WO2018087656A1

WO2018087656A1 - A new formulation for intranasal administration

Info

Publication number: WO2018087656A1
Application number: PCT/IB2017/056945
Authority: WO
Inventors: Antonio CHIARETTI; Benedetto Falsini; Giovanni Scambia; Riccardo RICCARDI; Luigi Manni
Original assignee: Università Cattolica del Sacro Cuore
Priority date: 2016-11-08
Filing date: 2017-11-07
Publication date: 2018-05-17
Also published as: EP3538129A1; IT201600112420A1

Abstract

The present invention relates to pharmaceutical compositions comprising NGF or molecules with NGF-like activity for use in the treatment of traumatic brain injury by means of intranasal administration, a method for treating traumatic brain injury by means of intranasal administration of NGF or molecules with NGF-like activity, and an associated pharmaceutical kit.

Description

"A NEW FORMULATION FOR INTRANASAL ADMINISTRATION"

DESCRIPTION

PRIOR ART

Traumatic brain injury (ΤΒΓ) is a common event in children and young adults, particularly in developed countries, and is responsible for a high level of morbidity and mortality. The clinical outcome of TBI is determined not only on the basis of the primary brain injury, but also on the basis of secondary brain damage. Following the traumatic event, a lesion forms which is the result of the mechanical destruction of the inter-neuronal connections and of the death of neurons, glial cells and vascular tissues at the trauma site. Damage to the cell membranes also causes a rise in their permeability, which leads to neuronal swelling, hypoperfusion of the tissue, and the triggering of a cascade of neurotoxic events.

Secondary brain damage is caused by the reperfusion of the damaged tissue, by the hypoxia subsequent to the interruption in breathing, and by the reduced blood flow towards the brain, all of which are events that can have devastating effects and that can have a significant influence on the development of the clinical picture.

Hypoxic-ischemic brain injury (ΉΙΒΓ) is one of the main complications of TBI and is often associated with a negative clinical and neurological outcome in survivors. HIBI causes a loss of brain neurons and can be associated with a progressive decline in cognitive and motor functions. In particular, newborns and children who suffer from these conditions experience long-term problems, which are most pronounced in respect of neurobehavioral and neuropsychological functions. At the moment, there are no effective therapies able to restore the neuronal loss and produce a substantial clinical improvement in patients who have suffered a TBI and/or HIBI. This results in a high health cost necessary to suitably assist both children affected by TBI and their families.

Nerve growth factor (NGF) is a neurotrophin that supports neurogenesis, neuronal growth, and differentiation and survival of neurons, and that can prove to be useful in treating the damage caused by TBI or other hypoxic-ischemic brain injuries, promoting the growth of new cells in the brain tissue and revascularisation of the ischemic areas. NGF allows neurites to grow and helps to restore the function of the damaged neurons. NGF present in the cerebrospinal fluid (CSF) is a useful indicator of brain damage after major head trauma, and an increase of NGF in the CSF correlates to a favorable neurological outcome.

Experimental studies have demonstrated that the intracerebroventricular (ICV) administration of NGF prevents the loss of phenotypes and conserves the damaged cholinergic neurons, increasing the synthesis of choline acetyltransferase and preventing atrophy of the basal prosencephalon. In addition, NGF administered intracerebroventricularly reduces the reductions in neuromotor performance and in spatial memory after TBI in rats (Dixon, Flinn et al. 1997). It has been reported in the literature that ICV infusion of NGF, in children affected by HIBI, has led to a reduction of the softened areas in the brain and an improvement in regional cerebral perfusion (Chiaretti, Genovese et al. 2005).

Regardless of the promising results, the main obstacles preventing the development of clinical therapies based on the use of NGF are the lack of ability of the molecules to diffuse through the blood-brain barrier (BBB), which limits the use of NGF to systemic administration, and the high invasiveness of the current methods of direct administration in the brain, for example intracerebroventricularly.

SUMMARY OF THE INVENTION

Nerve growth factor promotes the survival and growth of neurons, both of the central and peripheral nervous systems. In experimental animal models, it has been demonstrated that the administration of exogenous NGF is able to prevent or significantly reduce the acute loss of cholinergic cells and the subsequent deficiencies sustained as a result of metabolic, traumatic and hypoxic-ischemic lesions. The authors of the present invention have surprisingly found that the administration of NGF in accordance with the methods presented in the present description has a therapeutic effect on the short-, medium- and long-term consequences of hypoxic-ischemic syndromes produced as a result of changes to the vascular/circulatory system. The mechanisms involved in this function, and also the involved genes and metabolic pathways are still largely unknown, even today. The increase in the synthesis of NGF in the brain and the intraventricular administration of growth factors increase the number of new neurons and neuronal precursors produced in various structures, such as the subventricular zone (SVZ) and the subgranular zone (SGZ) of the dentate gyrus. A large portion of these neuronal precursors express a protein, referred to as doublecortin (DCX), which has been recently linked to the neurogenesis and migration of neuroblasts. A close link between NGF and DCX in neurogenesis has been demonstrated, since both the proteins are synthesized primarily in the hippocampus and prosencephalic neurons, and since both have a specific role in the neuroprotection and repair of nerve cells damaged by ischemic and traumatic injury. Recent evidence also suggests that NGF influences the migration of endothelial cells and neoangiogenesis after hypoxic-ischemic and traumatic brain injury (Chiaretti, Antonelli et al. 2008, Nico, Mangieri et al. 2008). The increased intracerebral availability of NGF thus appears to trigger the proliferation of brain capillary cells and also neoangiogenesis induced by neurons, stimulating production of the vascular endothelial growth factor (VEGF) and other vasoactive factors. In support of this experimental data it has been demonstrated that, in human tissues, NGF promotes the growth of capillaries and also arteriogenesis, which consists of the proliferation of pre-existing arterial connections in collateral arteries, whilst also providing a new capillary network, which supplies oxygenated blood to the ischemic tissues. Other studies have also demonstrated that NGF is able to promote the growth of pre-existing capillaries from sympathetic ganglia in developing rats and to improve cerebral circulation in children with hypoxic-ischemic brain injury. There are currently no pharmacological treatments able to improve the neurological losses brought about by injuries of this kind, particularly if sustained during the perinatal period. The patients affected by injuries of this kind immediately display severe motor and cognitive losses, which limit their quality of life, with significant economical and social repercussions. Any non-invasive treatment able to improve such losses could therefore have very important repercussions from both a clinical and social viewpoint.

