Eletriptan hydrobromide for the treatment of spinal cord injury and improvement of locomotor function

Abstract

The present invention discloses the use of eletriptan hydrobromide in the treatment of spinal cord injury and the improvement of locomotor function. Despite significant efforts in attempts to improve limited recovery following injury, only a multi-target combinatorial therapeutic approach will be effective given the complex nature of spinal cord injury. Utilizing a larval zebrafish drug discovery platform, eletriptan Hbr has been demonstrated herein to have locomotor restorative properties in a pro-regenerative model and confirmed to have a preservative effect in a rodent (pro-fibrotic) contusion model. This approach allows for the identification of promising new therapeutic applications for eletriptan Hbr, which, coupled with combinatorial therapeutic strategies and innovative engineering approaches, demonstrates great potential for spinal cord injury recovery.

Description

Technical Field

[0001] The present invention relates to the use of eletriptan hydrobromide in the treatment of spinal cord injury and the improvement of locomotive function.

Background Art

[0002] Spinal cord injury (SCI) leading to motor, sensory, and autonomic dysfunction causes neurological trauma that affects millions of people worldwide, and there is an urgent need to develop effective spinal cord repair strategies for clinical use (Kjell, J. & Olson, L. Rat models of spinal cord injury: from pathology to potential therapies. Dis Model Mech 9, 1125-1137, doi:10.1242 / dmm.025833 (2016)). The pathophysiology of SCI can be divided into primary and secondary mechanisms of injury. The primary injury occurs due to mechanical disruption of spinal cord tissue that induces axonal, vascular, and cell membrane alterations, leading to immediate neural tissue loss and demyelination (Boutonnet, M., Laemmel, E., Vicaut, E., Duranteau, J. & Soubeyrand, M. Combinatorial therapy with two pro-coagulants and one osmotic agent reduces the extent of the lesion in the acute phase of spinal cord injury in the rat. Intensive Care Med Exp 5, 51, doi:10.1186 / s40635-017-0164-z (2017), Donovan, J. & Kirshblum, S. Clinical Trials in Traumatic Spinal Cord Injury. Neurotherapeutics 15, 654-668, doi:10.1007 / s13311-018-0632-5 (2018), Duncan, G. J. et al. Locomotor recovery following contusive spinal cord injury does not require oligodendrocyte remyelination. Nat Commun 9, 3066, doi:10.1038 / s41467-018-05473-1 (2018)).After the initial trauma, secondary injury occurs due to disruption of the blood spinal cord barrier (BSCB), which leads to several mechanisms, namely, infiltration of inflammatory cells, release of inflammatory cytokines, and imbalanced release of excitatory neurotransmitters leading to excitotoxicity and ischemia (Boutonnet, M., Laemmel, E., Vicaut, E., Duranteau, J. & Soubeyrand, M. Combinatorial therapy with two pro-coagulants and one osmotic agent reduces the extent of the lesion in the acute phase of spinal cord injury in the rat. Intensive Care Med Exp 5, 51, doi:10.1186 / s40635-017-0164-z (2017), Donovan, J. & Kirshblum, S. Clinical Trials in Traumatic Spinal Cord Injury. Neurotherapeutics 15, 654-668, doi:10.1007 / s13311-018-0632-5 (2018), Zhou, X., He, X. & Ren, Y. Function of microglia and macrophages in secondary damage after spinal cord injury. Neural Regen Res 9, 1787-1795, doi:10.4103 / 1673-5374.143423 (2014), Yilmaz, T. & Kaptanoglu, E. Current and future medical therapeutic strategies for the functional repair of spinal cord injury. World J Orthop 6, 42-55, doi:10.5312 / wjo.v6.i1.42 (2015)).Therefore, secondary injury is multifactorial and characterized by the presence of reactive gliosis, edema, glial / axonal scarring, and inflammation that can cause central cavitation (Zhou, X., He, X. & Ren, Y. Function of microglia and macrophages in secondary damage after spinal cord injury. Neural Regen Res 9, 1787-1795, doi:10.4103 / 1673-5374.143423 (2014), Yilmaz, T. & Kaptanoglu, E. Current and future medical therapeutic strategies for the functional repair of spinal cord injury. World J Orthop 6, 42-55, doi:10.5312 / wjo.v6.i1.42 (2015), Picoli, C. C. et al. Pericytes Act as Key Players in Spinal Cord Injury. Am J Pathol 189, 1327-1337, doi:10.1016 / j.ajpath.2019.03.008 (2019)).

[0003] Mature SCI lesions present three main tissue compartments: a lesion core / fibrotic scar with non-neural tissue, an astrocytic scar surrounding the lesion core, and a surrounding region with residual neural tissue that is functional but reactive (O'Shea, T. M., Burda, J. E. & Sofroniew, M. V. Cell biology of spinal cord injury and repair. J Clin Invest 127, 3259-3270, doi:10.1172 / JCI90608 (2017)). Indeed, the SCI scar leads to a repair response (essential for preventing the propagation of cell damage) and a harmful response (limiting regrowth and tissue repair) that changes over time and is defined by the spatial location relative to the lesion (Gaudet, A. D. & Fonken, L. K. Glial Cells Shape Pathology and Repair After Spinal Cord Injury. Neurotherapeutics 15, 554-577, doi:10.1007 / s13311-018-0630-7 (2018), Hausmann, O. N. Post-traumatic inflammation following spinal cord injury. Spinal Cord 41, 369-378, doi:10.1038 / sj.sc.3101483 (2003), Bradbury, E. J. & Burnside, E. R. Moving beyond the glial scar for spinal cord repair. Nat Commun 10, 3879, doi:10.1038 / s41467-019-11707-7 (2019), Orr, M. B. & Gensel, J. C. Spinal Cord Injury Scarring and Inflammation: Therapies Targeting Glial and Inflammatory Responses. Neurotherapeutics 15, 541-553, doi:10.1007 / s13311-018-0631-6 (2018)).In fact, reactive astrocytes not only form glial scars that limit lesion expansion after injury, but also limit and inhibit axonal regeneration while confining inflammation to the lesion center (Gaudet, A. D. & Fonken, L. K. Glial Cells Shape Pathology and Repair After Spinal Cord Injury. Neurotherapeutics 15, 554-577, doi:10.1007 / s13311-018-0630-7 (2018), Cregg, J. M. et al. Functional regeneration beyond the glial scar. Exp Neurol 253, 197-207, doi:10.1016 / j.expneurol.2013.12.024 (2014)). Furthermore, microglia / macrophages not only enable phenotypic plasticity, but also produce cytotoxic factors that prolong and exacerbate the pro-inflammatory response that worsens lesion damage (i.e., secondary injury) (Gaudet, A. D. & Fonken, L. K. Glial Cells Shape Pathology and Repair After Spinal Cord Injury. Neurotherapeutics 15, 554-577, doi:10.1007 / s13311-018-0630-7 (2018), Zhang, B. et al. Reducing age-dependent monocyte-derived macrophage activation contributes to the therapeutic efficacy of NADPH oxidase inhibition in spinal cord injury. Brain Behav Immun 76, 139-150, doi:10.1016 / j.bbi.2018.11.013 (2019)).In addition, oligodendrocytes and oligodendrocyte precursor cells (OPCs) can not only die by apoptosis or necrosis, but also achieve differentiation and remyelination (Gaudet, A. D. & Fonken, L. K. Glial Cells Shape Pathology and Repair After Spinal Cord Injury. Neurotherapeutics 15, 554-577, doi:10.1007 / s13311-018-0630-7 (2018), Keirstead, H. S. et al. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci 25, 4694-4705, doi:10.1523 / JNEUROSCI.0311-05.2005 (2005), McTigue, D. M. & Tripathi, R. B. The life, death, and replacement of oligodendrocytes in the adult CNS. J Neurochem 107, 1-19, doi:10.1111 / j.1471-4159.2008.05570.x (2008)).Therefore, considering the complex interactions among multiple different cell types and the intracellular and extracellular microenvironments, eliminating even one of these cell types or responses completely is not effective for SCI repair (Gaudet, A. D. & Fonken, L. K. Glial Cells Shape Pathology and Repair After Spinal Cord Injury. Neurotherapeutics 15, 554-577, doi:10.1007 / s13311-018-0630-7 (2018), Bellver-Landete, V. et al. Microglia are an essential component of the neuroprotective scar that forms after spinal cord injury. Nat Commun 10, 518, doi:10.1038 / s41467-019-08446-0 (2019), Anderson, M. A. et al. Astrocyte scar formation aids central nervous system axon regeneration. Nature 532, 195-200, doi:10.1038 / nature17623 (2016)).To maintain the beneficial properties of the SCI scar and improve its repair response while targeting the negative aspects, a time-dependent strategy for combination therapy is needed (Gaudet, A. D. & Fonken, L. K. Glial Cells Shape Pathology and Repair After Spinal Cord Injury. Neurotherapeutics 15, 554-577, doi:10.1007 / s13311-018-0630-7 (2018), Bradbury, E. J. & Burnside, E. R. Moving beyond the glial scar for spinal cord repair. Nat Commun 10, 3879, doi:10.1038 / s41467-019-11707-7 (2019), Courtine, G. & Sofroniew, M. V. Spinal cord repair: advances in biology and technology. Nat Med 25, 898-908, doi:10.1038 / s41591-019-0475-6 (2019)).

