Intranasal administration of bdnf for the treatment of primary headaches

Abstract

The present invention relates to brain-derived neurotrophic factor (BDNF) for use in acute phase treatment or prophylactic treatment of a cluster headache or migraine attack in a subject, wherein the BDNF is administered intranasally to the subject.

Description

Technical Field

[0001] This invention relates to acute treatment or preventive treatment of primary headaches, particularly acute treatment or preventive treatment of migraines and cluster headaches. Background Technology

[0002] According to the third edition of the International Classification of Headache Disorders published in 2018, headaches can be classified into primary headaches, secondary headaches, neuropathic or facial pain, and other headaches.

[0003] Primary headache disorders (including migraine and cluster headache (CH)) are a leading cause of disability worldwide and are caused by independent pathological mechanisms, unlike secondary headaches which occur as a secondary symptom of other diseases or infections. Due to their unknown causes and the intense and persistent pain, primary headache disorders often become refractory to treatment.

[0004] Headache phenotypes differ among these primary headaches. Specifically, pain associated with migraines is typically located in the frontal, temporal, parietal, occipital, and upper neck regions, while pain in cluster headaches is primarily located in the periorbital, intraocular, periocular, or temporal regions.

[0005] Despite these differences, both are believed to be mediated by activation of the trigeminovascular system and are generally considered neurovascular diseases.

[0006] The abundant nociceptive nerve fiber plexus (unmyelinated C fibers and thin-myelinated Aδ fibers) originating from the trigeminal ganglion (TG) innervates intracranial vascular structures, including piacular vessels, arachnoid vessels, dural vessels, and the great arteries of the brain.

[0007] The axonal terminals of these aggregated nociceptive nerve fibers contain vasoactive neuropeptides, including calcitonin gene-related peptide (CGRP), substance P (SP), neurokinin A (NKA), vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase-activating peptide-38 (PACAP38). These peptides can be released upon stimulation of the trigeminal nerve, causing vasodilation of the dura mater and pia mater vessels, as well as potential cascade events leading to activation and sensitization of the trigeminal vascular system.

[0008] High concentrations of VIP, PACAP38, and CGRP in the serum of patients affected by migraine or CH have been reported (see, for example, Kubulas, A. et al., J Headache Pain 2023, v. 24, p. 34; Tanaka, M. et al., Cells2023, 12, 2649; Pellesi L et al., Front. Neurol. 2022, 13: 871176; Edvinsson, L. et al., J Headache Pain 2018, 19, 21; Riesco N. et al., Curr Pain Headache Rep. 2017, 21(4):18; Pellesi L et al., Cephalalgia. 2022, 42(8): 687-695). Furthermore, an increase in pro-inflammatory cytokines (e.g., IL1β) and a decrease in anti-inflammatory cytokines (e.g., IL-10) were observed in the serum of patients affected by migraine or chronic headache (see, for example, Fidan I. et al., J Neuroimmunology 2006, 171:184-188; Geng C. et al., J Clin Neurosci 2022, 98:213-218; Oliveira AB, et al., J Neuroimmunology 2017, 313:138-144; Chaudhry SR et al., Brain Stimul. 2019, 12(3): 643-651; Thuraiaiyah J et al., Cephallalgia. 2022, 42(14):1565-1588; Guo Z, et al., Neurobiol Pain 2022, 13; 12:100096; Uzar E, et al., Eur Rev Med Pharmacol Sci. 2011, 15(10): 1111-6).

[0009] Currently, the main strategies for treating both hepatitis C (CH) and migraine can be divided into two categories: acute-phase treatment aimed at interrupting a single attack, and preventative treatment aimed at reducing the frequency, duration, and intensity of attacks. Most patients receive both acute-phase treatment and preventative treatment simultaneously.

