NGF for treating spasticity

Abstract

The present invention relates to nerve growth factor (NGF) or a mutant protein thereof for use in the prevention or treatment of spasticity in a subject, wherein the NGF or mutant protein is preferably administered intranasally to said subject.

Description

[Technical Field]

[0001] The present invention relates to the prevention or treatment of spasticity in a subject. [Background technology]

[0002] Spasticity is a movement disorder characterized by increased muscle-tendon spasms caused by hyperexcitability of the muscle stretch reflex, along with a velocity-dependent increase in muscle tone (JW Lance, "Symposium synopsis," in Spasticity: Disordered Motor Control, R.G. Feldman, R.R. Young, and W.P. Koella, Eds., pp. 485-494, 1980). It can occur as a result of traumatic injury, chronic neurodegenerative conditions, or genetic abnormalities, which can cause destruction or dysfunction of brain regions and neural pathways responsible for controlling movement initiation and control. These include, for example, cerebral palsy, stroke, traumatic brain injury, amyotrophic lateral sclerosis, primary lateral sclerosis, multiple sclerosis (MS), and hereditary spastic paraplegia (HSP) (Mathewson et al., Phys Med Rehabil Clin N Am 2015, 26(1):57-67; Khundadze et al., Autophagy 2021, 17(11):3690-3706; Chang et al., Crit Rev Phys Rehabil Med 2013, 25(1-2):11-22).

[0003] Spasticity is a highly disabling condition for patients because it affects the ability to perform basic daily activities, such as hygiene, dressing, walking, and sleeping. Without long-term treatment, spasticity can lead to pain, permanent joint deformities, urinary tract infections, chronic constipation, peripheral neuropathy, and pressure ulcers.

[0004] Therefore, treatment of this condition is essential to improve quality of life and avoid serious medical complications. Currently available treatment options are limited and only aim to relieve symptoms and enable individuals to tolerate the least possible discomfort and limitation with few side effects.

[0005] Available treatments include systemic medications and interventional procedures. Systemic drug therapies include centrally acting substances such as baclofen, anticonvulsants such as benzodiazepines and gabapentin, and peripherally acting substances such as dantrolene.Interventional procedures include local injections of botulinum toxin, phenol or alcohol, and insertion of an intrathecal baclofen pump.

[0006] Unfortunately, all available treatments are characterized by adverse effects and risks that may outweigh the potential benefits they may offer. Systemic medications have the potential to be beneficial for patients with generalized spasticity, but they can cause many unwanted effects, such as generalized muscle relaxation, sedation, fatigue, and in some cases, can lead to tolerance and dependence.

[0007] Interventional therapies are generally associated with few systemic side effects if patients comply and the procedure is performed correctly, but serious complications can arise if the procedure is performed incorrectly, resulting, for example, from the diffusion of drugs to other parts of the body, and therefore must be performed by appropriately trained professionals. Summary of the Invention

[0008] Therefore, there is a strong need to develop new effective, durable, and safe therapeutic approaches for the prevention or treatment of spasticity. Furthermore, there is a need to identify new treatments that address not only the symptoms of this condition but also the underlying pathological mechanisms.

[0009] Summary of the Invention As described in the Examples, the present inventors have found that intranasal administration of NGF delivers particularly high concentrations to dysfunctional brain regions in patients with spasticity.

[0010] The inventors have also surprisingly found that intranasal administration of NGF is effective in restoring activity in these areas, thereby reducing spasticity and ultimately restoring motor function in patients.

[0011] Furthermore, the effectiveness of intranasally administered NGF against spasticity is independent of the nature of the underlying disease that induces spasticity. Indeed, we have found that NGF restores motor activity in both animal models of mechanically induced brain injury (TBI model) and associated neuronal physiopathological damage (chemogenetic model).

[0012] Therefore, based on the available data, NGF, unlike the therapeutic options available to date, is capable of reducing the symptomatology of patients suffering from spasticity and at least partially reversing the movement disorders associated with this spasticity.