[0004] Treatment options, whether standard care or experimental, have had limited success in bringing about good neurological and functional recovery in severely injured patients. Ghosh and Pearse (2015) suggested that the glutamine, NA, DA, and 5-HT pathways are involved in the initiation and regulation of locomotor movement, and that experimental studies have provided evidence of the contribution of 5-HT in the regulation of the rhythm and coordination of movements via the central pattern generator. Experiments using the non-selective 5-HT receptor agonist, gepirone, have shown the induction of locomotor-like movements in the presence of the selective 5-HT2 antagonists, SB204741 and SB242084. Ghosh and Pearse did not disclose eletriptan, a 5-HT 1B / 1D receptor agonist, when used alone without 5-HT2 antagonists in the treatment of spinal cord injury and the improvement of locomotor function.

[0005] Eletriptan is a triptan drug first disclosed in WO 92 / 06973 (Pfizer) as a 5-HT1B / 1D receptor agonist for the treatment of migraine and for the prevention of migraine recurrence. WO 92 / 06973 does not disclose the use of eletriptan in spinal cord injury and improvement of locomotor function.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

[0008] In one aspect, the present disclosure relates to the use of eletriptan hydrobromide in the treatment of spinal cord injury and the improvement of locomotor function.

[0009] Embodiment 1. This patent application discloses eletriptan hydrobromide or a pharmaceutical composition thereof for use in the treatment of spinal cord injury.

[0010] Embodiment 2. Eletriptan hydrobromide or a pharmaceutical composition thereof for use in the improvement of locomotor function after spinal cord injury.

[0011] Embodiment 3. Eletriptan hydrobromide or a pharmaceutical composition thereof for use according to Embodiment 1, wherein the spinal cord injury is in the acute or subacute phase.

[0012] Embodiment 4. Eletriptan hydrobromide or a pharmaceutical composition thereof for use in the regulation of inflammation associated with spinal cord injury.

[0013] Embodiment 5. Eletriptan hydrobromide or a pharmaceutical composition thereof for use in the protection against spinal cord tissue Blood leakage.

[0017] Embodiment 6 . A method for treating spinal cord injury in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of eletriptan hydrobromide or a pharmaceutical composition thereof.

[0019] Embodiment 7 . The method according to the embodiment, wherein the subject is a warm-blooded vertebrate, preferably a mammal, more preferably a human. 6 BRIEF DESCRIPTION OF THE DRAWINGS

[0020] For easier understanding of the present application, figures representing preferred forms of implementation are attached to the accompanying documents, which are not intended to limit the technology disclosed herein.

Figure 1

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DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention relates to the use of eletriptan hydrobromide in the treatment of spinal cord injury and improvement of locomotor function.

[0022] As used herein, spinal cord injury (SCI) refers to any damage to the spinal cord or nerve that results from trauma (e.g., motor vehicle accident) or disease or degeneration (e.g., cancer) and causes a temporary or permanent change in the function of the spinal cord or nerve. Symptoms include those in the parts of the body where the spinal cord functions below the level of the injury LocomotionIt may include partial or complete loss of function, sensation, or autonomic function. The most severe spinal cord injuries affect systems that regulate bowel or bladder control, breathing, heart rate, and blood pressure. Most patients with spinal cord injuries experience chronic pain.

[0023] As used herein, the terms "subject," "host," and "patient" are used interchangeably. As used herein, a subject is preferably a mammal such as a non - primate (e.g., cow, pig, horse, cat, dog, rat, mouse, etc.) or a primate (e.g., monkey and human), most preferably a human.

[0024] As used herein, a "therapeutically effective amount" refers to the amount of an agent (e.g., the amount of eletriptan Hbr for use in the present invention) that, when administered to a subject suffering therefrom, provides at least one therapeutic benefit in the treatment or management of a target disease or disorder. Further, a therapeutically effective amount with respect to an agent for use in the present invention means the amount of the agent, alone or in combination with other therapies, that provides at least one therapeutic benefit in the treatment or management of a disease or disorder.

[0025] "Administering" refers to the physical introduction of an agent to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration for the compounds disclosed herein include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes by injection or infusion. The phrase "parenteral administration" as used herein means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intrathecal, intracavitary, intrasaccular, intradermal, intraperitoneal, subcutaneous, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as electroporation in vivo. In some embodiments, the compound is administered orally, for example, via a non - parenteral route. Other non - parenteral routes include topical, epidermal, or mucosal administration routes, for example, intranasal, sublingual, or topical. Administration may be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0026] One useful strategy for accelerating the discovery of new therapeutic agents for clinical use is to find new uses for existing drugs, i.e., the drug repositioning strategy (Hall, C. J. et al. Repositioning drugs for inflammatory disease - fishing for new anti-inflammatory agents. Dis Model Mech 7, 1069-1081, doi:10.1242 / dmm.016873 (2014)). In fact, drugs that have been found to have a therapeutic effect in one disease may potentially be effective in another disease, and since the drugs have already undergone preclinical and clinical trials, this strategy can shorten the development period, reduce costs, reduce risks, and improve the success rate (Hall, C. J. et al. Repositioning drugs for inflammatory disease - fishing for new anti-inflammatory agents. Dis Model Mech 7, 1069-1081, doi:10.1242 / dmm.016873 (2014), Buckley, C. E. et al. Drug reprofiling using zebrafish identifies novel compounds with potential pro-myelination effects. Neuropharmacology 59, 149-159, doi:10.1016 / j.neuropharm.2010.04.014 (2010)).

