, segmental dystonia, and generalized dystonia.

82. The method of claim 79, wherein the excessive muscle tone is selected from spasticity, rigidity, and tonic defiance.

83. The method of any one of claims 45 to 82, wherein the NGF is formulated into a pharmaceutical composition for intranasal administration, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable excipient.

84. The method of claim 83, wherein the NGF is present in the pharmaceutical composition at a concentration of 5 µg / ml to 1 mg / ml, more preferably 10 µg / ml to 400 µg / ml, and even more preferably 15 µg / ml to 200 µg / ml.

85. The method of claim 82 or 86, wherein the pharmaceutical composition comprises, or is composed of, NGF, sodium chloride, phosphate buffer, and water.

86. The method of any one of claims 45 to 85, wherein the NGF is human NGF, more preferably recombinant human NGF.

87. The method of any one of claims 45 to 86, wherein the human NGF comprises or consists of the amino acid sequence of SEQ ID NO:

1.

88. The method of any one of claims 45 to 86, wherein the human NGF comprises or consists of the amino acid sequence of SEQ ID NO: 2.

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