[0013] An object of the present invention therefore relates to NGF or a mutant protein thereof for use in the prevention or treatment of spasticity in a subject. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows the biodistribution of NGF in rat brain regions after intranasal administration, measured over 24 hours by ELISA as described in Example 1. Results are expressed as a percentage of total absorption in the rat brain over 24 hours after administration. [Figure 2] Figure 2 shows the "rotarod latency" (% vs. sham animals) in rats with chemogenetic blockade of cholinergic neurons (ChAT off) or sham rats (sham) treated with intranasal vehicle (ChAT off and sham, respectively) or rhNGF (ChAT off + NGF and sham + NGF, respectively), as described in Example 2. [Figure 3]Figure 3 shows "Rotarod latency" (% vs. sham animals) in mice with traumatic brain injury or sham treated with intranasal vehicle (TBI or sham, respectively) or rhNGF (TBI + NGF and sham + NGF, respectively), as described in Example 3. [Figure 4] Figure 4 shows the time, in seconds, required to turn (Panel A) and descend from the post (Panel B) in sham mice (Sham) or mice with traumatic brain injury treated with vehicle (TBI / Veh) or intranasal administration of rhNGF starting on day 1 (TBI / rhNGF d1) or day 7 (TBI / rhNGF d7) after TBI, as described in Example 4. §P<0.05, §§P<0.01, §§§P<0.001, and §§§§P<0.0001 vs. Sham, and *P<0.05, **P<0.01, ****P<0.0001 vs. TBI / Vehicle. Data are presented as mean ± SEM (n=20 / group). [Figure 5] Figure 5 shows the results of the open field test in sham mice (Sham) or mice with traumatic brain injury treated with vehicle (TBI / Veh) or intranasal administration of rhNGF starting on day 1 (TBI / rhNGF d1) or day 7 (TBI / rhNGF d7) after TBI, as described in Example 4. Specifically, panel A shows representative trajectories, panel B shows the average distance traveled in cm, and panel C shows the movement speed in cm / sec. §§P<0.01, §§§P<0.001, and §§§§P<0.0001 vs. Sham, **P<0.01, ***P<0.001, and ****P<0.0001 vs. TBI / Vehicle. Data are presented as mean ± SEM (n=20 / group). [Figure 6]Figure 6 shows mechanical allodynia, expressed as paw withdrawal response (g), measured in sham mice (Sham) or mice with traumatic brain injury treated with vehicle (TBI / Veh) or intranasal administration of rhNGF starting on day 1 (TBI / rhNGF d1) or day 7 (TBI / rhNGF d7) after TBI, as described in Example 4. §§P<0.01, §§§P<0.001, and §§§§P<0.0001 vs. Sham, *P<0.05, **P<0.01, and ****P<0.0001 vs. TBI / Vehicle. Data are expressed as mean ± SEM (n=20 / group). [Figure 7-1] Figure 7 shows the results of footprint testing in sham mice (Sham) or mice with traumatic brain injury treated with vehicle (TBI / Veh) or intranasal administration of rhNGF starting on day 1 (TBI / rhNGF d1) or day 7 (TBI / rhNGF d7) after TBI, as described in Example 4. In particular, panel A shows parameters measured in footprint recording and footprint analysis, with the dotted line representing direction of progression (DoP). Vertical black arrows indicate stride length analysis, horizontal black arrows indicate sway length, and diagonal black arrows indicate overlap. Panel B shows stride length measured in cm. §P<0.05, §§P<0.01, §§§P<0.001, and §§§§P<0.0001 vs. Sham; *P<0.05, **P<0.01, and ****P<0.0001 vs. TBI / Vehicle. Data are expressed as mean ± SEM (n=10 / group). [Figure 7-2]Figure 7 shows the results of footprint testing in sham mice (Sham) or mice with traumatic brain injury treated with vehicle (TBI / Veh) or intranasal administration of rhNGF starting on day 1 (TBI / rhNGF d1) or day 7 (TBI / rhNGF d7) after TBI, as described in Example 4. Panel C shows sway length measured in cm. §P<0.05, §§P<0.01, §§§P<0.001, and §§§§P<0.0001 vs. Sham, *P<0.05, **P<0.01, and ****P<0.0001 vs. TBI / Vehicle. Data are presented as mean ± SEM (n=10 / group). Panel D shows overlap measured in cm. §P<0.05, §§P<0.01, §§§P<0.001, and §§§§P<0.0001 vs. sham, *P<0.05, **P<0.01, and ****P<0.0001 vs. TBI / vehicle. Data are expressed as mean ± SEM (n=10 / group). [Figure 8] FIG. 8 shows the time in seconds required to descend from the post in sham mice (Sham) or mice with traumatic brain injury when treated with vehicle (TBI / Veh) or intermittent intranasal administration of rhNGF from day 7 post-TBI (TBI / rhNGF d7), as described in Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0015] A first object of the present invention relates to nerve growth factor (NGF) or a mutant protein thereof for use in the prevention or treatment of spasticity in a subject. Preferably, the NGF or mutant protein is administered to the subject intranasally.

[0016] Preferably, the spasticity is caused by a condition selected from cerebral palsy, stroke, traumatic brain injury, amyotrophic lateral sclerosis, primary lateral sclerosis, multiple sclerosis (MS), and hereditary spastic paraplegia (HSP).