[0010] Regarding acute drug treatment, the most commonly used medication during CH and migraine attacks is subcutaneous sumatriptan, a serotonin receptor agonist for 5-HT1B and 5-HT1D, which provides rapid pain relief within 20 minutes of injection in most subjects. This drug has been shown to be safe in healthy patients, but is contraindicated in cases of cardiovascular, metabolic, and cerebrovascular disease (Hoskin KL, Goadsby PJ, Exp Neurol 1998, 150:45-516; Humphrey PP et al., Cephalalgia 1994, 14:401-410; Nature Reviews, 2018, doi: 10.1038 / nrdp.2018.6).

[0011] Available preventative treatments include the administration of verapamil, corticosteroids, or lithium; however, these treatments have limited efficacy or have shown side effects with long-term use (Nature Reviews, 2018, doi: 10.1038 / nrdp.2018.6).

[0012] Therefore, there remains a great need for new and more effective treatments for cluster headaches and migraines to overcome the limitations of current known treatment strategies.

[0013] Brain-derived neurotrophic factor (BDNF) is one of the most important growth factors in the central nervous system (CNS) (Barde YA et al. Brain derived neurotrophic factor. Prog BrainRes 1987; 71: 185-189.), and it plays a crucial role in CNS development and neuronal plasticity. BDNF is involved in a variety of brain diseases (Autry AE et al., Brain derived neurotrophic factor and neuropsychiatric disorders. Pharmacol Rev 2012; 64: 238-258), including schizophrenia, intellectual disability, and autism, as well as depression and other mood disorders (Bjorkholm C, Monteggia LM. BDNF - a key transducer of antidepressant effects. Neuropharmacology 2016; 102: 72-79.). It is also a crucial neuromodulator in pain transmission.

[0014] In a two-center prospective study, patients with migraine and CH showed significantly higher serum BDNF levels during migraine attacks compared with patients without headache, patients with tension headache, and healthy controls (Fischer, M., Wille, G., Klien, S. et al. Brain-derived neurotrophic factor in primary headaches. J Headache Pain 13, 469-475 (2012)). Summary of the Invention

[0015] The inventors have now unexpectedly discovered that intranasal BDNF is effective in reducing trigeminal hyperalgesia in a primary headache model associated with trigeminal neurovascular system activation based on nitroglycerin (NTG) administration, which represents cluster headache (CH) and migraine.

[0016] In particular, in an NTG-induced rat model, intranasal administration of BDNF inhibited the enhancement of facial rubbing behavior following formalin stimulation, which mimics the typical hyperalgesia of subjects with cluster headaches or migraines, and it was also effective in reducing the levels of vasoactive neuropeptides and inflammatory mediators involved in the pathophysiology of migraines and cluster headaches.

[0017] Specifically, it induced significant reductions in VIP, PACAP38, and CGRP, a significant decrease in IL-1β, and a significant increase in IL-10.

[0018] The above results support the role of BDNF in both the acute treatment and preventative treatment of CH or migraine attacks.

[0019] Therefore, the first object of the present invention is BDNF for acute treatment or preventive treatment of migraine or cluster headache attacks in a subject, wherein the BDNF is administered intranasally to the subject.

[0020] Another object of the present invention is a pharmaceutical composition comprising BDNF for the acute treatment or preventive treatment of migraine or cluster headache attacks in a subject, the composition being administered intranasally to the subject. Attached Figure Description

[0021] Figure 1 A schematic diagram of the experimental design for Example 1 is shown.

[0022] Figure 2The time (in seconds) spent on friction is shown for the “CT”, “NTG”, “NTG+BDNF1”, “BDNF1”, and “NTG+SUMA” rat groups (subplots A and D), the “CT”, “NTG”, “NTG+BDNF2”, “BDNF2”, and “NTG+SUMA” rat groups (subplots B and E), and the “NTG”, “NTG+BDNF1”, “NTG+BDNF2”, and “NTG+SUMA” rat groups (subplots C and F), as described in Example 1. Data were tested for normality using the Shapiro-Wilk test and were considered normally distributed. One-way ANOVA was followed by a Tukey multiple comparison test. .