The authors of the present invention have shown for the first time in humans, on the basis of experimental data, that the intranasal administration of NGF to a patient of pediatric age suffering from severe traumatic brain injury has led to the recovery of numerous brain functions with associated neuronal regeneration (see Figure 1 : CEREBRAL PET), and have also surprisingly found that the intranasal administration of NGF as described here has a therapeutic effect on the short-, medium- and long-term consequences of hypoxic-ischemic syndromes caused by changes to the vascular/circulatory system, resulting in an improvement of cerebral perfusion and a subsequent rise in basic metabolic activity of the brain parenchyma, as shown in figures 1-3.

Regardless of the fact that pre-clinical and clinical literature exists demonstrating the pro-angiogenic properties of NGF, it has never before been hypothesized, let alone demonstrated that the delivery of neurotrophin at cerebral level could provide such results on the functionality of the vascular system and on the metabolic activity of the nervous tissue, with subsequent restoration also of neurological functions. Although it is difficult to speculate on the biological mechanisms that lead to this effect, it is not plausible to attribute it to neo- angiogenesis (the effect being too acute and premature), but rather to causes, not yet clearly identified, as a result of which NGF is able to influence the supply and reperfusion of the damaged brain parenchyma, both at fluid-dynamic level and also with regard to the ability to improve the amount of oxygen and nutrients transported in the blood to the cerebral cells, restoring their "viability". There is no literature prior to that reported in the present patent application that demonstrates an objective and quantifiable improvement of the cerebral perfusion and metabolism after therapy with NGF.

This effect of NGF administered intranasally is innovative and particular insofar as drugs having neuroprotective action and used in the therapy of hypoxic- ischemic brain injury (i.e. antiepileptic drugs, dietary supplements, antiplatelet drugs, anticoagulants, antiedemic drugs, etc.) are currently unable to stimulate simultaneously cerebral perfusion and also an improvement in the glucidic metabolism of brain cells, as demonstrated by NGF (figures 1-3).

The invention therefore relates to the use of NGF administered intranasally as active ingredient able to induce an improvement in blood perfusion by way of the brain parenchyma and, as a result of this effect, also able to indirectly stimulate a rise in the glucidic metabolism by way of the brain cells with a subsequent partial or total restoration of the neurophysiological functionalities altered by the vascular damage or by the hypoxic event.

The invention therefore also relates to the use of NGF as active ingredient for use in therapy of pathologies that recognize an etiopathogenesis in respect of the cardiovascular system able to determine hypoxic-ischemic suffering of tissues in general and of the brain in particular.

Such administration, in the doses and in the ways described hereinafter, has allowed the drug to be carried to the brain parenchymal in a non-invasive way; a relatively quick diffusion of NGF in the central nervous system; with significant therapeutic effects and with subsequent reduction or lowering of the possible side effects associated with the action of NGF on peripheral nociceptive and autonomic neurons.

Administrating NGF intranasally is a quick and safe way of transporting drugs into areas of the CNS that are difficult to access on account of the presence of the blood-brain barrier. This process does not require particular equipment, patient collaboration or preparation, is minimally invasive, is quick, and has an excellent safety profile, guaranteeing optimum bioavailability of the drug, in view of virtually absent and in any case minimal adverse effects or side effects. The invention is absolutely novel, since there are currently no pharmacological preparations available for use in the treatment of conditions or pathologies that require the therapeutic effect of neuronal regeneration, such as the treatment of the neurological outcomes of primary hypoxic-ischemic brain injuries or such injuries caused by brain trauma. The composition for the use described in the invention and the kit described here represent not only the first pharmaceutical product aimed at treating the above-described clinical pictures, but also the first pharmacological therapeutic approach that can be placed on the market for intra-hospital and extra- hospital treatment of the neurological outcomes of hypoxic-ischemic brain injuries.

The present invention therefore relates to a pharmaceutical composition comprising NGF and/or one or more molecules with NGF-like activity and at least one pharmaceutically acceptable carrier for use in the treatment of traumatic brain injuries, of conditions derived therefrom, or of sporadic and non-sporadic neurodegenerative pathologies by intranasal administration to a human patient.

The present invention also relates to a therapeutic treatment of traumatic brain injuries, of conditions derived therefrom, or of sporadic and non-sporadic neurodegenerative pathologies by intranasal administration to a human patient of a pharmaceutical composition comprising NGS and/or one or more molecules with NGF-like activity.