[0017] The terms "treatment" and "prophylaxis" as used herein refer to eradicating / ameliorating or preventing / delaying the onset of one or more of the symptoms associated with a disorder, respectively. Preferably, the subject is a human subject.

[0018] According to one embodiment, the subject has been diagnosed with spasticity caused by a condition selected from cerebral palsy, stroke, traumatic brain injury, amyotrophic lateral sclerosis, primary lateral sclerosis, multiple sclerosis (MS), and hereditary spastic paraplegia (HSP), and the NGF or mutant protein thereof is for use in treating the spasticity in the subject, preferably by intranasal administration to the subject.

[0019] According to an alternative embodiment, the subject has been identified as being at risk of developing spasticity caused by a condition selected from cerebral palsy, stroke, traumatic brain injury, amyotrophic lateral sclerosis, primary lateral sclerosis, multiple sclerosis (MS), and hereditary spastic paraplegia (HSP), and the NGF or mutant protein thereof is for use in preventing the spasticity in the subject prior to the onset of the spasticity, preferably by intranasal administration to the subject.

[0020] Preferably, the NGF is human NGF. Preferably, the human NGF has the amino acid sequence of SEQ ID NO:1 below. SEQ ID NO:1: SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVR Alternatively, the human NGF has the amino acid sequence of SEQ ID NO:2 below. SEQ ID NO:2: SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA Alternatively, the human NGF is a mixture of NGFs having the sequences of SEQ ID NO:1 and SEQ ID NO:2.

[0021] Human NGF of SEQ ID NO: 2 has an amino acid sequence that differs slightly from NGF of SEQ ID NO: 1 by the presence of two additional amino acids at the C-terminus. Both forms of NGF are found in human cells and are therefore considered to be wild-type human NGF.

[0022] Thus, when reference is made in this application to "human NGF" or "wild-type human NGF," it means the human NGF of SEQ ID NO:1 or SEQ ID NO:2. Of the two forms of wild-type NGF, human NGF of SEQ ID NO: 1 is particularly preferred. Indeed, the present inventors have found that this particular form of NGF has particularly advantageous biological activity compared to NGF of SEQ ID NO: 2, and exhibits high neuroprotective activity against nerve cells.

[0023] Preferably, the NGF is produced by recombinant DNA technology, and is preferably human recombinant NGF (rhNGF). Methods for producing rhNGF are known to those skilled in the art, for example, the methods described in International Publication Nos. 0022119 and 2013092776.

[0024] Preferably, the NGF has a purity of greater than 70%, more preferably greater than 80%, 90%, 95%, 98%, or 99%. The purity of NGF can be determined by conventional means known to those skilled in the art, such as by HPLC analysis.

[0025] The term "NGF mutein" refers to a biologically active mutein of NGF, and means an NGF protein having an amino acid sequence with one or more amino acid mutations, preferably substitutions, such that the therapeutic activity of wild-type NGF is maintained.

[0026] Preferably, said mutein is a mutein of wild-type human NGF as defined above. Particularly preferred muteins for use according to the present invention are muteins of NGF characterized by greater than 80%, more preferably greater than 90%, even more preferably greater than 95%, and most preferably greater than 98% sequence identity with wild-type human NGF.

[0027] Preferably, the mutein is a mutein of wild-type human NGF characterized by at least one mutation, preferably a substitution of an amino acid at position 61 of the sequence of wild-type human NGF with another amino acid for proline. In a particularly preferred embodiment, proline at position 61 is substituted with serine.

[0028] Preferably, the mutein is a mutein of human NGF characterized by at least one mutation in the amino acid sequence associated with reduced nociceptive activity. More preferably, the mutein is characterized by at least one mutation, preferably a substitution of an amino acid at any of positions 95 to 101 of wild-type human NGF. Even more preferably, the mutein is characterized by a substitution of arginine at position 100 of wild-type human NGF with another amino acid. More preferably, arginine at position 100 in wild-type human NGF is substituted with glutamic acid.

[0029] Preferably, the mutant protein is a mutant protein of wild-type human NGF characterized by at least a substitution of proline at position 61 with another amino acid, preferably serine, and a substitution of arginine at position 100 in wild-type human NGF with another amino acid, preferably glutamic acid.