[0027] Zebrafish is emerging as a particularly versatile vertebrate model for simple and cost-effective drug screening (Hall, C. J. et al. Repositioning drugs for inflammatory disease - fishing for new anti-inflammatory agents. Dis Model Mech 7, 1069-1081, doi:10.1242 / dmm.016873 (2014), Rennekamp, A. J. & Peterson, R. T. 15 years of zebrafish chemical screening. Curr Opin Chem Biol 24, 58-70, doi:10.1016 / j.cbpa.2014.10.025 (2015), Early, J. J. et al. An automated high-resolution in vivo screen in zebrafish to identify chemical regulators of myelination. Elife 7, doi:10.7554 / eLife.35136 (2018), MacRae, C. A. & Peterson, R. T. Zebrafish as tools for drug discovery. Nat Rev Drug Discov 14, 721-731, doi:10.1038 / nrd4627 (2015)). To achieve the results disclosed herein, the inventors used the larval zebrafish drug screening platform previously developed by their laboratory to identify new molecules with therapeutic properties for SCI indications and identified eletriptan Hbr, which has locomotor recovery properties. Eletriptan Hbr is a 5-hydroxytryptamine 1-receptor subtype B / D (5-HT1 B / 1 D ) and F (5-HT1 F) is an FDA-approved drug with a highly selective affinity for (Capi, M. et al. Eletriptan in the management of acute migraine: an update on the evidence for efficacy, safety, and consistent response. Ther Adv Neurol Disord 9, 414-423, doi:10.1177 / 1756285616650619 (2016), Tepper, S. J., Rapoport, A. M. & Sheftell, F. D. Mechanisms of action of the 5-HT1B / 1D receptor agonists. Arch Neurol 59, 1084-1088, doi:10.1001 / archneur.59.7.1084 (2002)). For the first time herein, potential new therapeutic indications for the use of this molecule are disclosed. A repurposing strategy was adopted, and zebrafish larvae (a regeneration-promoting model) were utilized as an in vivo drug screening platform to identify eletriptan Hbr, and then we confirmed the preservation of the therapeutic effect in a mouse contusion (fibrosis-promoting) model by demonstrating the ability of eletriptan Hbr to improve spinal cord function repair.

[0028] Method Description of ethics All experiments involving animals were conducted in accordance with European Community guidelines (Directive 2010 / 63 / EU) and Portuguese law on animal care (DL113 / 2013) and were approved by the Instituto de Medicina Molecular Internal Committee (ORBEA) and the Portuguese Animal Ethics Committee (DGAV). All efforts were made to minimize the number of animals used and to reduce the suffering of the animals used in this disclosure.

[0029] Animals Zebrafish SCI model: Tg(mnx1:GFP ml2 ) (abbreviated as hb9): GFP was maintained and raised under certain conditions by following the standard guidelines for fish care and management protocols (Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio). (2000)). Zebrafish larvae were used for phenotype-based screening for SCI.

[0030] Mouse SCI model: Adult female C57BL / 6J mice (8 - 10 weeks old; Charles River) were used in this study. Mice were housed 3 - 4 per cage and maintained on a 12-hour light / dark cycle with unrestricted food and water during the study period.

[0031] Phenotype-based screening for SCI Using the larval zebrafish phenotype-based screening, a compound pool from a small molecule library (PHARMAKON 1600 - MicroSource Discovery Systems, Inc., USA) was screened as previously described by Diana et al. 2019 (Chapela, D. et al. A zebrafish drug screening platform boosts the discovery of novel therapeutics for spinal cord injury in mammals. Sci Rep 9, 10475, doi:10.1038 / s41598-019-47006-w (2019)). Briefly, zebrafish larvae were grown in EM containing 1 μM methylene blue until 5 days post-fertilization (dpf). At 5 dpf, the zebrafish larval spinal cord was transected at the level of the anal pore (Diana et al. 2019 (Chapela, D. et al. A zebrafish drug screening platform boosts the discovery of novel therapeutics for spinal cord injury in mammals. Sci Rep 9, 10475, doi:10.1038 / s41598-019-47006-w (2019)))。At 1 dpi, larvae were randomly distributed into 6-well plates containing EM + 10 mM HEPES and exposed to chemical compounds added to the medium for 24 hours. At 2 dpi, behavioral evaluation was performed using DanioVision (trademark) (manufactured by Noldus Information Technology, Netherlands), an automated tracking system for zebrafish larvae. Larvae were allowed to swim freely in 96-well plates containing EM + 10 mM HEPES (1 larva / well), and the swimming activity of the larvae was tracked for 90 minutes with a 10-minute light-dark cycle (i.e., 3 light cycles and 3 dark cycles). The acquired tracking data were analyzed using Ethovision X.T. 10 software (manufactured by Noldus, Wageningen, Netherlands), and only the swimming activity obtained during the 3 dark periods was analyzed (de Esch, C. et al. Locomotor activity assay in zebrafish larvae: influence of age, strain and ethanol. Neurotoxicol Teratol 34, 425-433, doi:10.1016 / j.ntt.2012.03.002 (2012)).

[0032] Contusion spinal cord injury and postoperative care Using an Infinite Horizon Impactor (manufactured by PSI), moderate-severe contusion-type injuries were induced in adult C57BL / 6 female mice (10 - 12 weeks old) (Tep, C. et al. Oral administration of a small molecule targeted to block proNGF binding to p75 promotes myelin sparing and functional recovery after spinal cord injury. J Neurosci 33, 397 - 410, doi:10.1523 / JNEUROSCI.0399 - 12.2013 (2013)). All surgeries were performed under aseptic conditions. Briefly, under deep anesthesia with ketamine and xylazine (120 mg / kg and 16 mg / kg, i.p., respectively), dorsal laminectomy at the ninth thoracic vertebra (T9) level was performed on the mice. After fixing the lateral processes of the eighth thoracic vertebra (T8) and the tenth thoracic vertebra (T10) with a stainless-steel impactor tip, a controlled force of 75 kdyne was applied as a defined impact to the exposed spinal cord (Chapela, D. et al. A zebrafish drug screening platform boosts the discovery of novel therapeutics for spinal cord injury in mammals. Sci Rep 9, 10475, doi:10.1038 / s41598 - 019 - 47006 - w (2019), Tep, C. et al. Oral administration of a small molecule targeted to block proNGF binding to p75 promotes myelin sparing and functional recovery after spinal cord injury. J Neurosci 33, 397 - 410, doi:10.1523 / JNEUROSCI.0399 - 12.2013 (2013)).Next, the muscle and skin were closed using 4.0 polyglycolic acid (PGA) absorbable sutures (Safil, G1048213). In this study, mice were excluded if the actual displacement value was outside the range of 500 - 700 μm, or if the actual force after impact exceeded 2SD 75 Kdyne. Immediately after injury, 0.5 ml of sterile saline was injected subcutaneously into the mice, and then injected daily for 5 days. During this experiment, the mice received gentle bladder compression twice a day until they urinated on their own. In this SCI model at the time of injury, since a 10% weight loss was generally observed, the body weight was monitored daily until day 15 post-injury (15 dpi), and then weekly for the duration of the study, and high-calorie pellets (Supreme Mini-Treats™ S05478 and S05472) were provided as nutritional supplements.

[0033] Drug treatment in a mouse SCI model The dose of eletriptan Hbr (manufactured by Sigma-Aldrich, PZ0011) used was the same as the human market dose (Nair, A. B. & Jacob, S. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm 7, 27 - 31, doi:10.4103 / 0976 - 0105.177703 (2016)), and all treatment doses were aliquoted using a coding system to maintain double-blind measures. This code was revealed only at the end of all behavioral tests. The mice were randomly assigned to each experimental group (SCI + vehicle and SCI + eletriptan Hbr). Vehicle and eletriptan Hbr were administered by intraperitoneal injection (i.p.) daily from 1 hour post-injury (hpi) and then until day 15 post-injury (15 dpi).