[0023] Figure 3 Serum concentrations (pg / ml) of CGRP (subplot A), PACAP38 (subplot D), and VIP (subplot G) are shown in the “CT,” “NTG,” “NTG+BDNF1,” “BDNF1,” and “NTG+SUMA” rat groups; serum concentrations (pg / ml) of CGRP (subplot B), PACAP38 (subplot E), and VIP (subplot H) are shown in the “CT,” “NTG,” “NTG+BDNF2,” “BDNF2,” and “NTG+SUMA” rat groups; and serum concentrations (pg / ml) of CGRP (subplot C), PACAP38 (subplot F), and VIP (subplot I) are shown in the “NTG,” “NTG+BDNF1,” “NTG+BDNF2,” and “NTG+SUMA” rat groups. The Kruskal-Wallis test was followed by the Dunn post-hoc test. .

[0024] Figure 4The following data are presented: mRNA expression analysis of CGRP (sub-figure A), VIP (sub-figure D), PACAP38 (sub-figure G), IL-1β (sub-figure J), ​​and IL-10 (sub-figure M) in the trigeminal ganglion of the “CT”, “NTG”, “NTG+BDNF1”, “BDNF1”, and “NTG+SUMA” rat groups; mRNA expression analysis of CGRP (sub-figure B), VIP (sub-figure E), PACAP38 (sub-figure H), IL-1β (sub-figure K), and IL-10 (sub-figure N) in the trigeminal ganglion of the “CT”, “NTG”, “NTG+BDNF2”, “BDNF2”, and “NTG+SUMA” rat groups; and mRNA expression analysis of CGRP (sub-figure C), VIP (sub-figure F), PACAP38 (sub-figure I), IL-1β (sub-figure L), and IL-10 (sub-figure O) in the trigeminal ganglion of the “NTG”, “NTG+BDNF1”, “NTG+BDNF2”, and “NTG+SUMA” rat groups. The data were found to be normally distributed after the Shapiro-Wilk test, so a one-way ANOVA was applied followed by a post-hoc Tukey multiple comparison test. .

[0025] Figure 5 The following data are presented: mRNA expression analysis of CGRP (sub-figure A), VIP (sub-figure D), PACAP38 (sub-figure G), IL-1β (sub-figure J), ​​and IL-10 (sub-figure M) in the medulla oblongata-pons region of the “CT”, “NTG”, “NTG+BDNF1”, “BDNF1”, and “NTG+SUMA” rat groups; mRNA expression analysis of CGRP (sub-figure B), VIP (sub-figure E), PACAP38 (sub-figure H), IL-1β (sub-figure K), and IL-10 (sub-figure N) in the medulla oblongata-pons region of the “CT”, “NTG”, “NTG+BDNF2”, “BDNF2”, and “NTG+SUMA” rat groups; and mRNA expression analysis of CGRP (sub-figure C), VIP (sub-figure F), PACAP38 (sub-figure I), IL-1β (sub-figure L), and IL-10 (sub-figure O) in the medulla oblongata-pons region of the “NTG”, “NTG+BDNF1”, “NTG+BDNF2”, and “NTG+SUMA” rat groups. The data in A, B, C, E, G, H, I, J, K, and L are normally distributed after the Shapiro-Wilk test; therefore, a one-way ANOVA is applied followed by a post-hoc Tukey multiple comparison test. The data in D, F, M, N, and O are not normally distributed after the Shapiro-Wilk test; therefore, the Kruskal-Wallis test is applied followed by a Dunn post-hoc test. .