The present invention furthermore relates to a kit for the intranasal administration of NGF and/or molecules with NGF-like activity comprising one or more aliquots of a pharmaceutical composition comprising NGF and/or molecules with NGF-like activity and one or more devices for the topical administration of drugs to the nasal mucosa by atomization of said drugs.

DETAILED DESCRIPTION OF THE DRAWINGS

Figure 1 shows transverse PET images of brain sections with 18F-FDG taken before treatment and after four cycles of intranasal administration of NGF, showing a significant increase in brain metabolism in many valued cortical regions. In fact, as can be seen in this figure (cerebral PET), there is a progressive increase in brain metabolism from the time TO (before starting therapy by means of intranasal administration of NGF), to the times T1 , T2, T3 and T4 relating, respectively, to the checks performed after the first, second, third and, lastly, fourth administration of NGF.

Figure 2 shows the development of cerebral perfusion before and after intranasal administration of NGF. The initial SPECT scan (TO, before treatment with NGF) revealed moderate hypoperfusion in the anterior right-hand temporal region and in the left-hand portion of the front cortex, and also in the cerebral hemispheres. In the second SPECT (T1 , after the first treatment with NGF), the cerebral perfusion was slightly improved in the front right-hand and left-hand cortex, and also in the right-hand portion of the anterior temporal cortex, and there was an improvement in both cerebral hemispheres. In the third and fourth SPECT (T2 and T3), a further improvement was observed in the right-hand and left-hand front cortex and in the right-hand anterior temporal cortex. In the last SPECT (T4, at the end of the treatment with NGF), the regional perfusion was improved further in the right-hand and left-hand front cortex, in the right-hand anterior temporal cortex, in the left hand occipital cortex, and in both cerebral hemispheres. The visual interpretation of the SPECT images was confirmed by a semi-quantitative analysis performed using SPM8 (Statistical Parametric Mapping 8) and VOIs (Volumes of Interest) in relation to the grey matter of both hemispheres and the cerebellum. This figure, which relates to the cerebral perfusion data, also shows a progressive increase in cerebral perfusion from the time TO (before the therapy by means of intranasal administration of NGF was started) to the times T1 , T2, T3 and T4 relating, respectively, to the checks performed after the first, second, third and, lastly, fourth administration of NGF.

Figure 3 shows nuclear magnetic resonance images taken before and after intranasal administration of NGS and showing both a significant reduction of the extent of parenchymal brain lesions and an enlargement of the ventricular system, with a corresponding enlargement of the periencephalic spaces, evidencing an improved trophism of the brain parenchyma. This figure relates to cerebral NMR before (a-d) and after treatment with intranasal NGF (e-h). The images are shown with various cuts of the brain parenchyma: axial (a, e), axial FLAIR (b), axial T1w (c, g), and coronal T2w (d, h), so as to be able to examine the entire brain parenchyma in greater detail. The NMR before treatment with NGF shows areas of pathological T2w and FLAIR hyperintensity (a, b, outlined arrows) and of T1w hypointensity, which is also pathological at the basal ganglia (c, outlined arrow), and also the presence of hydrocephalus d, white arrows), a reduction of hydrocephalus (h, white arrows) and a subsequent enlargement of the thickness of the periventricular grey matter, which confirms a reduction of the softened areas of the brain parenchyma with subsequent improvement of cellular trophism. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a pharmaceutical composition comprising NGF and/or one or more molecules with NGF-like activity and at least one pharmaceutically acceptable carrier for use in the treatment of pathologies caused by changes to the vascular/circulatory cerebral system, such as traumatic brain injuries or also neurodegenerative diseases caused by changes to the vascular/circulatory system.

The pathologies according to the present invention are generally pathologies characterized also by the mechanical and/or pathological destruction (for example degenerative) of the interneuronal connections and death of neurons, glial cells and optionally also vascular tissues at the site affected by such destruction. A non- limiting example of pathologies or conditions according to the present description is constituted by pathologies/conditions deriving from a traumatic event involving the central nervous system, such as traumatic brain injuries (TBIs) or pathologies/conditions associated therewith or directly derived therefrom, such as hypoxic/ischemic brain injuries (HIBIs), or hypoxic-ischemic encephalopathy in neonatal patients, infantile cerebral paralysis, or other pathologies/conditions caused by mechanical traumas or by cerebral hypoxia/ischemia.

Brain hypoxic-ischemic injuries mean injuries caused by ischemic episodes following a reduction of the amount of oxygen supplied to the brain. These injuries can be primary or secondary effects of head trauma.

In the present invention the therapeutic treatment with NGF is limited to pathologies caused by changes to the vascular/circulatory system and results in an objective and quantifiable improvement of cerebral perfusion and metabolism.

TBI, for the purposes of the present description and as reported in the literature, is constituted by damage to the brain caused by the application of an external physical force which leads to a temporary or permanent structural and functional impairment of the brain. In addition, traumatic brain injuries represent risk factors for sporadic neurodegenerative diseases. The pathologies/conditions associated with TBIs according to the invention thus also include sporadic neurodegenerative diseases (more specifically not resulting from genetic causes).

Injuries such as hypoxic/ischemic encephalopathy secondary damage, perinatal damage, infantile cerebral paralysis, brain injuries caused by cerebral ischemia of various type, including brain aneurysm ruptures, brain arteriovenous malformations, and brain sinus thrombosis, are also included.