[0034] Basso Mouse Scale (BMS) assessment The open-field locomotor activity was evaluated by a BMS assessment system (Tep, C. et al. Oral administration of a small molecule targeted to block proNGF binding to p75 promotes myelin sparing and functional recovery after spinal cord injury. J Neurosci 33, 397-410, doi:10.1523 / JNEUROSCI.0399-12.2013 (2013); Basso, D. M. et al. Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains. J Neurotrauma 23, 635-659, doi:10.1089 / neu.2006.23.635 (2006)). The open field used was a circular platform with a diameter of 85 cm and a height of 30.5 cm, placed in a quiet test room with normal lighting. Two weeks before surgery, the mice were acclimated to the test platform for 5 minutes daily.As previously described, BMS tests (BMS scoring and sub-scoring) were performed (Chapela, D. et al. A zebrafish drug screening platform boosts the discovery of novel therapeutics for spinal cord injury in mammals. Sci Rep 9, 10475, doi:10.1038 / s41598-019-47006-w (2019), Tep, C. et al. Oral administration of a small molecule targeted to block proNGF binding to p75 promotes myelin sparing and functional recovery after spinal cord injury. J Neurosci 33, 397-410, doi:10.1523 / JNEUROSCI.0399-12.2013 (2013)). Briefly, mice were tested before surgery to obtain baseline pre-operative locomotor values that were expected to be the maximum BMS score value. To determine functional recovery after treatment, the BMS score of each mouse was judged at 1, 3, 7, 14 dpi and then weekly until the end of the experiment. Judgments of BMS scores and sub-scores were blinded to the treatment group and were always performed by two assessors who recorded hindlimb locomotion for 4 minutes per mouse. When the scores differed between assessors, the final score obtained was the average of both scores.

[0035] Acetone evaporation assay The acetone evaporation assay was used to measure sensitivity to cold stimuli during injury (Deuis, J. R., Dvorakova, L. S. & Vetter, I. Methods Used to Evaluate Pain Behaviors in Rodents. Front Mol Neurosci 10, 284, doi:10.3389 / fnmol.2017.00284 (2017)). First, mice were acclimated to a platform with a wire mesh for 4 minutes, and then 50 μl of acetone was dropped onto the plantar surface of the hind paw using a 1 ml syringe. Five trials were performed on each hind paw at 2-minute intervals between trials. In each trial, a timer was used to measure the time required to move or lick the hind paw in 60 seconds and recorded for analysis (Golden, J. P. et al. RET signaling is required for survival and normal function of nonpeptidergic nociceptors. J Neurosci 30, 3983-3994, doi:10.1523 / JNEUROSCI.5930-09.2010 (2010)).

[0036] Perfusion and tissue processing At the end of the behavioral tests, mice were perfused transcardially with 0.9% NaCl followed by a 4% paraformaldehyde (PFA) solution in 0.1 M phosphate-buffered saline (PBS, pH 7.4). After rinsing overnight with PBS, the spinal cords were cryoprotected in 30% sucrose for 3 days and then frozen in optimal cutting temperature (OCT) compound (Sakura Finetek, USA) as blocks 3 mm rostral to 3 mm caudal to the center of the injury (6 mm total). Each block was then sectioned transversely (10 μm) on a cryostat, mounted on slides in alternating 10 sets, and stored at -20 °C until needed (Ma, M., Basso, D. M., Walters, P., Stokes, B. T. & Jakeman, L. B. Behavioral and histological outcomes following graded spinal cord contusion injury in the C57Bl / 6 mouse. Exp Neurol 169, 239-254, doi:10.1006 / exnr.2001.7679 (2001) 、Hoschouer, E. L., Finseth, T., Flinn, S., Basso, D. M. & Jakeman, L. B. Sensory stimulation prior to spinal cord injury induces post-injury dysesthesia in mice. J Neurotrauma 27, 777-787, doi:10.1089 / neu.2009.1182 (2010) )。

[0037] Immunohistochemistry For immunohistochemistry in sections, OCT was removed from frozen sections using PBS (30 minutes). Sections were washed with 0.5% PBS-Triton X-100 and blocked in blocking solution (5% goat serum in PBS containing 0.1% Triton X-100) for 2 hours at room temperature. Spinal cord sections were then incubated overnight at 4 °C with primary antibodies against GFAP (1:500; ThermoFisher Scientific, 13-0300), anti-PDGFRβ (1:200; Abcam, ab32570), anti-CD31 / PECAM-1 (1:100; RD Systems, AF3628), anti-F4 / 80 (1:500; Abcam, ab6640) and anti-P2Y12 (1:500; AnaSpec, AS-55043A), washed in 0.1% PBS-Triton X-100 and PBS, and reincubated overnight at 4 °C with AlexaFluor 568 (1:500; ThermoFisher Scientific, A11011 or A11057), AlexaFluor 488 (1:500; ThermoFisher Scientific, A11006 or A11008) secondary antibodies. For quantification of activated microglia, sections were counterstained with DAPI. Sections were then washed in PBS and mounted in fluorescence mounting medium containing DABCO.

[0038] FluoroMyelin Green Staining and White Matter Remnant Analysis To quantify demyelination, a set of sections spanning the entire block and placed 100 μm apart were stained with FluoroMyelin™ Green (ThermoFisher Scientific, F34651) for 1 hour. Z-stack compositions were acquired with a motorized inverted wide-field fluorescence microscope at 10x magnification (Zeiss Cell Observer, Carl Zeiss MicroImaging). Using Fiji software, the cross-sectional area of white matter sparing (WMA) and the total cross-sectional area of the tissue section (TCA) were measured, and then the proportional cross-sectional area from 1100 μm rostral to 1100 μm caudal relative to the center of the lesion was calculated (WMA / TCA). The center was identified as the tissue section with the smallest WMA / TCA, which had the smallest area of fluorescent green-stained white matter at the periphery. Analyses of lesions were performed by researchers via coded sections and blinded to treatment or outcome groups (Hoschouer, E. L., Finseth, T., Flinn, S., Basso, D. M. & Jakeman, L. B. Sensory stimulation prior to spinal cord injury induces post-injury dysesthesia in mice. J Neurotrauma 27, 777-787, doi:10.1089 / neu.2009.1182 (2010)).

[0039] Quantification of fibrous scar A set of sections was stained with anti-PDGFRβ and anti-GFAP to delineate the contours of the boundaries of the fibrous scar region. Z-stack compositions were acquired with a motorized inverted wide-field fluorescence microscope at 20x magnification (Zeiss Cell Observer, Carl Zeiss MicroImaging). Using Fiji software, the contours were traced manually, and PDGFRβ +The area and total cross-sectional area were calculated, and then the percentage of the fibrotic scar area relative to the total cross-sectional area was quantified. The rostral and caudal extent of the fibrotic lesion was determined by examination. The length of the fibrotic lesion and the extent of PDGFRβ + expression increase (respectively) were calculated by multiplying the number of sections containing tissue with increased expression by the distance (100 μm) between each section. + The range of PDGFRβ expression was calculated.

[0040] Quantification of activated microglia To quantify activated microglia, a set of spinal cord sections were stained with anti-P2Y12 (a microglia-specific marker) and anti-F4 / 80 (a pan-macrophage marker) and counterstained with DAPI. A Z-stack composition was acquired using a 20x motorized inverted wide-field fluorescence microscope (Zeiss Cell Observer, Carl Zeiss MicroImaging). After manually setting the threshold and parameters, the number of F4 / 80 + and P2Y12 + cells was quantified using custom Multichannel Cell counter4TIFF software that calculates the number of cells.