[0026] Figure 6 The following data are presented: mRNA expression analysis of CGRP (sub-figure A), VIP (sub-figure D), PACAP38 (sub-figure G), IL-1β (sub-figure J), ​​and IL-10 (sub-figure M) in the hypothalamus of the “CT”, “NTG”, “NTG+BDNF1”, “BDNF1”, and “NTG+SUMA” rat groups; mRNA expression analysis of CGRP (sub-figure B), VIP (sub-figure E), PACAP38 (sub-figure H), IL-1β (sub-figure K), and IL-10 (sub-figure N) in the hypothalamus of the “CT”, “NTG”, “NTG+BDNF2”, “BDNF2”, and “NTG+SUMA” rat groups; and mRNA expression analysis of CGRP (sub-figure C), VIP (sub-figure F), PACAP38 (sub-figure I), IL-1β (sub-figure L), and IL-10 (sub-figure O) in the hypothalamus of the “NTG”, “NTG+BDNF1”, “NTG+BDNF2”, and “NTG+SUMA” rat groups. The data in D, E, F, G, H, I, J, K, L, and M are normally distributed after the Shapiro-Wilk test; therefore, a one-way ANOVA is applied followed by a post-hoc Tukey multiple comparison test. The data in A, B, C, N, and O are not normally distributed after the Shapiro-Wilk test; therefore, the Kruskal-Wallis test is applied followed by a Dunn post-hoc test. . Detailed Implementation

[0027] The first objective of this invention is brain-derived neurotrophic factor (BDNF) for acute or preventative treatment of cluster headaches or migraine attacks in subjects, wherein the BDNF is administered intranasally to the subjects.

[0028] The term "preventive treatment" as used in this article refers to treatments administered at regular intervals to reduce the frequency, duration, and intensity of CH or migraine attacks, thereby minimizing the need for acute treatment.

[0029] The term "acute phase treatment" as used in this article refers to treatment that interrupts or stops a single attack of migraine or CH at regular intervals.

[0030] Preferably, the object is a human object.

[0031] Preferably, the BDNF is human BDNF, and more preferably, it is recombinant human BDNF (rhBDNF).

[0032] Preferably, the total amount of BDNF administered each time is 50 μg to 1 mg, more preferably 200 μg to 800 μg, and even more preferably 250 μg to 650 μg, distributed in equal doses to each nostril.

[0033] Preferably, the BDNF used according to the present invention is applied once to three times a day for a treatment period of at least 7 days, preferably at least 60 days, and more preferably at least 90 days.

[0034] The administration regimen may vary depending on the patient and is selected by a person skilled in the art based on the characteristics of the patient, the severity of the disease, and the tests performed during treatment.

[0035] The evaluation of the above factors is within the knowledge and expertise of those skilled in the art.

[0036] Another object of the present invention relates to a pharmaceutical composition comprising BDNF for the acute treatment or preventive treatment of cluster headache or migraine attacks in a subject, wherein the composition is administered intranasally to the subject.

[0037] Preferably, the pharmaceutical composition according to the invention comprises an effective amount of BDNF and at least one pharmaceutically acceptable excipient, said excipient preferably selected from solvents, thickeners, mucosal adhesives, buffers, antioxidants, preservatives and penetration enhancers.

[0038] Preferably, the pharmaceutical composition according to the invention is a liquid intranasal pharmaceutical composition.

[0039] Preferably, the BDNF is human BDNF, and more preferably, it is recombinant human BDNF (rhBDNF).

[0040] Preferably, the concentration of BDNF in the pharmaceutical composition according to the invention is from 300 μg / ml to 1.5 mg / ml, more preferably from 500 μg / ml to 1.3 mg / ml.

[0041] Preferably, the solvent is water.

[0042] Preferably, the buffer is a phosphate buffer.

[0043] A particularly preferred liquid intranasal pharmaceutical composition according to the invention comprises BDNF, a phosphate buffer, and water, preferably consisting of BDNF, a phosphate buffer, and water.

[0044] The pharmaceutical compositions according to the invention can be suitably formulated using appropriate methods known in the art or by methods disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton, Pa.

[0045] In another aspect, the present invention relates to a method for acute treatment or preventive treatment of cluster headache or migraine attacks in a subject, the method comprising administering a therapeutically effective amount of BDNF as described above into the subject's nose.

[0046] Preferably, the BDNF used in the method of the present invention is in the form of a pharmaceutical composition as defined above.

[0047] The present invention will be further described in the following embodiments, which do not limit the scope of the invention as defined in the claims.

[0048] Example

[0049] The inventors evaluated the effect of intranasal administration of rhBDNF in an experimental animal model of primary headache associated with activation of the trigeminal neurovascular system based on nitroglycerin (NTG) administration, the model representing cluster headache and migraine.