According to the present description, pathologies caused by changes to the vascular/circulatory system include, for example, hypoxic-ischemic syndromes which include, as will be defined in greater detail hereinafter, traumatic injuries; hemorrhagic injuries; atherosclerotic injuries; cardiogenic injuries; hypertensive injuries.

At pediatric level, such pathologies include, for example, fetal and/or neonatal hypoxia/asphyxiation; major head trauma; acute cerebral hemorrhage, poisoning (carbon monoxide, cyanide); brain sinus thrombosis; coagulation factor deficiency; or also genetic diseases.

In adults, examples of such pathologies include ictus; TIA; chronic vascular encephalopathy; major head trauma; acute brain hemorrhage; poisoning (carbon monoxide, cyanide); atherosclerosis; atrial fibrillation; arterial hypertension; embolism; Alzheimer's disease.

In addition, the formulation according to the invention is also intended for use in the treatment of neurodegenerative pathologies/conditions generally also brought about by genetic causes. Non-limiting examples of neurodegenerative diseases according to the invention include Parkinson's disease, Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis (ALS), multiple sclerosis, and muscular dystrophy.

The formulation of the present invention, in its doses and methods of administration, is suitable for the therapeutic treatment of pathologies/conditions of this kind insofar as it allows the drug to be carried to the brain parenchyma in a non-invasive manner; allows relatively quick diffusion of NGF in the central nervous system; allows minimal systemic diffusion of the drug, with significant therapeutic effects and with a subsequent reduction or lowering of the possible side effects associated with the action of NGF on peripheral nociceptive and autonomic neurons.

As is known to a person skilled in the art, nerve growth factor (NGF) is a neurotrophin able to stimulate repair of damage of the nervous system by means of its trophic action on the neurons and ancillary cells as a result of its action promoting neurogenesis and vascularisation and perfusion of the damaged brain parenchyma.

The authors of the invention have surprisingly found, as shown in figures 1- 3, that NGF is able to influence the supply and reperfusion of the damaged brain parenchyma, both at fluid-dynamic level and also by means of its ability to improve the amount of oxygen and nutrients transported by blood to the brain cells, restoring their "viability", and is therefore able at the same time to stimulate cerebral perfusion and also an improvement in the glucidic metabolism of the brain cells.

In accordance with one embodiment of the invention, the following can be used in the composition for the use described herein: mouse NGF (nerve growth factor), extracted from tissues or recombinant; recombinant human NGF, molecules with NGF-like activity which act on cells that express the receptors (TrkA and p75) of NGF, molecules which promote endogenic production or the activity of endogenically produced NGF, or mixtures thereof.

In accordance with a further embodiment, molecules with NGF-like activity or molecules with an amino acid/protein structure similar to that of NGF or compounds able to function as agonists of NGF receptors can be used optionally in combination with one or more of the molecules described above.

The term 'molecules with NGF-like activity' means: protein molecules, natural or synthetic peptides, natural or synthetic chemical compounds that act by directly activating (through the ligand-receptor bond) or indirectly influencing the biological activity of the receptors of NGF, more specifically the tyrosine receptor kinase A (TrkA) and the p75 pan-neurotrophic receptor (p75NTR), alone or in combination with one another in any stoichiometric ratio. In addition, what are meant are molecules with neurotrophic activity similar to that exerted by NGF, interpreted as an ability to induce cellular survival and/or proliferation and/or growth and/or differentiation, both in neuronal cells and in non-neuronal cells that express receptors for NGF, and also activity in promoting vascular trophism, angiogenesis and blood perfusion of tissues, in accordance with that reported in the literature (Nico, Mangieri et al. 2008). The cited substances can be produced by direct synthesis of peptides from amino acids, by extraction and/or purification from biological tissues or cells, derived from biotechnologies, such as recombinant DNA techniques, or can be acquired from producers of these substances. Such molecules can be, for example: besides the protein NGF, as per the protein sequence reported in the UNIPROT references (www.uniprot.org) P01138 (http://www.uniprot.org/uniprot/P01138) and/or P01139

(http://www.uniprot.org/uniprot/P01139), any peptide fragment of the same or any size having neurotrophic activity similar to that of the protein as a whole; proteins or peptides with agonistic activity relative to the receptors of NGF, which stimulate the induction of neurotrophic and/or angiogenic effects similar to those induced by NGF itself, including anti-TrkA or anti-p75NTR antibodies which, by means of the bond with the above-mentioned receptors, induce activation of the endocellular signal pathways thereof (LeSauteur, Maliartchouk et al. 1996); natural or synthetic chemical compounds, such as those reported in the literature by (Scarpi, Cirelli et al. 2012), amitriptyline , kinase K252a inhibitor, gambogic acid amide.

According to the invention, the composition can be formulated as a liquid composition or as a lyophilized composition + carrier so as to be able to be restored just before use by the user.

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

In accordance with one embodiment of the invention, the composition shall comprise, as sole active ingredient, NGF and/or one or more molecules with NGF- like activity.

In one embodiment, the pharmaceutical composition according to the invention is thus formed of NGF and/or one or more molecules with NGF-like activity and at least one pharmaceutically acceptable carrier.

In accordance with the present invention, the human patient can be an adult patient, of any age, or can be a patient of pediatric age or even neonatal age.

The intranasal administration will be performed preferably by means of suitable devices for topical administration, which act by means of atomization of the drugs through the nasal mucosa, preferably by the formation of atomized particles with a diameter between 10 and 50 micrometers.