[0030] Particularly preferred mutant proteins according to the present invention have the amino acid sequences of SEQ ID NOs: 3 to 6 below. SEQ ID NO:3: SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWEFIRIDTACVCVLSRKAVR SEQ ID NO:4: SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWEFIRIDTACVCVLSRKAVRRA SEQ ID NO:5: SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDSNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWEFIRIDTACVCVLSRKAVR SEQ ID NO:6: SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDSNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWEFIRIDTACVCVLSRKAVRRA The above-mentioned mutein maintains the same biological activity as wild-type human NGF, but is described as being able to induce lower nociceptive sensitivity compared to the corresponding wild-type human NGF, and is therefore particularly advantageous for use according to the present invention.Preferably, the above-mentioned mutein of human NGF is produced by recombinant DNA technology.Methods for producing the mutein of rhNGF according to the present invention by recombinant DNA technology are known to those skilled in the art, and include, for example, the method described in International Publication No. 2019 / 207106.

[0031] Preferably, NGF or mutein for use according to the present invention is administered to a subject once daily or once every two / three days throughout the treatment period. Preferably, the treatment period is 7 to 300 days, preferably 60 to 240 days, more preferably 100 to 200 days.

[0032] More preferably, the NGF or mutant protein for use according to the present invention is administered to the subject 1 to 3 times daily for a treatment period of 7 to 300 days, preferably 60 to 240 days, more preferably 100 to 200 days.

[0033] A preferred administration schedule is a continuous administration schedule, in which the NGF or mutein is administered according to the same schedule throughout the treatment period. Alternatively, several cycles of treatment may be administered, whereby a preferred dosing schedule is an intermittent dosing schedule, in which two or more treatment periods are alternated with drug-free periods.

[0034] The inventors have surprisingly observed that in cases of spasticity induced by a traumatic event, such as stroke or traumatic brain injury, treatment with NGF is more effective if initiated once symptoms of spasticity have fully developed.

[0035] Thus, when the nerve growth factor (NGF) or mutant protein thereof is for use in treating spasticity caused by a condition selected from stroke or traumatic brain injury in a subject, the NGF or mutant protein is preferably administered to the subject starting 6 to 10 months, more preferably 8 to 10 months, more preferably 9 to 10 months, and even more preferably 9 months after the onset of the stroke or traumatic brain injury. Preferably, in all of the above preferred embodiments, the NGF or mutant protein is administered to the subject intranasally.

[0036] Preferably, when NGF or a mutant protein thereof is administered intranasally, the amount of NGF or a mutant protein thereof per administration is 5 μg to 1 mg, more preferably 10 μg to 400 μg, and even more preferably 15 μg to 200 μg.

[0037] The effective amount of NGF or mutant protein to be used in each administration, the duration of treatment, and the number of daily administrations will be selected by one skilled in the art based on the characteristics of the subject being treated, the severity of the spasticity, and the evaluation tests performed during treatment.

[0038] A further object of the present invention relates to a pharmaceutical composition for intranasal administration comprising NGF or a mutant protein as defined above and at least one pharmaceutically acceptable excipient suitable for intranasal use.

[0039] Preferably, the pharmaceutical composition for intranasal administration of the present invention is a liquid intranasal composition. Preferably, the pharmaceutical composition according to the present invention comprises an effective amount of the above-mentioned NGF or mutant protein and at least one pharmaceutically acceptable excipient suitable for intranasal use, preferably selected from solvents, viscosity-increasing substances, mucoadhesive substances, buffers, antioxidants, preservatives, and penetration enhancers.

[0040] Preferably, the concentration of said NGF or mutant protein in the liquid intranasal composition according to the invention is between 5 μg / ml and 1 mg / ml, more preferably between 10 μg / ml and 400 μg / ml, even more preferably between 15 μg / ml and 200 μg / ml.

[0041] Preferably, the solvent is water. Preferably, the mucoadhesive substance is glycerol, more preferably at a concentration of 0.05% w / v to 0.2% w / v, more preferably 0.1% w / v.

[0042] Preferably, the antioxidant is methionine, more preferably at a concentration of 0.005 mg / ml to 0.02 mg / ml, more preferably 0.01 mg / ml. Preferably, the surfactant is Kolliphor P188, more preferably at a concentration of 0.05% w / v to 0.2% w / v, more preferably 0.1% w / v.

[0043] Preferably, the buffer is a phosphate buffer. Preferably, the penetration enhancer is n-dodecyl-β-D-maltoside, more preferably at a concentration of 0.1% w / v to 1% w / v, more preferably 0.5% w / v.

[0044] Particularly preferred liquid intranasal compositions according to the present invention comprise, and preferably consist of, NGF or a mutant protein as described above, sodium chloride, phosphate buffer, and water. Another particularly preferred liquid intranasal composition according to the present invention comprises, and preferably consists of, NGF or a mutant protein as described above, sodium chloride, phosphate buffer, Kolliphor P188, L-methionine, and water.