[0041] Statistical analysis All graph displays and data analyses from the larval zebrafish SCI model were performed using Prism 8 software (GraphPad Software, Inc., San Diego, CA, USA). The statistical tests used were two-sided tests. Comparison of mean values between different groups in zebrafish SCI studies was performed using an unpaired Student t-test with Welch's correction. Data analysis from the mouse SCI model was performed using SigmaPlot 14, using two-way analysis of variance (ANOVA) or repeated measures two-way ANOVA, followed by Bonferroni post hoc tests. A P-value less than 0.05 was considered significant. All data were represented as mean ± standard error of the mean (SEM).

[0042] Results Eletriptan Hbr rescues locomotor dysfunction in a zebrafish larval spinal cord transection injury model. To accelerate the discovery of promising new therapeutics for spinal cord injury (SCI), we used a phenotypic assay that enabled screening of a chemical compound pool from an FDA-approved small molecule library. In this phenotype-based screening, we blindly administered chemical compounds (25 μM) on day 1 post-injury (dpi), and we performed behavioral assessments 24 hours later (i.e., 2 dpi) as previously described by Chapela D. et al. 2019. Small molecules were first blindly selected if there was a statistically significant improvement in the total distance moved and / or rotation angle parameters, which were selected as indicators of locomotor function. We then narrowed the selection by defined exclusion criteria (having a patented or reported therapeutic indication for SCI; having significant reported toxicity or being unable to cross the blood-brain barrier). Notably, eletriptan Hbr was one of the most promising candidates identified through this drug discovery platform. Eletriptan Hbr is known as a second-generation triptan drug for the acute treatment of migraine, with or without aura, in the human adult, and interestingly, it was shown to rescue locomotor dysfunction in this zebrafish larval spinal cord transection injury model (Figure 1A, A’), emerging as a promising SCI therapeutic drug candidate to be tested in mammalian SCI models.

[0043] Eletriptan Hbr improves motor function in mice with T9 contusion-type injuries. To verify the therapeutic effect of eletriptan Hbr on SCI indications in a non-regenerative model, it was decided to test its efficacy in an SCI rodent model. Therefore, a contusive injury was performed on C57BL / 6 female mice using an Infinite Horizon (IH) Impactor, and the Basso Mouse Scale (BMS) test (Basso, D. M. et al. Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains. J Neurotrauma 23, 635-659, doi:10.1089 / neu.2006.23.635 (2006)) was used to determine the locomotor ability of the animals after injury. First, the mice were acclimated to an open-field platform, and 15 days later, the mice were injured with a moderate-severe T9 contusion (75 kdyne) (Figure 2A). Immediately after injury and at the biomechanical exclusion criteria, the mice were randomly assigned to each experimental group (SCI + vehicle and SCI + eletriptan Hbr). The treatment dose (vehicle or eletriptan Hbr) was administered by daily intraperitoneal (i.p.) injection from 1 hour post-injury (1 hpi) and then until 15 days post-injury (15 dpi) (Figure 2A). There was no difference in the injury force or displacement applied by the IH impactor between the SCI + vehicle and SCI + eletriptan Hbr experimental groups (Figure 4). For the determination of locomotor recovery at the time of injury, the BMS score and subscores were measured for 42 days (Figure 3B - B'). The mean BMS scores in eletriptan Hbr-treated mice and vehicle-treated mice increased after 1 day post-injury (1 dpi) and reached a plateau between 28 - 35 days post-injury (28 - 35 dpi) and between 21 - 28 days post-injury (21 - 28 dpi), respectively. The BMS scores in eletriptan Hbr-treated mice were consistently higher, although not significantly, over the long term compared to vehicle-treated mice (Figure 3B).Notably, one out of 13 eletriptan Hbr-treated mice achieved a BMS score of 6 (i.e., showed frequent plantar steps with some hindlimb coordination), and the remaining 12 achieved a BMS score of 5 at 42 days post-injury (42 dpi) (i.e., showed frequent or consistent plantar steps). At the same time point, the BMS scores of vehicle-treated mice were 4 - 5, and only 3 out of 9 animals achieved occasional plantar steps. Furthermore, 85% of eletriptan Hbr-treated mice also showed frequent plantar steps with parallel placement of the feet of both hindlimbs at initial ground contact, and 23% exhibited parallel placement of at least one of the hindlimb feet during lift.

[0044] The mean BMS subscores were consistently higher in eletriptan Hbr-treated mice than in vehicle-treated mice from 7 to 42 days post-injury (7 - 42 dpi), and significantly higher from 35 days post-injury (35 dpi) (Figure 3B’) to 42 days post-injury (42 dpi) (Figure 3B’).

[0045] At 42 days post-injury (42 dpi), 8 out of 9 vehicle-treated mice showed severe trunk instability such as swaying, tremors or impending collapse of the hindlimbs, and only 1 animal achieved mild trunk stability. Furthermore, eletriptan Hbr-treated mice showed a reduction in events that interrupted walking movements such as convulsions and falls, and 46% of the animals in this experimental group achieved mild trunk stability (Figure 3C).

[0046] Furthermore, we also determined the state of bladder dysfunction, which is a common consequence observed after SCI (Donovan, J. & Kirshblum, S. Clinical Trials in Traumatic Spinal Cord Injury. Neurotherapeutics 15, 654-668, doi:10.1007 / s13311-018-0632-5 (2018); Dias, D. O. et al. Reducing Pericyte-Derived Scarring Promotes Recovery after Spinal Cord Injury. Cell 173, 153-165.e122, doi:10.1016 / j.cell.2018.02.004 (2018)). During manual bladder compression, we scored from 0 (normal bladder without urine, i.e., animals with the ability to urinate spontaneously) to 3 (large bladder with a large amount of stored urine). Eletriptan Hbr-treated mice showed a statistically significantly smaller amount of stored urine (i.e., a smaller score) on day 1 post-injury (1 dpi), but this effect was lost after this time point and there was no difference between the experimental groups until the end of the study (Figure 3D).

[0047] Allodynia is defined as hypersensitivity, a painful response to a normally non-noxious cold stimulus, which is commonly associated with SCI (Deuis, J. R., Dvorakova, L. S. & Vetter, I. Methods Used to Evaluate Pain Behaviors in Rodents. Front Mol Neurosci 10, 284, doi:10.3389 / fnmol.2017.00284 (2017)). In this study, we determined the sensitivity to cold stimuli using the acetone evaporation test at 14 days post-injury (14 dpi) and 42 days post-injury (42 dpi) by quantifying the number and duration of episodes of the nocifensive response induced by evaporative cooling. The mean of eletriptan Hbr-treated mice showed a non-significant but consistent decrease in cumulative response time and a decrease in the number of episodes of cold allodynia compared to vehicle-treated mice (Figure 3E, E').

[0048] Eletriptan Hbr is thought to prevent demyelination near the terminal extent of the lesion. To analyze the effect of eletriptan Hbr on the demyelinated state during injury, FluoroMyelin™ green fluorescent myelin staining was used to compare the remaining white matter area to the total cross-sectional area between experimental groups (SCI + eletriptan Hbr and SCI + vehicle) (Figure 5A). The mean of the remaining white matter to the total cross-sectional area in eletriptan Hbr-treated mice was not significantly higher but consistently higher than that in vehicle-treated mice at the lesion center and extending 700 μm rostral and caudal from the center of the lesion (Figure 5B).