An example of devices suitable for intranasal administration of the composition of the invention is constituted by devices of the MAD (mucosal atomizer device) type or by devices that act with similar mechanisms and are commercially available. The application of the MAD to a syringe enables atomization of the drug as a whole, thus guaranteeing optimal absorption thereof at the nasal mucosa and reducing dispersion to the external environment and at pulmonary level to a minimum. Its structure makes it a device that is simple to use and extremely effective.

The administration of NGF intranasally by means of an MAD device is a valid and accessible way of granting this active ingredient access at CNS level. In fact, NGF is carried through the nasal mucosa by means of the nose-brain pathway at fluid level, reaching areas usually inaccessible to the vast majority of drugs.

The device loaded with the composition as described here can thus be used for the administration of NGF (or the molecules having NGF-like action) at the nasal mucosa and olfactory epithelium, with the objective of carrying it through the olfactory pathways and/or nerve endings, towards the brain parenchyma. The device, combined with the drug, can therefore be used in clinical practice with the objective of inducing neuronal regeneration in the brain, for example for the treatment of the outcomes of brain hypoxic-ischemic injuries or neurodegenerative diseases, treating pathologies or conditions that currently cannot be treated pharmacologically. The compositions described here can be administered in one or more daily doses, with amounts of from 0.05 to 0.4 mg of NGF and/or molecules with NGF-like action per kg of body weight of the patient per dose.

The administration can be performed for example 1 , 2, 3, 4, 5, 6, 7 or more times per day, depending on the patient's needs, and each time the dose can be, as described above, from 0.05 to 0.4 mg of NGF and/or molecules with NGF-like action per kg of body weight of the patient.

In one embodiment, for example, the composition can be administered 2-4 times per day, for example 3 times per day, at a dose of 0.05-0.2 mg of NGF and/or molecules with NGF-like action per kg of body weight of the patient per administration.

For example, the composition can be administered 3 times per day in a dose of 0.1 mg of NGF and/or molecules with NGF-like action per kg of body weight of the patient.

The composition can be administered in any of the ways and doses described above for one or more therapeutic cycles.

Such therapeutic cycles for example can last between one and two weeks.

By way of example, the pharmaceutical composition can be provided in the form of a number (for example any number from 10 to 20) of pre-prepared aliquots, each containing, by way of non-limiting example, 1 ml of physiological solution or any other suitable carrier, in which 0.5-4 mg of NGF are diluted. Such aliquots can be contained in phials, or also, in an embodiment offering particular ease of use, in pre-dosed syringes suitable for connection to an MAD device. The embodiment of the aliquots is provided so as to give a possibility of administering a dose between 0.05 and 0.4 mg of NGF and/or molecules with NGF-like action per kg of body weight of the patient. In accordance with a possible embodiment, the composition of the invention can comprise, for example, 1 ml aliquots containing 1 mg of NGF and/or molecules with NGF-like action, this type of formulation being particularly suitable for children with a body weight around 10 kg. Alternatively the composition can comprise 1 ml aliquots of a suitable carrier with 2 mg of NGF and/or molecules with NGF-like action to be used, for example, for children with a body weight around 20 kg, and so on. In the knowledge of the dose advised in the present description, preparing suitable aliquots in the pharmaceutical composition by body weight range is a simple implementation for a person skilled in the art.

The possibility of administering drugs intranasally, with the objective of carrying them towards the brain, bypassing the blood-brain barrier, was already suggested more than a decade ago and is currently used for the administration of some types of sedative or anesthetic drugs, for which, by means of intranasal administration, it is possible to reduce the dose and limit them, and therefore also the possible side effects. Studies relating to the administration of NGF intranasally in animals (rats) subjected to experimental TBI are also known and have been published, but, for various reasons, do not present valid conditions on which to base the development of similar treatment in human patients, of both adult and pediatric age. Firstly, the doses used in the above mentioned studies cannot be related to those reported in the present description. The dose range used in rats is between 0.5 and 1.3 micrograms/kg, and administration is performed in a single treatment cycle with one-time daily administration for a duration varying from 3 to 14 days. Doses of this kind, although they influence some biochemical parameters in relation to the development of neuronal suffering (for example accumulation of beta-amyloid protein, phosphorylation of the protein tau, rise in aquaporin, etc.), they do not always induce "clinical" improvements. The use of these methods of administration in addition does not make it possible to assess the possibility of the onset of side effects (neuropathic pain, hyperalgesia) already demonstrated in previous preclinical studies and clinical trials with peripheral administration of NGF. Instead, in the clinical study performed by the applicants, the dose administered by single cycle was from 0.05 to 0.4 mg/kg of body weight, in particular 0.1 mg/kg administered for a period of 10 days, and the administration in more daily doses was repeated for 4 cycles. As a result of this absolutely innovative administration protocol not developed on the basis of previous preclinical studies, a significant improvement both of metabolism and cerebral perfusion and of the clinical characteristics of the patient treated by the applicants (see examples hereinafter) was observed. The fact that, whereas in the studies on patients with peripheral administration of NGF significant side effects were reported (myalgia, abdominal pain, hyperalgesia), in the study performed by the applicants on patients with TBI treated with high doses (approximately 0.1 mg/kg of body weight) of NGF administered intranasally, no side effects attributable to the effect of NGF on the neurons of the peripheral nervous system developed, is therefore of significant importance and was not in any way foreseeable on the basis of the studies on animals. The invention also relates to a kit for the intranasal administration of NGF and/or molecules with NGF-like activity comprising one or more aliquots of a pharmaceutical composition comprising NGF and/or molecules with NGF-like activity and one or more devices for topical administration, by atomization, of drugs through the nasal mucosa, in which kit said composition and said devices correspond to any one of the embodiments described above. In accordance with one embodiment, the kit can be suitable for defined body weight ranges, and said one or more aliquots can be already pre-loaded in containers to be applied to, or forming part of, devices for topical administration as defined above.