[0045] Another particularly preferred liquid intranasal composition according to the present invention comprises, and preferably consists of, NGF or a mutant protein as described above, sodium chloride, phosphate buffer, Kolliphor P188, L-methionine, glycerol, n-dodecyl-β-D-maltoside, and water.

[0046] Preferably, the liquid intranasal composition according to the present invention comprises, and preferably consists of, the following ingredients: - the above-mentioned NGF or its mutant protein at a concentration of preferably 0.3 to 2 mg / ml, more preferably 0.5 to 1.5 mg / ml; NaH2PO4*H2O, preferably at a concentration of 5-8 mg / ml, more preferably 6.9 mg / ml NaCl, preferably at a concentration of 5 to 6.5 mg / ml, more preferably 5.84 mg / ml Kolliphor P188, preferably at a concentration of 0.05% w / v to 0.2% w / v, more preferably 0.1% w / v L-methionine, preferably at a concentration of 0.05 mg / ml to 0.2 mg / ml, more preferably 0.1 mg / ml - optionally n-dodecyl-β-D-maltoside, preferably at a concentration of 0.1% w / v to 1% w / v, more preferably 0.5% w / v, and / or glycerol, preferably at a concentration of 0.05% w / v to 0.2% w / v, more preferably 0.1% w / v; - water Pharmaceutical compositions according to the present invention may be suitably formulated using any suitable method known in the art or as disclosed in Remington's Pharmaceutical Sciences (latest edition), Mack Publishing Company, Easton Pa.

[0047] Preferably, the pharmaceutical composition of the present invention is for use in the prevention or treatment of spasticity in a subject, as described above, and said composition is administered intranasally to the subject. In a further aspect, the present invention relates to a method for the prevention or treatment of spasticity in a subject, preferably comprising the step of intranasally administering to the subject NGF or a mutant protein thereof, as described above, in a therapeutically effective amount.

[0048] Preferably, in the method according to the invention, said NGF or mutant protein is administered as described above. Preferably, the NGF or mutant protein used in the methods of the present invention is in the form of a pharmaceutical composition, as described above.

[0049] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Example [Example]

[0050] The biodistribution of NGF in rat brain regions after a single intranasal administration was evaluated. A formulation containing 1.2 mg / mL of rhNGF was administered intranasally to rats once. Rats were euthanized at various time points corresponding to 2, 4, 8, and 24 hours after treatment.

[0051] Samples from the parietal cortex, hypothalamus, thalamus, striatum, hippocampus, brainstem, frontal cortex, and medial septum were collected for rhNGF quantification by ELISA, and cumulative absorption (2–24 h) was calculated.

[0052] Before analysis, brain samples were homogenized in ice using an UltraTurrax and centrifuged to collect the supernatant. A commercially available ELISA kit (RayBiotech, catalog ELH-BNGF) was used according to the manufacturer's instructions. The calibration range for NGF determination was established between 20.5 and 5000 pg / mL.

[0053] As shown in Figure 1, NGF uptake was evident in all brain tissues, but the protein was particularly concentrated in regions involved in locomotor activity, particularly the hypothalamus and thalamus. [Example]

[0054] The role of NGF in counteracting spasticity and the associated decline in balance and motor coordination was evaluated in a rat chemical genetic model representative of the neuronal abnormalities observed in HSP. Briefly, selective blockade of cholinergic striatal neurons was achieved by stereotactic injection of a Cre-dependent adeno-associated virus (AAV-hSynDIO-hM4Di-mCherry) into the relevant brain region, followed by neuronal deactivation by clozapine-N-oxide (CNO) treatment, which acts against a specific promoter used to selectively regulate cholinergic striatal neuronal function. This is described in Aldrin-Kirk et al., Neurobiology of Disease 2018(109), pp. 148-162.

[0055] In this experiment, we assessed the selective contribution of cholinergic neurons in the development of motor dysfunction using the rotarod test and demonstrated the functional relevance of the potential restoration of corticostriatal connectivity promoted by intranasal administration of rhNGF (50 μg / kg for 3 days).

[0056] As shown in Figure 2, blockade of cholinergic neurons (ChAT off) reduced rotarod latency to approximately 50% of the value measured in sham animals. Treatment with NGF (ChAT off + NGF) restored motor balance to approximately 75% of the value measured in sham animals. No relevant changes in motility were observed in sham animals treated with NGF (Sham + NGF). Measurements were performed 24 hours after the final treatment. [Example]

[0057] The effects of NGF were also evaluated in a mouse TBI model. Animal care was performed in accordance with the Italian (DL116 / 92) and European Commission (OJof ECL358 / 1 18 / 12 / 86) regulations for the protection of experimental animals. Every effort was made to reduce both the number of animals and their suffering in the experiments.