[0050] Nitroglycerin administration is a well-known animal and human model of migraine because it induces a spontaneous-like migraine attack in patients with migraine (Demartini C. et al., Nitroglycerin as a comparative experimental model of migraine pain: From animal to human and back. Prog Neurobiol. 2019 Jun;177:15-32).

[0051] NTG is also known to induce CH-like attacks during the active phase of CH in patients (Sances G. et al., Reliability of the nitroglycerin provocative test in the diagnosis of neurovascular headaches. Cephalalgia. 2004 Feb;24(2):110-9).

[0052] These observations support the validity of NTG-induced attacks in migraine and CH subjects as a relevant model for studying the neurobiological mechanisms involved in these primary headaches.

[0053] Example 1

[0054] Male Sprague-Dawley rats (Charles River Laboratories, Italy srl, Calco, Lecco) weighing 200–270 g were housed in cages of two for one week prior to the start of the experiment under controlled light (12-hour light / dark cycle; light on at 6:00 AM) and standard environmental conditions (temperature 20–22°C, humidity 55%–60%). Rats had free access to feed and tap water. Animal care was conducted in accordance with Italian (DL 116 / 92) and European Commission (OJ of EC L358 / 1 18 / 12 / 86) regulations on the protection of laboratory animals. Every effort was made to minimize both the number of animals and their suffering during the experiment.

[0055] according to Figure 1 The experimental design shown in the figure randomly assigned animals to the following treatment groups, with each group containing 9 animals:

[0056] 1) NTG Treatment with NTG 5 mg / kg, ip (5 ip injections in total, delivered every other day) and BDNF carrier (4 intranasal injections in total, delivered on the day when NTG injection is not performed);

[0057] 2) CT Treatment with NTG carrier (5 total intranasal injections, delivered every other day) and BDNF carrier (4 total intranasal injections, delivered on the day NTG injections are not performed);

[0058] 3) NTG+BDNF 1 Treatment with NTG 5 mg / kg, ip (5 ip injections in total, delivered every other day) and high dose rhBDNF (80 μg / kg) (4 intranasal injections in total, delivered on the day NTG injection was not performed);

[0059] 4) NTG+BDNF 2 Treatment included NTG 5 mg / kg, ip (5 ip injections in total, delivered every other day) and low-dose rhBDNF (40 μg / kg) (4 intranasal injections in total, delivered on the day NTG injection was not performed);

[0060] 5) BDNF 1Treatment with NTG carrier (5 total intranasal injections, delivered every other day) and high dose rhBDNF (80 μg / kg) (4 total intranasal injections, delivered on the same day if NTG carrier injection was not performed);

[0061] 6) BDNF 2 Treatment with NTG carrier (5 total intranasal injections, delivered every other day) and low-dose rhBDNF (40 μg / kg) (4 total intranasal injections, delivered on the same day if NTG carrier injection was not performed);

[0062] 7) NTG+SUMA Treatment with NTG 5 mg / kg, ip (5 ip injections in total, delivered every other day) and sumatriptan (5 mg / kg, ip; Imigran, 6 mg / 0.5 ml, delivered as a single injection 1 hour after the last NTG injection).

[0063] Specifically, the animals in the above experimental groups were intraperitoneally (ip) injected every other day for 9 days with 5 mg / kg nitroglycerin (NTG) (Bioindustria LIM, Novi Ligure, Italy) (groups 1, 3, 4, and 7) or its carrier (NTG carrier) (groups 2, 5, and 6), for a total of 5 injections. The final volume of NTG or its carrier injected into the animals was 1.5 mL / kg.

[0064] The NTG solution was prepared from a stock solution of 5.0 mg / 1.5 mL nitroglycerin dissolved in 27% ethanol and 73% propylene glycol; the resulting NTG solution was diluted in saline (0.9% NaCl) to achieve a final concentration of approximately 6% ethanol and 16% propylene glycol. The NTG carrier solution was an aqueous solution of 0.9% NaCl containing approximately 6% ethanol and 16% propylene glycol.