In accordance with a further embodiment, said one or more aliquots can be pre- calibrated for pediatric administration or for administration in adults and can be subdivided into single doses per body weight range comprising a suitable amount of NGF and/or molecules with NGF-like activity.

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

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

By way of non-limiting example, the kit of the invention can comprise a number (for example any number from 10 to 20) of pre-prepared aliquots, each containing, by way of non-limiting example, 1 ml of physiological solution, in which 1-2 mg of NGF and/or molecules with NGF-like action, are diluted, which, for the 10 pre-dosed syringes, gives the possibility of administering 0.1 mg/kg of NGF in total. Such aliquots can be already contained in sterile syringes optionally already equipped with the MAD device for intranasal administration of the neurotrophin. In accordance with a possible embodiment, the kit of the invention for example can comprise 10 syringes with 1 mg of NGF for children of body weight around 10 kg, or 10 syringes with 2 mg of NGF for children of body weight around 20 kg, or a kit of 20 syringes with 1 mg of NGF for children of body weight around 20 kg. In the knowledge of the dose advised in the present description, preparing a suitable pharmaceutical kit for body weight ranges is a simple implementation for a person skilled in the art. In this way, it would be possible to safeguard the intranasal administration of a suitable amount of NGS able to stimulate the intracerebral receptors for NGF (TrkA and p75) present in the majority of serotoninergic and cholinergic areas of the brain. Kits of this kind, in addition, would allow the intranasal administration of NGF at home and by the parents of children (given the simplicity and safety of the methodology), significantly improving the quality of life of these children.

In addition, the composition, the kits, and the method of the invention can be used not only for patients (including patients of pediatric or neonatal age) suffering from neurological deficiencies caused by major head trauma, but also by all children affected by cerebral damage caused by hypoxic-ischemic encephalopathy brought about by perinatal damage and by patients affected generally by infantile cerebral paralysis and secondary brain injuries and cerebral ischemia of various nature (for example brain aneurysm rupture, brain arteriovenous malformations and brain sinus thrombosis).

EXAMPLES

The details of a pediatric clinical case and examples of formulations suitable for therapeutic treatment in accordance with the invention will be reported hereinafter.

The clinical case reported below was subjected to a therapeutic treatment that had been approved by the Ethics Committee of the Catholic University of the Sacred Heart in Rome and by the parents of the child, who gave their informed consent.

1. Clinical case

A child of four years, 22 kg body weight, was taken to the Paediatric Intensive Care Unit (PICU) of the A. Gemelli university hospital in Rome after a cardiac arrest, following a major head trauma caused by a car accident. Cardiopulmonary resuscitation was performed immediately on the child, and a tracheal tube fitted. After 7 minutes of cardiorespiratory arrest, a return of spontaneous circulation was established and the child was brought to our Pediatric Intensive Care Unit, where the neurological examination revealed a GCS of 4. The TAC total-body revealed the presence of blood in the lateral ventricles of the brain, deep and diffuse hemorrhagic petechiae at white matter level, as experienced with diffuse axonal injury, occipital fracture, left pulmonary contusion, and bilateral pleural effusion. In accordance with our protocol for management of post- resuscitation cardiac arrest, a mild therapeutic hypothermia (34°C) was established for 24 hours. After four days from the trauma, the sedation was interrupted and the child opened their eyes, but without any respiratory trigger. The emergency MRI of the spinal column revealed a frontal cortical-subcortical hemorrhagic contusion, and signs of axonal injury at the corpus callosum trunk and splenium. For the progression of the brain injuries, the absence of spontaneous respiratory trigger, and the appearance of complete tetraplegia, the patient was subjected to a tracheostomy, with positioning of a gastrostomy. On day 28 following the head trauma, the complete absence of respiratory efforts and respiratory triggers was confirmed, and thus the child was completely dependent on the mechanical ventilator. The child was, however, able to open their eyes spontaneously, but demonstrated an absence of swallowing and an inability to speak. Communication was possible solely by eye movements and opening of the mouth. The somatosensory evoked potentials (SSEPs) showed a significant functional loss of the upper limbs, presumably linked to the damage to the encephalic trunk. Six months after the head trauma, the child was transferred to a neurorehabilitation ward for rehabilitative care.

2. Neurological and functional examination

Once admitted to the neurorehabilitation unit, the neurological examination revealed a relatively comatose child with complete tetraplegia, severe communicative and neuropsychological impairment, and respiratory insufficiency. The child was fed by gastrostomy. The child was unable to control the bladder and intestinal functions due to the magnitude of the injury to the spinal cord. There was an increase in muscle tone at the lower limbs and also verbal dyspraxia. It was difficult to make an accurate assessment, considering the age of the child, the concomitant brain injuries, and the severe disability with regard to communication, however a sensomotoric assessment was performed with application of the "ASIA-IMSLP Classification", which revealed a loss of sensitivity of the skin and movement below level C2. The ASIA scale yielded a result of "A", which confirmed complete lesion of the spinal cord. The EEG recordings revealed basic theta activity with poor electrical organization. The functional assessment was performed using the SCIM (spinal cord independence measure), version III and gave a final score of 11 (from 0 to 100, where 100 is total independence of movements). Focused management of the spasticity in specific muscular groups with botulinum toxin was performed, with no improvement. The patient was also subjected to numerous cycles of physiotherapy, but without any sign of improvement either in communicative capabilities or motor capabilities.