[0049] The range of increased PDGFR-β expression levels is reduced by eletriptan Hbr treatment. The fibrous compartment of the scar is PDGFRβ, which creates a core of fibroblast-like cells. + It is composed of a subset of perivascular cells and a high-density deposition of extracellular matrix molecules (Dias, D. O. et al. Reducing Pericyte-Derived Scarring Promotes Recovery after Spinal Cord Injury. Cell 173, 153-165.e122, doi:10.1016 / j.cell.2018.02.004 (2018)). More recently, it has been shown that reducing pericyte-derived scarring promotes functional recovery during spinal cord injury in mice (Dias, D. O. et al. Reducing Pericyte-Derived Scarring Promotes Recovery after Spinal Cord Injury. Cell 173, 153-165.e122, doi:10.1016 / j.cell.2018.02.004 (2018)), so it was decided to analyze the effect of eletriptan Hbr on the fibrotic scar state by determining the area and length of the fibrotic scar.

[0050] For this analysis, double immunohistochemistry was performed with GFAP, which enables delineation of the contour of the glial-restricted border of the scar, and the pericytes marker PDGFRβ for labeling the fibrous elements of the scar (Figs. 6A–C, 6A’–C’, 6A’’–C’’). This approach delineated the contour of the fibrous scar compartment and measured its area at the center of the lesion, and PDGFRβ + the extent in length of the lesion core, and further the PDGFRβ + in the sham mice by comparison with the level of staining, PDGFRβ + the range of increase in the level of immunostaining could be defined. At the center of the lesion, at 42 days post-injury (42 dpi), there was no statistically significant difference in the area of the fibrotic lesion core between the eletriptan Hbr-treated and vehicle-treated mice. There was no statistically significant difference in the extent of the PDGFRβ + lesion core between the vehicle and eletriptan Hbr treatment groups, but there was a statistically significant reduction in the range of increase in the level of PDGFRβ + immunostaining in the mice treated with eletriptan Hbr compared to the vehicle-treated mice (Figs. 6D–F).

[0051] Treatment with eletriptan Hbr reduces the extent of tissue with an abnormal number of PDGFRβ + cells associated with vasculature To infer whether the increase in the PDGFRβ + level detected at the ends of the lesion extent was associated with blood vessels, we decided to perform double immunohistochemistry using the pericyte marker PDGFRβ and the endothelial cell marker CD31 (PECAM-1) (Fig. 7). The increase in PDGFRβ expression detected in the vehicle-treated mice and reduced in the eletriptan Hbr-treated mice in sections 1000 μm from the center was associated with CD31 + endothelial cells (Figs. 7A–C’’’).

[0052] Microglia are affected by eletriptan Hbr It is known that microglia are very important for SCI repair. Without microglia, demyelination and pathological MDM infiltrates are enhanced, glial scar formation is disrupted, and motor dysfunction worsens (Brennan, F. H., Hall, J. C. E., Guan, Z. & P.G., P. Microglia limit lesion expansion and promote functional recovery after spinal cord injury in mice. Cold Spring Harbor Laboratory, doi:https: / / doi.org / 10.1101 / 410258 (2018)). In this context, it was decided to determine the effect of eletriptan Hbr on the microglial state at the time of injury by performing double immunohistochemistry with P2Y12 (a microglial marker) and F4 / 80 (a pan-macrophage marker). P2Y12 + Microglia showed a branched morphology with long processes in sham mice, while in vehicle-treated mice, microglia showed shorter processes and were sparsely detected at the lesion center (Figs. 8A–C’’). In vehicle-treated mice, P2Y12 + Microglia were rarely detected at the lesion center and, when present, microglia were mainly observed in clusters at the lesion periphery or in the remaining white matter with an amoeboid shape (Figs. 8B, B’’; 8C, C’’). Eletriptan Hbr-treated mice also showed microglia in clusters with an amoeboid shape and shorter processes at the lesion periphery or in the remaining white matter, but in some sections, longer branched processes were also observed near or at the center, a feature not observed in vehicle-treated mice (Figs. 8B, B’’; 8C, C’’). In sections 1000 μm from the center, P2Y12 + Microglia were present throughout the gray and white matter and showed a morphology with some shorter processes and a more reactive shape in the gray matter (mainly on the dorsal side of the spinal cord) in both vehicle-treated and eletriptan Hbr-treated mice compared to sham animals (Figs. 8A, A’; 8B, B’’; 8C, C’’). P2Y12+ F4 / 80 + There was no statistically significant difference in the number of (activated microglia) cells, although this number was consistently higher at 400 μm rostral and caudal to the center in eletriptan Hbr-treated mice (Figure 8D).

[0053] Spinal cord injury (SCI) has a very complex nature. The best system for investigating the potential of compounds that interfere with complex physiological processes is to determine their effects in vivo, and it is well known that zebrafish larvae are becoming an especially versatile vertebrate model for phenotypic drug screening in vivo (Hall, C. J. et al. Repositioning drugs for inflammatory disease - fishing for new anti-inflammatory agents. Dis Model Mech 7, 1069-1081, doi:10.1242 / dmm.016873 (2014), Rennekamp, A. J. & Peterson, R. T. 15 years of zebrafish chemical screening. Curr Opin Chem Biol 24, 58-70, doi:10.1016 / j.cbpa.2014.10.025 (2015)).

[0054] Using the in vivo larval zebrafish phenotype-based screening previously established in our laboratory (i.e., using the regeneration promotion model) (Chapela, D. et al. A zebrafish drug screening platform boosts the discovery of novel therapeutics for spinal cord injury in mammals. Sci Rep 9, 10475, doi:10.1038 / s41598-019-47006-w (2019)), promising compounds with SCI rescue properties from an FDA-approved small molecule library are identified herein for the first time. We then verify and determine the preservation of the therapeutic effect of this compound in an in vivo mouse contusion (fibrosis-promoting) model of SCI.

[0055] From our previously validated zebrafish drug discovery platform, we selected eletriptan Hbr as one of the most promising candidates with the potential for spinal cord recovery that rescues motor dysfunction in both total distance moved and rotation angle parameters. Importantly, the improvements in the total distance and rotation angle parameters not only indicated that eletriptan Hbr rescued the swimming ability of SCI larvae, but also seemed to improve motor direction control respectively.

[0056] After the selection of eletriptan Hbr by this zebrafish approach, this compound was administered daily to a T9 contusion mouse model from 1 hour post-injury (1hpi [hour upon injury]) and then until 15 days post-injury (15dpi) during the acute and subacute injury phases. Thus, not only was it confirmed that eletriptan Hbr had a preserved effect on the improvement of locomotor behavior in the fibrosis-promoting model, but it was also possible to determine its effect on demyelination, fibrotic scar formation, and inflammatory processes at 42 days post-injury (42dpi). Importantly, this time point corresponds to the chronic phase time in rodents where reactive astrogliosis and macrophage / microglia-induced inflammation were present. (Gaudet, A. D. & Fonken, L. K. Glial Cells Shape Pathology and Repair After Spinal Cord Injury. Neurotherapeutics 15, 554-577, doi:10.1007 / s13311-018-0630-7 (2018)) 。

[0057] In particular, eletriptan Hbr was shown to significantly improve locomotor ability in the BMS assessment, improve trunk stability in mice, and reduce the number of severe events. Furthermore, administration of eletriptan Hbr consistently, although not significantly, decreased the cumulative response time in the acetone evaporation test and reduced the number of episodes of cold allodynia, suggesting that this compound may reduce cold allodynia in the SCI situation. Indeed, despite being used in several studies to measure cold sensitivity, the acetone evaporation test has several limitations, namely, it is difficult to ensure that an accurate amount of acetone is consistently dripped each time, and the cold stimulus varies (Deuis, J. R., Dvorakova, L. S. & Vetter, I. Methods Used to Evaluate Pain Behaviors in Rodents. Front Mol Neurosci 10, 284, doi:10.3389 / fnmol.2017.00284 (2017); Brenner, D. S., Golden, J. P. & Gereau, R. W. A novel behavioral assay for measuring cold sensation in mice. PLoS One 7, e39765, doi:10.1371 / journal.pone.0039765 (2012)). This method is easy to perform but only quantifies the magnitude of the response instead of measuring the minimum temperature that elicits a response (Brenner, D. S., Golden, J. P. & Gereau, R. W. A novel behavioral assay for measuring cold sensation in mice. PLoS One 7, e39765, doi:10.1371 / journal.pone.0039765 (2012)).