[0065] On days when NTG injection was not performed, rhBDNF (stock solution 2.43 mg / ml) or its carrier (BDNF carrier, 50 mM sodium dihydrogen phosphate dihydrate, 100 mM sodium chloride, pH 7.0) was administered intranasally at a final dose of 40 μg / kg (groups 4 and 6; volume range 3.54 to 4.85 μl, depending on rat weight, unilateral nostril) or 80 μg / kg (groups 3 and 5; volume range 6.72 to 9.6 μl, depending on rat weight, distributed into both nostrils), for a total of 4 administrations. Figure 1After mildly anesthetizing each rat for 4 to 5 minutes with 4% isoflurane in 5% oxygen (flow rate 1.5 L / min), the rats were placed in a supine position; rhBDNF dissolved in phosphate buffer (25 mM sodium dihydrogen phosphate dihydrate, 100 mM sodium chloride, pH 7.0) was delivered intranasally as described below.

[0066] Using your dominant hand, load the predetermined amount of compound into the micropipette P10. Place the filled pipette tip at a 45-degree angle near the rat's left nostril. Position the droplet close enough to the nostril for the rat to inhale. After 20 seconds of inhalation through the left nostril, repeat the same procedure to form another small droplet for the rat to inhale the compound through the other nostril.

[0067] Rats in the carrier group received equal amounts of 6.72 to 9.6 μl of BDNF carrier (25 mM sodium dihydrogen phosphate dihydrate, 100 mM sodium chloride, pH 7.0) (dispensed into both nostrils) according to the same procedure based on rat weight.

[0068] In group 7, the effect of sumatriptan (SUMA) was evaluated as a positive control for the putative effect of rhBDNF.

[0069] On day 9, two hours after the last administration of NTG or the carrier, the animals underwent an orofacial formalin test as described below, and were subsequently euthanized with a lethal dose of an anesthetic (thiopental sodium, 150 mg / kg, ip), followed by decapitation, to evaluate the gene expression (especially mRNA expression) of neuropeptides (CGRP, PACAP38, VIP) and cytokines (IL-1β, IL-10) in the medulla oblongata-pons region, hypothalamus region, and trigeminal ganglion (by RT-PCR), as well as the concentration of said neuropeptides in serum (by ELISA).

[0070] Statistical analysis

[0071] On the basis of previous studies (Greco R., et al., Effects of kynurenic acid analogue 1(KYNA-A1) in nitroglycerin-induced hyperalgesia: Targets and anti-migrainemechanisms. Cephalalgia. 2017 Nov;37(13):1272-1284; Greco R., et al., Characterization of the peripheral FAAH inhibitor, URB937, in animal Models of acute and chronic migraine. Neurobiol Dis. 2021 Jan;147:105157; Greco R. et al., FAAH inhibition as a treatment for migraine: A pre-clinical study. Neurobiol Dis. 2020 Feb;134:104624.; MA., et al., The effects of levetiracetam, sumatriptan, and caffeine in a rat model of trigeminal pain: interactions in 2-component combinations. Anesth Analg. 2015 Jun;120(6):1385-93.) Prior power analysis was performed to calculate the minimum sample size (GPower 3.1) required to achieve statistical power of 0.90 at an α level of 0.05, thus estimating 6 to 9 rats per experimental group. Normality of the data was tested using the Kolmogorov-Smirnov test or the Shapiro-Wilk test. Parametric tests (one-way ANOVA followed by a post-hoc Tukey multiple comparison test) or nonparametric tests (Kruskal-Wallis test followed by a post-hoc Dunn test for multiple comparisons) were applied depending on the data distribution. Probability levels less than 5% were considered significant. Statistical significance was set at p < 0.05. All statistical analyses were performed using GraphPad Prism software (version 8). Data are expressed as mean ± standard deviation (SD).