3. Intranasal administration of NGF

Six months after the head trauma, with the comatose state persisting alongside complete tetraplegia, in the absence of any clinically noticeable improvement in spite of the conventional and approved therapeutic strategies and also in the absence of any other possible therapeutic approach, treatment with NGF administered intranasally was considered. Such treatment was approved by the Ethics Committee of the Catholic University of the Sacred Heart in Rome and by the parents of the child, who gave their informed consent. The child received a total of 4 cycles of intranasal NGF. Each treatment was repeated at intervals of one month. With each cycle, NGF was administered in a dose of 0.1 mg/kg (2 mg of NGF diluted in 20 ml of saline solution, three times per day for 10 consecutive days) by means of an MAD (mucosal atomization device), used to administer drugs by atomization thereof in the nasal cavity. Such an amount of NGF was considered sufficient to reach and stimulate the NGF receptors, primarily the tyrosine kinase of receptor A (TrkA), in the majority of the cholinergic and serotoninergic brain areas. Before the administration of NGF, the nostrils were washed with 1 ml of physiological solution, then were sprayed, so as to avoid any interference with the absorption of the drug.

4. Clinical results

After the treatment with NGF, significant improvements in some cognitive processes were observed, particularly in the planning of a communication strategy, attention and verbal comprehension (in the "PinG-Parole in gioco" wordplay test, a score within the normal range was achieved). After the intranasal administration of NGF, the patient demonstrated a significant improvement also in facial gestures (many new expressions added, such as joy, fear, astonishment, etc.), with an initial ability to furrow the eyebrows, wrinkle the nose, cry with tears (all absent in the pre- treatment phase), improved symmetry of the smile and improved communicative expression. The child was able to have a better relationship with loved ones and with the health workers, and consequently became less frustrated and had an enhanced mood. The verbal dyspraxia demonstrated a progressive improvement, particularly in respect of the control of oral motility, such as opening the mouth, motility of the tongue, chewing and swallowing. In addition, the possibility to feed oneself improved; in fact, the child was able to eat small amounts of food and the parents communicated an improvement in the ability to distinguish between different tastes. Alongside the improvement of the verbal dyspraxia, improvements were also observed in phonation abilities, with formulation of more explicit sounds. In addition, during the cycles of therapy with NGF, a progressive and constant improvement in movements of the head (primarily lateral rotational movements and, to a lesser extent, flexion-extension). Recently, some movements of the fingers of the hand have been observed during playtime, with characteristics of voluntary control and with improved muscle tone and trophism. Another important improvement was that of intestinal function: in fact, the child now has no need to be stimulated to evacuate the system. At the end of therapy with NGF, the patient has also demonstrated some spontaneous respiratory actions when disconnected from the mechanical ventilator. During these efforts, the appearance of coughing and hiccupping reflexes has also been observed, which were absent beforehand.

5. PET results Transverse PET images of brain sections with 18F-FDG taken before the treatment and after four cycles of intranasal administration of the NGF showed a significant increase in brain metabolism in many cortical regions assessed (Figure 1).

6. SPECT results

The brain SPECT revealed the development of the cerebral perfusion before and after intranasal administration of NGF. At the start of the treatment with NGF, brain hypoperfusion was present, evident most clearly at the frontal and temporal cortex dx, in the cerebral cortex sx, and at the brain hemispheres. After the treatment with NGF intranasally, a progressive and significant improvement of the cerebral perfusion was observed in the areas described above, with the exception of the brain hemispheres, where, instead, zones of vascular hypoperfusion still persisted. The visual interpretation of the SPECT images was confirmed by a semiquantitative analysis, using the statistical parametric mapping 8 (SPM8) system and the volumes of interest (VOIs) positioned at the grey brain matter (Figure 2)

7. Electroencephalogram (EEG) studies

The EEG examinations were performed before and after the treatment with intranasal administration of NGF and revealed a constant and progressive reduction of slow-wave activity with partial recovery of a normal electrophysiological rhythm.

8. Event-related potential (ERP) studies

ERPs were recorded at the start and at the end of the treatment with NGF, in accordance with a technique already standardized. We found that, compared to the start, the ERPs in both eyes demonstrated a significant increase in the phase/latency ratio (>90° percentile) of the test-retest variability established in healthy children and in children with serious pathologies of the optical nerve caused by glioma, with a change of 60 and 100 degrees respectively for the right and left eye. This change corresponds to an advance of latency response between 20 and 30 milliseconds, indicating faster and more efficient visual cortical responses to light after treatment with NGF.

9. Determination of nerve growth factor and doublecortin in the cerebrospinal fluid

In order to assess whether the intranasal administration of NGF allows a modification of the concentration of NHF in the cerebrospinal fluid (CSF), the NGF ELISA was performed on samples of CSF collected by lumbar puncture, performed under deep sedation of the patient, before (pre-treatment) and at the end of the administration of the NGF. The levels of the NGF were significantly increased in the CSF after intranasal administration of NGF. In fact, after the first and fourth treatment, an increase of 30% and of 44% of the NGF content in the CSF was observed, respectively. It has already been demonstrated how the levels of the protein doublecortin (DCX), associated with neurogenesis, appear to rise following intraventricular infusion of NGF, this being correlated with an improved result in children with major head trauma. The presence and variation of the concentrations of DCX in the fluid was thus analyzed, by means of Western blot, before and after intranasal administration of NGF. The levels of DCX in the CSF were increased in both the post-treatment samples compared to their respective pre-treatment controls. In addition, similarly to that observed for the NGF protein, the initial content of DCX in the CSF has risen over time, as demonstrated by a comparison with the pre-treatment samples.