[0058] In humans, sometimes the degree of dysfunction does not necessarily correlate with the degree of tissue damage. In fact, contusion-type injuries often lead to a complete loss of movement and sensation despite the presence of tissue remaining at the site of injury (Oudega, M. Molecular and cellular mechanisms underlying the role of blood vessels in spinal cord injury and repair. Cell Tissue Res 349, 269-288, doi:10.1007 / s00441-012-1440-6 (2012)). Although there were no statistically significant differences between treatment groups, eletriptan Hbr was shown to consistently promote higher myelin maintenance near the terminal extent of the lesion.

[0059] After SCI, there is the formation of fibrous scars that inhibit axonal regrowth and at the same time limit the infiltration of immune cells into the spinal cord parenchyma (Zhu, Y. et al. Hematogenous macrophage depletion reduces the fibrotic scar and increases axonal growth after spinal cord injury. Neurobiol Dis 74, 114-125, doi:10.1016 / j.nbd.2014.10.024 (2015)). Importantly, moderately inhibiting pericyte-derived scars not only maintains wound healing and reduces inflammation and reactive astrogliosis, but also enables axonal regeneration and improves functional recovery (Dias, D. O. et al. Reducing Pericyte-Derived Scarring Promotes Recovery after Spinal Cord Injury. Cell 173, 153-165.e122, doi:10.1016 / j.cell.2018.02.004 (2018)).

[0060] Interestingly, eletriptan Hbr significantly reduced the range of increase in + the level of PDGFRβ immunostaining, but not the PDGFRβ +It is disclosed herein that the fibrotic lesion core area or extent was not reduced.

[0061] As disclosed in this patent application, PDGFRβ detected at the ends of the lesion extent + An increase in levels is associated with blood vessels, and eletriptan Hbr inhibits PDGFRβ + Cells, i.e., CD31 + PDGFRβ with cells + It was suggested that it reduces the extent of the lesioned tissue having an increase in the number of, and probably has an effect on ischemic conditions.

[0062] In response to injury, microglia are activated and enable the regeneration of severed axons through the release of neurotrophic factors and debris removal (Brennan, F. H., Hall, J. C. E., Guan, Z. & P.G., P. Microglia limit lesion expansion and promote functional recovery after spinal cord injury in mice. Cold Spring Harbor Laboratory, doi:https: / / doi.org / 10.1101 / 410258 (2018)) . However, neurotoxicity can also be promoted by the secretion of inflammatory cytokines and the production of free radicals (Brennan, F. H., Hall, J. C. E., Guan, Z. & P.G., P. Microglia limit lesion expansion and promote functional recovery after spinal cord injury in mice. Cold Spring Harbor Laboratory, doi:https: / / doi.org / 10.1101 / 410258 (2018)).

[0063] Due to the very important role of microglia in SCI repair and recovery, the inventors decided to investigate the effect of eletriptan Hbr on the inflammatory process. Interestingly, mice treated with eletriptan Hbr were shown to have microglia with a morphology similar to that characteristic of moderately activated microglia, showing hypertrophy with shorter processes (Brennan, F. H., Hall, J. C. E., Guan, Z. & P.G., P. Microglia limit lesion expansion and promote functional recovery after spinal cord injury in mice. Cold Spring Harbor Laboratory, doi:https: / / doi.org / 10.1101 / 410258 (2018))In fact, amoeba-like microglia were observed in the clusters, but some mice showed longer branched processes near the lesion center in spinal cord sections. This was not observed in vehicle-treated mice in which only strongly activated microglia with an amoeba-like morphology, characteristic of a strong molecular inflammatory response (Gaudet, A. D. & Fonken, L. K. Glial Cells Shape Pathology and Repair After Spinal Cord Injury. Neurotherapeutics 15, 554-577, doi:10.1007 / s13311-018-0630-7 (2018)), were detected.

[0064] Eletriptan Hbr is a serotonin receptor agonist with high affinity for the 5-HT1B, 5-HT1D, and 5-HT1F receptors, known for its significant clinical efficacy in the treatment of migraine (Capi, M. et al. Eletriptan in the management of acute migraine: an update on the evidence for efficacy, safety, and consistent response. Ther Adv Neurol Disord 9, 414-423, doi:10.1177 / 1756285616650619 (2016)). Notably, for the first time herein, a new indication for eletriptan Hbr is disclosed, and its locomotor recovery properties are shown in two different animal models of SCI (promoting transverse regeneration and promoting contusion fibrosis). Finally, eletriptan Hbr in combination therapy with other molecules, as well as eletriptan Hbr with engineering techniques and specific time-dependent interventions, has great potential in the context of SCI.

[0065] Several features are described below that can be used independently of each other or in any combination with any other features. However, no individual feature can address any of the problems discussed above, or may only be able to address one of the problems discussed above. Some of the problems discussed above cannot be fully addressed by any of the features described in this specification. Although headings are provided, information regarding a particular heading that is not found in the section with that heading may be found elsewhere in this specification.

[0066] Description of Embodiments Here, the preferred embodiments of the present application are described in detail, but these are not intended to limit the scope of the present application.

[0067] Embodiment 1. This patent application discloses eletriptan hydrobromide or a pharmaceutical composition thereof for use in the treatment of spinal cord injury.

[0068] Embodiment 2. Eletriptan hydrobromide or a pharmaceutical composition thereof for use in improving locomotor function after spinal cord injury.

[0069] Embodiment 3. The eletriptan hydrobromide or a pharmaceutical composition thereof according to Embodiment 1 for use where the spinal cord injury is in the acute phase or the subacute phase .

[0070] Embodiment 4. Eletriptan hydrobromide or a pharmaceutical composition thereof for use in modulating inflammation associated with spinal cord injury.

[0071] Embodiment 5. Eletriptan hydrobromide or a pharmaceutical composition thereof for use in protecting against spinal cord tissue leakage blood .

[0075] Embodiment 6 . A method of treating spinal cord injury in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of eletriptan hydrobromide or a pharmaceutical composition thereof.

[0077] Embodiment 7 .An embodiment in which the subject is a warm-blooded vertebrate, preferably a mammal, more preferably a human 6 Method according to

[0078] Suitable unit forms of administration of a pharmaceutical composition comprising eletriptan Hbr include, by way of non-limiting example, forms administered orally and forms administered via parenteral routes, non-limiting examples of which include inhalation, subcutaneous administration, intramuscular administration, intravenous administration and intradermal administration.

[0079] In some embodiments, the pharmaceutical composition for oral administration may be in the form of tablets, pills, powders, hard gelatin capsules, soft gelatin capsules, and / or granules. In some embodiments of such pharmaceutical compositions, the disclosed compound and / or a pharmaceutically acceptable salt of the disclosed compound is mixed with one or more inert diluents, non-limiting examples of which include starch, cellulose, sucrose, lactose, and silica. In some embodiments, such pharmaceutical compositions may further comprise one or more substances other than diluents, such as (by way of non-limiting example) lubricants, colorants, coatings, or varnishes.