[0072] - Oral and facial formalin test

[0073] Each rat was moved to the oral-facial formalin test chamber and, 5 minutes later, received a subcutaneous injection of formalin (1.5%, 50 μL) (a 37% formaldehyde aqueous solution) into the right upper lip. Immediately after the formalin injection, each rat was placed in an observation chamber (a 30×30×30 cm glass chamber with mirrored sides), and its nocifensive behavior was recorded for 45 minutes using a camera positioned 50 cm from the chamber (Greco R. et al., Characterization of the peripheral FAAH inhibitor, URB937, in animal models of acute and chronic migraine. Neurobiol Dis. 2021 Jan;147:105157).

[0074] Facial friction was measured (offline) by counting the number of seconds an animal spent grooming the injection area with its ipsilateral forepaw or hind paw during 0 to 3 minutes (Phase I, reflecting acute pain) or 12 to 45 minutes (Phase II, reflecting hyperalgesic pain) after formalin injection. The observation time was divided into 15 blocks of 3 minutes each.

[0075] Surprisingly, Figure 2 The results shown in A through F demonstrate that even the lowest tested dose of rhBDNF (NTG+BDNF2) was significantly effective in reducing NTG-induced pain sensitivity (hyperalgesia) (NTG); unexpectedly, the NTG+BDNF1 and NTG+BDNF2 rat groups showed a substantial reduction in NTG-induced pain sensitivity, comparable to rats treated with sumatriptan (NTG+SUMA).

[0076] These findings are significant because intranasally administered BDNF unexpectedly exerted a similar effect to sumatriptan, a synthetic drug with adverse side effects that have prevented its long-term use for acute or preventative treatment of migraine and CH attacks.

[0077] - Serum concentrations of VIP, PACAP38 and CGRP biomarkers

[0078] To assess whether rhBDNF also upregulates the expression of vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide-38 (PACAP38), and calcitonin gene-related peptide (CGRP) biomarkers, and has an effect on increasing pro-inflammatory cytokines (e.g., IL1β) and decreasing anti-inflammatory cytokines (e.g., IL-10) (these phenomena have been widely reported in the serum of patients affected by migraine or CH), the rats were euthanized by decapitation as described above at the end of the orofacial formalin test, and blood samples were immediately collected into serum tubes containing coagulation activator and separating gel, and centrifuged at 1500 × g for 15 min at room temperature (RT). Serum neuropeptide levels were measured using commercial ELISA kits (α-CGRP: Elabsciences, Houston, TX, USA; PACAP38 and VIP: Neo Biothech, Nanterre, France).

[0079] Figure 3 The results shown in A through I indicate that both tested concentrations of intranasally administered rhBDNF (NTG+BDNF1, NTG+BDNF2) unexpectedly suppressed serum levels of VIP, PACAP38, and CGRP, which are significantly involved in the activation of the trigeminal nervous system in patients with migraine and cluster headache, to baseline, thereby significantly reducing serum levels of these biomarkers to those in sumatriptan-treated rats (NTG+SUMA).

[0080] These serum data suggest that intranasal rhBDNF can modulate the imbalance between vasoactive neuropeptides and nociceptive neuropeptides associated with neurogenic inflammation in the pathogenesis of migraine and CH.

[0081] - Genes of VIP, PACAP38, and CGRP biomarkers in the medulla oblongata-pons region, hypothalamus, and trigeminal ganglion Express.

[0082] Since the specific brain regions involved in the onset of headache are the medulla oblongata-pons region, hypothalamus, and trigeminal ganglion, when animals were euthanized as described above, these regions on the same side as the formalin injection site were dissected, rinsed in sterile 0.9% NaCl cold solution, placed in cryovials, and stored at -80°C until rt-PCR processing was performed to target the expression (i.e., mRNA expression) of the genes CGRP, PACAP38, VIP, interleukin-1β (IL-1β), and interleukin-10 (IL-10).

[0083] Following total RNA extraction (performed using the Aurum Fatty / Fibrous Tissue RNA Kit, 7326830, BioRad), the absorbance ratio (260 / 280 nm) of all RNA samples varied between 1.9 and 2.0, indicating no significant protein contamination, including protein contamination from blood sources.