[0058] Male C57BL / 6 mice (Envigo, Italy) weighing 18–20 g were lightly anesthetized with isoflurane and placed in a prone position on a porous support. The head was not immobilized. To establish TBI, a 120 g weight was dropped from a height of 25 cm onto the skull of the mouse, inducing consistent brain damage and establishing severe motor dysfunction. Sham mice were subjected to the same procedure as described for TBI mice, but without the weight drop.

[0059] The mice were visually inspected for signs of spasticity in the limbs, such as clonus (repeated muscle spasms), prolonged spasms, twitching (rapid involuntary contractions), and hyperreflexia. Once spasticity symptoms were well established, treatment with NGF (50 μg / kg / day for 3 days) was initiated.

[0060] The ability of NGF to reduce spasticity and restore muscle functionality as a measure of motor coordination impairment was assessed using the rotarod test. The test consisted of two walking sessions on a rotating cylinder, separated by a 1-hour rest period. After a 30-second acclimation period, the rotation speed was gradually increased from 3 to 30 rpm for a maximum test time of 5 minutes. The latency to fall (seconds) was recorded, and the final data were expressed as a percentage of the latency relative to sham. As reported in Figure 3, induction of TBI (TBI) reduced rotarod latency to approximately 60% of the sham value. Treatment with NGF (TBI + NGF) was able to restore motor balance to approximately 80% of the sham value. No effect of NGF itself was observed in sham treatment (sham + NGF). Measurements were performed 24 hours after the final treatment. [Example]

[0061] Male C57BL / 6 mice (Envigo, Italy), weighing 18–20 g, were housed three per cage under controlled lighting (12-h light / dark cycle; lights on at 6:00 A.M.) and standard environmental conditions (ambient temperature 20–22°C, humidity 55–60%) for at least 1 week prior to the start of the experiment. Mouse chow and tap water were available ad libitum. Animal care was in accordance with Italian (DL116 / 92) and European Commission (OJof ECL358 / 1 18 / 12 / 86) regulations for the protection of laboratory animals. Every effort was made to reduce both the number of animals and their suffering during the experiment.

[0062] Severe TBI, commonly associated with spasticity symptoms, was induced according to the Marmarou drop weight model according to the following protocol. Mice were lightly anesthetized with isoflurane and placed in a prone position on a porous support. The head was not immobilized. After a midline longitudinal incision, the skull was exposed to locate the impact area and placed under a metal tube apparatus, with the opening positioned directly above the animal's head. Injury was induced by dropping a cylindrical metal weight (250 g) from a height of 2 cm through a vertical metal guide tube, based on the method described by Khalin et al. (Khalin I et al., (2016), Neural Regen Res 11(4):630-635). The impact point was between the anterior coronal suture (bregma) and the posterior coronal suture (mandibular suture). Immediately after injury, the skin was closed with surgical wound clips, and the mice were returned to their cages to recover from anesthesia. Sham mice underwent the same procedure as described for TBI mice, but without the weight drop.

[0063] Mice were visually observed for signs of spasticity, such as clonus (repeated muscle spasms), prolonged spasms, twitching (rapid involuntary contractions), and hyperreflexia. Mice showed spastic hypertonia from 24 to 48 hours after injury.

[0064] Mice were treated once daily with vehicle or rhNGF, administered by the intranasal route once daily starting on day 1 or 7 post-TBI until day 28. In detail, the animals were divided into the following groups: - False (n=20) - TBI + vehicle: treated once daily with 20 μl of vehicle (TBI / vehicle; n=20) - TBI + rhNGF: Treatment with 50 μg / kg of rhNGF once daily starting on day 1 after TBI (TBI / rhNGF d1; n=20) - TBI + rhNGF: treatment with 50 μg / kg of rhNGF once daily starting on day 7 after TBI (TBI / rhNGF d7; n=20) Vehicle or rhNGF administration was performed as follows: Using the dominant hand, 10 μl of compound or vehicle was loaded into a P20 micropipette. The tip of the filled pipette was positioned near the left nostril of the mouse at a 45° angle. The droplet was positioned close enough to the mouse's nostril so that the mouse could inhale the droplet. Immediately after the mouse inhaled this droplet, the remaining liquid in the pipette tip was expelled, forming another small droplet that the mouse absorbed into the same nostril approximately 2–3 seconds later. The mouse was maintained in this position for 15 seconds after administration.