10. NMR examination

Nuclear magnetic resonance, performed before and after the intranasal

administration of NGF, demonstrated a significant reduction both of the extent of the parenchymal brain injuries and of the enlargement of the ventricular system, with a corresponding enlargement of the periencephalic spaces, evidenced by improved trophism of the brain parenchyma (Figure 3).

11. Formulation of NGF for intranasal administration used in the clinical case

The nasal preparations were liquid, semi-solid, or solid preparations to be administered in the nasal cavities to obtain a systemic or local effect. The nasal preparation with NGF used in the treatment of the clinical case described above contained a single active ingredient in isotonic solution and was provided in a single dose, in a syringe with a system for nasal nebulation. The nasal solution was produced by the pharmacy of the Agostini Gemelli university hospital using:

1 mg aliquots of lyophilized mouse NGF;

carrier (physiological solution sterile 0.9% NaCI);

3 ml luer-lock BD syringe for coupling with the MAD Nasal (mucosal atomization device) system for the administration of drugs through the nasal and oropharyngeal mucosas.

The lyophilized NGF was restored using 0.9% NaCI sterile physiological solution by means of an aseptic technique, in a sterile environment, under a vertical laminar flow hood, as indicated by the Good Preparation Practices of the Official Pharmacopoeia, edition XII. The solution was then divided into sterile syringes, each containing 1 ml of solution formed by 0.07 mg of NGF in physiological solution (0.9% NaCI). The prepared preparations were then introduced into sterile bags to ensure, at the moment of use, the absence of microbiological contamination and were labeled, noting all of the data requested by the Good Preparation Practices of the Official Pharmacopoeia, edition XII.

BIBLIOGRAPHY

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Chiaretti, A., O. Genovese, R. Riccardi, C. Di Rocco, D. Di Giuda, P. Mariotti, S.

Pulitano, M. Piastra, G. Polidori, G. S. Colafati and L. Aloe (2005). "Intraventricular nerve growth factor infusion: a possible treatment for neurological deficits following hypoxic-ischemic brain injury in infants." Neurol Res 27(7): 741-746.

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Claims

WHAT IS CLAIMED IS

1. A pharmaceutical composition comprising NGF and/or one or molecule with NGF-like activity and at least one pharmaceutically effective carrier for use in the treatment of traumatic brain lesions, of brain ischemic hypoxic lesions, of conditions derived from said lesions or of sporadic and non-sporadic neurodegenerative pathologies by intranasal administration to a human patient.

2. The composition for use according to claim 1 wherein said human patient a pediatric or neonatal patient.

3. The composition for use according to claim 1 or 2 wherein said intranasal administration is carried out with devices for the topical administration of drugs to the nasal mucosa by atomization of said drugs.

4. The composition for use according to claim 3 wherein said device is a MAD- like device (Mucosal Atomizer Device).

5. The composition for use according to any one of claims from 1 to 4 wherein said composition is administered in one or more daily doses comprising from 0.05 a 0.4 mg of NGF and/or molecules with NGF-like activity per kg of body weight of said patient.

6. The composition for use according to claim 5 wherein said one or more daily doses comprise from 0.05 to 0.2 mg of NGF and/or molecules with NGF-like activity per kg of body weight of said patient.

7. The composition for use according to any one of claims from 5 or 6 wherein said daily doses are divided in 1 to 5 aliquots.

8. The composition for use according to any one of claims from 1 to 7 wherein said one or more daily doses are administered for one or more therapeutic cycle if a duration of time comprised from 1 and 2 weeks.

9. The composition for use according to any one of claims from 1 to 8 wherein said brain ischemic hypoxic lesions are selected from hypoxic-ischemic brain injuries, hypoxic-ischemic encephalopathies, Infantile cerebral palsy, or other pathologies/conditions caused by mechanical traumas or by brain hypoxy/ischemia, or other lesions implying a neuronal loss, and wherein said neurodegenrative diseases are selected from Parkinson's disease, Alzheimer's disease, Huntington's chorea, Amyotrophic Lateral Sclerosis (ALS), hypoxic-ischemic encephalopathy secondary damages, perinatal damages, brain lesions caused by cerebral ischemia including brain aneurysm ruptures, brain arteriovenous malformations and brain sinus thrombosis.

10. A kit for the intranasal administration of NGF and/or molecules with NGF-like activity comprising one or more aliquots of a pharmaceutical composition comprising of NGF and/or molecules with NGF-like activity and or more device for the topical administration of drugs to the nasal mucosa by atomization of said drugs.

1 1. The kit according to claim 10 wherein said one or more aliquots are for pediatric administration or for adult administration and are divided in single doses for specific body weight ranges, each of said doses comprising a suitable quantity of NGF and/or molecules with NGF-like activity.

12. The kit according to claim 10 wherein said aliquots are multi-dosage aliquots that can be divided in suitable dosages according to the patient's body weight.

13. The kit according to anyone of claims from 10 to 12 wherein said more devices are MAD-like devices (Mucosal Atomizer Device).