[0084] For quantitative real-time reverse transcriptase PCR (qPCR), a 20 μl reaction mixture (SsoFast EvaGreenSupermix, Bio-Rad) was used, which contained 1.2 μl primers (0.6 μl forward primer and 0.6 μl reverse primer), 1 μl cDNA (obtained via iScript cDNA Synthesis Kit, 1708891, Bio-Rad), and 10 μl Syber Green Master and nuclease-free water (variable) mixture, according to Table 1.

[0085] Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), whose expression remained constant across all experimental groups, was used for normalization. All samples were measured in duplicate and... The method was used to study differences in gene expression levels (Greco et al., 2022). The primers described above have the sequences shown in Table 1 below (Merck), and are input from Primer3 ( https: / / primer3.ut.ee / )get.

[0086]

[0087]

[0088] Table 1: List of primers used for PCR and their SEQ ID No.

[0089] Each PCR reaction was performed in triplicate using the following parameters: polymerase activation, 95°C for 30 seconds (cycle 1); denaturation, 95°C for 15 seconds (cycle 40); annealing, 60°C for 30 seconds (cycle 40); extension, 72°C for 20 seconds (cycle 40), followed by melting curve analysis. LightCycler® 480 Software release 1.5.1.62SP3).

[0090] Surprisingly, trigeminal ganglia were observed in animals treated with rhBDNF (NTG+BDNF1, NTG+BDNF2). Figure 4 A to O), medulla oblongata-pons region ( Figure 5 A to O) and the hypothalamus region ( Figure 6Significantly decreased expression of VIP, PACAP38, CGRP, and IL-1β genes in A to O, accompanied by increased expression of IL10 gene.

[0091] It is worth noting that in animals treated with rhBDNF (NTG+BDNF1 and NTG+BDNF2), Figures 4 to 6 The brain transcriptional changes of VIP, PACAP38, and CGRP shown in the figure are related to Figure 3 The decrease in serum levels of PACAP38, VIP, and CGRP shown in the figure suggests that rhBDNF may be involved in the expression of mediators of migraine and CH.

[0092] In summary, these data demonstrate that intranasal administration of the rhBDNF is a feasible and multi-functional treatment method for the acute or preventative treatment of migraine and cluster headache patients via a safe, non-invasive delivery route.

Claims

1. Brain-derived neurotrophic factor (BDNF) for acute or preventative treatment of cluster headache or migraine attacks in subjects, wherein the BDNF is administered intranasally to the subjects.

2. The BDNF of claim 1, wherein the BDNF is a human BDNF, preferably a recombinant human BDNF (rhBDNF).

3. The BDNF used according to claim 1 or 2, wherein the BDNF is applied once or three times daily for a treatment period of at least 7 days, preferably at least 60 days, and more preferably at least 90 days.

4. The BDNF applied according to claims 1 to 3, wherein the total amount of BDNF applied each time is 50 μg to 1 mg, preferably 200 μg to 800 μg, more preferably 250 μg to 650 μg, distributed in equal doses to each nostril.

5. A pharmaceutical composition comprising BDNF and at least one pharmaceutically acceptable excipient for the acute treatment or preventative treatment of cluster headache or migraine attacks in a subject, wherein the composition is administered intranasally to the subject.

6. The pharmaceutical composition according to claim 5, wherein the BDNF is human BDNF, preferably recombinant human BDNF (rhBDNF).

7. The pharmaceutical composition according to claim 5 or 6, wherein the BDNF is contained in the composition at a concentration of 300 μg / ml to 1.5 mg / ml, preferably at a concentration of 500 μg / ml to 1.3 mg / ml.

8. The pharmaceutical composition according to claims 5 to 7, wherein the pharmaceutical composition is a liquid intranasal pharmaceutical composition.

9. The pharmaceutical composition according to claims 5 to 8, comprising BDNF, a phosphate buffer, and water, preferably consisting of BDNF, a phosphate buffer, and water.

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