[0065] Various behavioral tests were performed as described below to assess motor and locomotor impairments at various time points after TBI induction. Data were analyzed using GraphPad Prism version 8.04 (GraphPad Software). Data from behavioral experiments were expressed as mean ± SEM. Two-way ANOVA followed by Tukey's post-hoc test was used to analyze differences between groups, with treatment (drug or vehicle) and test time (days 1, 3, 7, 14, 21, and 28 after TBI) as analysis factors.

[0066] - Pole Exam A 50 cm vertical steel pole was placed inside the cage and covered with adhesive tape to create a rough surface. The animal was placed head-up on top of the pole. The latency to look down and the total latency to descend were measured.

[0067] The results are shown in Figures 4a and 4b. As can be seen, TBI / vehicle mice required more time to turn and less time to descend the pillar compared to sham.

[0068] Intranasal rhNGF (50 μg / kg) administered on both days 1 or 7 after TBI significantly improved pole test performance in a time-dependent manner, with a more pronounced effect on descent latency.

[0069] Unexpectedly, treatment initiated on day 7 significantly normalized the time to descent, resulting in a significant improvement over treatment initiated on day 1. This is the most significant parameter for assessing motor activity impairments associated with spasticity. This is surprising, given the general consensus that early treatment should be more effective in preventing damage and the establishment of spasticity following TBI. These data support the fact that treatment with NGF is more effective when initiated once symptoms of spasticity have fully developed. This time point in mice corresponds to approximately 6-10 months after the traumatic event in human subjects (Dutta et al., Life Sciences 2016(152), pp. 244-248).

[0070] - Open field test The open field test apparatus was a large cubic box measuring 1 m long x 1 m wide x 1 m high. The top of the cube was left uncovered. Animals were placed in the center of the bottom and allowed to move around and explore their environment, with movement recorded over the course of minutes to hours. After the experiment, a computer tracking program analyzed the animal's movement over time. Movement speed and total distance traveled were determined as a measure of the animal's motor impairment.

[0071] TBI / vehicle mice showed a significant decrease in distance traveled (1504.1 ± 451.32 cm, p < 0.0001, 28 days after TBI) and velocity (2.5 ± 0.86 cm / sec, p < 0.0001, 28 days after TBI) compared to sham mice (4995.9 ± 1066.04 cm and 7.63 ± 1.27 cm / sec) (Figure 5A, Figure 5B, and Figure 5C).

[0072] In contrast, TBI / rhNGF d1 mice (distance traveled: 3837.75 ± 1161.4 cm, p < 0.0001; velocity: 4.79 ± 1.27 cm / sec, p < 0.0001, 28 days after TBI) and TBI / rhNGF d7 mice (distance traveled: 4779.05 ± 1479.1 cm, p < 0.0001; velocity: 6.19 ± 0.99 cm / sec, p < 0.0001, 28 days after TBI) showed significantly improved locomotor activity compared to TBI / vehicle mice (Figures 5A, 5B, and 5C).

[0073] Also in this study, treatment beginning on day 7 unexpectedly provided greater improvement than early treatment on day 1, restoring test parameters to values ​​virtually identical to those observed in sham animals.

[0074] - von Frey Mechanical allodynia is measured using a modified version of the up-down method of Chaplan et al. (Chaplan et al., (1994), J. Neurosci. Methods 53, 55-63) using a series of calibrated von Frey nylon filaments (Stoelting, Wood Dale, IL, USA) ranging from 0.002 to 2 g.

[0075] The elevated mesh platform for mechanical allodynia used in the study was a large test board (perforated metal platform) with a Plexiglas box for animal acclimation prior to behavioral analysis.

[0076] Manually applied von Frey filaments for mechanical allodynia are single nylon fibers that provide an approximately logarithmic scale of actual force and a linear scale of perceived intensity. When the tip of a fiber of a given length and diameter is pressed perpendicular to the skin, the applied force increases as the researcher continues to probe until the fiber bends.

[0077] The results reported in Figure 6 show that a significant decrease in the limb withdrawal response (g) was observed in TBI / vehicle mice 7–28 days after trauma induction (0.26 ± 0.28 g, p < 0.0001, 28 days after TBI) compared to the sham group (1.29 ± 0.49 g), and interestingly, mechanical allodynia was significantly reduced in TBI / rhNGF d1 (0.98 ± 0.55 g, p = 0.0001) and TBI / rhNGF d7 (1.35 ± 0.49 g, p < 0.0001) compared to TBI / vehicle animals.

[0078] - Footprint Test The footprinting apparatus typically consisted of a 50-60 cm long track with a dark goal box at the end. After drug administration, the front and hind paws of the mice were painted with various colors (e.g., red for the front paws and green for the hind paws). The mice were then asked to walk on absorbent paper.

[0079] TBI / vehicle mice exhibited abnormal patterns in stride length (3.19 ± 1.15 cm, p = 0.069, 28 days post-TBI) (Figures 7A and 7B), sway length (1.03 ± 0.51 cm, p < 0.0001, 28 days post-TBI) (Figures 7A and 7C), and overlap (0.26 ± 0.18 cm, p = 0.001, 28 days post-TBI) (Figures 7A and 7D). These irregularities in the sequence of steps were the result of spasmodic contractions interfering with the regular progression of the stride.

[0080] TBI / vehicle mice exhibited robust joint movements, frequent trajectory changes during the swing phase, short steps, and frequent crossed forelimb positions. Conversely, treatment of mice initiated on day 7 (TBI / rhNGF d7) significantly normalized gait patterns, improving stride length (5.59 ± 1.58 cm, p = 0.0064, 28 days after TBI), sway length (2.76 ± 0.69 cm, p < 0.0001, 28 days after TBI), and overlap (1.05 ± 0.19 cm, p < 0.0001, 28 days after TBI) (Figures 7A-D). Surprisingly, early treatment with NGF initiated on day 1 after TBI (TBI / rhNGF d) did not significantly improve gait patterns in this study (Figures 7A-D). [Example]

[0081] To evaluate the efficacy of intermittent intranasal administration of NGF on spasticity-related movement disorders induced by a traumatic event and to explore the effects of a longer treatment period, a mouse model of severe TBI identical to the model described in Example 4 was used in further experiments.

[0082] Mice were treated with vehicle or rhNGF (50 μg / Kg) for 61 days, administered by intranasal route starting on day 7 post-TBI, with an intermittent treatment period consisting of 3 consecutive days of treatment alternating with a 7-day washout period until day 61 post-TBI.

[0083] In detail, the animals were divided into the following groups: - False: (False / Veh; n=20) - Sham + NGF: (50 μg / Kg) Started on day 7 after TBI (Sham / rhNGF; n=20) - TBI+medium: (20μl) (TBI / veh;n=20) - TBI + rhNGF: (50 μg / Kg) Started on day 7 after TBI (TBI / rhNGF d7; n=20) Intranasal administration of rhNGF and vehicle was performed as described in Example 4. The same behavioral tests as those described in Example 4 were performed. In all tests, intermittent treatment with rhNGF resulted in improvements in test parameters at all time points up to approximately 40 days. However, after this time point, it was observed that the motor activity impairment associated with spasticity in this animal model spontaneously returned to basal levels, thereby losing significance.

[0084] The results obtained demonstrate that prolonged administration of rhNGF by intermittent administration is effective in treating spasticity. Figure 8 shows representative results obtained in the pole test.

Claims

1. A pharmaceutical composition for intranasal administration for use in the prevention or treatment of spasticity in a subject, comprising nerve growth factor (NGF) or a mutant protein thereof and at least one pharmaceutically acceptable excipient, wherein the mutant protein has the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, and the NGF or mutant protein is administered intranasally to the subject.

2. The pharmaceutical composition for use according to claim 1, wherein the spasticity is caused by a condition selected from cerebral palsy, stroke, traumatic brain injury, amyotrophic lateral sclerosis, primary lateral sclerosis, multiple sclerosis (MS), and hereditary spastic paraplegia (HSP).

3. The pharmaceutical composition for use according to claim 1, wherein the NGF or a mutant protein thereof 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.

4. A pharmaceutical composition for use according to claim 1, comprising, or preferably comprising, NGF or a mutant protein thereof, sodium chloride, phosphate buffer, and water.

5. The pharmaceutical composition for use according to claim 1, wherein the NGF is human NGF, and more preferably recombinant human NGF.

6. The pharmaceutical composition for use according to claim 5, wherein the human NGF has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

7. The pharmaceutical composition for use according to any one of claims 1 to 6, wherein the NGF or mutant protein is administered once to three times a day for a treatment period of 7 to 300 days, preferably 60 to 240 days, more preferably 100 to 200 days.

8. A pharmaceutical composition for use according to any one of claims 1 to 6, wherein the amount of NGF or mutant protein per single administration is 5 μg to 1 mg, more preferably 10 μg to 400 μg, and even more preferably 15 μg to 200 μg.

9. The pharmaceutical composition for use according to any one of claims 1 to 6, wherein the spasticity in the subject is caused by a stroke or traumatic brain injury, and the NGF or mutant protein is administered to the subject starting 6 to 10 months, more preferably 8 to 10 months, more preferably 9 to 10 months, and even more preferably 9 months after the onset of the stroke or traumatic brain injury.

Images