[0080] In this specification, embodiments of the present invention are described with respect to many specific details which may vary from one implementation to another. Thus, the sole and exclusive indicator of what the present invention is, and what is intended by the applicants to be the present invention, is a series of claims arising from this application, taking the specific form in which such claims arise, including amendments thereto. The definitions expressly set forth herein for terms contained in such claims shall govern the meaning of such terms as used in the claims. Accordingly, no limitation, element, characteristic, feature, advantage or attribute not expressly recited in a claim should in any way limit the scope of such claim. Accordingly, this specification and the drawings are to be regarded in an illustrative rather than a limiting sense. (References) 1 Kjell, J. & Olson, L. Rat models of spinal cord injury: from pathology to potential therapies. Dis Model Mech 9, 1125-1137, doi:10.1242 / dmm.025833 (2016). 2 Boutonnet, M., Laemmel, E., Vicaut, E., Duranteau, J. & Soubeyrand, M. Combinatorial therapy with two pro-coagulants and one osmotic agent reduces the extent of the lesion in the acute phase of spinal cord injury in the rat. Intensive Care Med Exp 5, 51, doi:10.1186 / s40635-017-0164-z (2017). 3 Donovan, J. & Kirshblum, S. Clinical Trials in Traumatic Spinal Cord Injury. Neurotherapeutics 15, 654-668, doi:10.1007 / s13311-018-0632-5 (2018). 4 Duncan, G. J. et al. Locomotor recovery following contusive spinal cord injury does not require oligodendrocyte remyelination. Nat Commun 9, 3066, doi:10.1038 / s41467-018-05473-1 (2018). 5 Zhou, X., He, X. & Ren, Y. Function of microglia and macrophages in secondary damage after spinal cord injury. Neural Regen Res 9, 1787-1795, doi:10.4103 / 1673-5374.143423 (2014). 6 Yilmaz, T. & Kaptanoglu, E. Current and future medical therapeutic strategies for the functional repair of spinal cord injury. World J Orthop 6, 42-55, doi:10.5312 / wjo.v6.i1.42 (2015). 7 Picoli, C. C. et al. Pericytes Act as Key Players in Spinal Cord Injury. Am J Pathol 189, 1327-1337, doi:10.1016 / j.ajpath.2019.03.008 (2019). 8 O'Shea, T. M., Burda, J. E. & Sofroniew, M. V. Cell biology of spinal cord injury and repair. J Clin Invest 127, 3259-3270, doi:10.1172 / JCI90608 (2017). 9 Gaudet, A. D. & Fonken, L. K. Glial Cells Shape Pathology and Repair After Spinal Cord Injury. Neurotherapeutics 15, 554-577, doi:10.1007 / s13311-018-0630-7 (2018). 10 Hausmann, O. N. Post-traumatic inflammation following spinal cord injury. Spinal Cord 41, 369-378, doi:10.1038 / sj.sc.3101483 (2003). 11 Bradbury, E. J. & Burnside, E. R. Moving beyond the glial scar for spinal cord repair. Nat Commun 10, 3879, doi:10.1038 / s41467-019-11707-7 (2019). 12 Orr, M. B. & Gensel, J. C. Spinal Cord Injury Scarring and Inflammation: Therapies Targeting Glial and Inflammatory Responses. Neurotherapeutics 15, 541-553, doi:10.1007 / s13311-018-0631-6 (2018). 13 Cregg, J. M. et al. Functional regeneration beyond the glial scar. Exp Neurol 253, 197-207, doi:10.1016 / j.expneurol.2013.12.024 (2014). 14 Zhang, B. et al. Reducing age-dependent monocyte-derived macrophage activation contributes to the therapeutic efficacy of NADPH oxidase inhibition in spinal cord injury. Brain Behav Immun 76, 139-150, doi:10.1016 / j.bbi.2018.11.013 (2019). 15 Keirstead, H. S. et al. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci 25, 4694-4705, doi:10.1523 / JNEUROSCI.0311-05.2005 (2005). 16 McTigue, D. M. & Tripathi, R. B. The life, death, and replacement of oligodendrocytes in the adult CNS. J Neurochem 107, 1-19, doi:10.1111 / j.1471-4159.2008.05570.x (2008). 17 Bellver-Landete, V. et al. Microglia are an essential component of the neuroprotective scar that forms after spinal cord injury. Nat Commun 10, 518, doi:10.1038 / s41467-019-08446-0 (2019). 18 Anderson, M. A. et al. Astrocyte scar formation aids central nervous system axon regeneration. Nature 532, 195-200, doi:10.1038 / nature17623 (2016). 19 Courtine, G. & Sofroniew, M. V. Spinal cord repair: advances in biology and technology. Nat Med 25, 898-908, doi:10.1038 / s41591-019-0475-6 (2019). 20 Hall, C. J. et al. Repositioning drugs for inflammatory disease - fishing for new anti-inflammatory agents. Dis Model Mech 7, 1069-1081, doi:10.1242 / dmm.016873 (2014). 21 Buckley, C. E. et al. Drug reprofiling using zebrafish identifies novel compounds with potential pro-myelination effects. Neuropharmacology 59, 149-159, doi:10.1016 / j.neuropharm.2010.04.014 (2010). 22 Rennekamp, A. J. & Peterson, R. T. 15 years of zebrafish chemical screening. Curr Opin Chem Biol 24, 58-70, doi:10.1016 / j.cbpa.2014.10.025 (2015). 23 Early, J. J. et al. An automated high-resolution in vivo screen in zebrafish to identify chemical regulators of myelination. Elife 7, doi:10.7554 / eLife.35136 (2018). 24 MacRae, C. A. & Peterson, R. T. Zebrafish as tools for drug discovery. Nat Rev Drug Discov 14, 721-731, doi:10.1038 / nrd4627 (2015). 25 Capi, M. et al. Eletriptan in the management of acute migraine: an update on the evidence for efficacy, safety, and consistent response. Ther Adv Neurol Disord 9, 414-423, doi:10.1177 / 1756285616650619 (2016). 26 Tepper, S. J., Rapoport, A. M. & Sheftell, F. D. Mechanisms of action of the 5-HT1B / 1D receptor agonists. Arch Neurol 59, 1084-1088, doi:10.1001 / archneur.59.7.1084 (2002). 27 Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio). (2000). 28 Chapela, D. et al. A zebrafish drug screening platform boosts the discovery of novel therapeutics for spinal cord injury in mammals. Sci Rep 9, 10475, doi:10.1038 / s41598-019-47006-w (2019). 29 de Esch, C. et al. Locomotor activity assay in zebrafish larvae: influence of age, strain and ethanol. Neurotoxicol Teratol 34, 425-433, doi:10.1016 / j.ntt.2012.03.002 (2012). 30 Tep, C. et al. Oral administration of a small molecule targeted to block proNGF binding to p75 promotes myelin sparing and functional recovery after spinal cord injury. J Neurosci 33, 397-410, doi:10.1523 / JNEUROSCI.0399-12.2013 (2013). 31 Nair, A. B. & Jacob, S. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm 7, 27-31, doi:10.4103 / 0976-0105.177703 (2016). 32 Basso, D. M. et al. Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains. J Neurotrauma 23, 635-659, doi:10.1089 / neu.2006.23.635 (2006). 33 Deuis, J. R., Dvorakova, L. S. & Vetter, I. Methods Used to Evaluate Pain Behaviors in Rodents. Front Mol Neurosci 10, 284, doi:10.3389 / fnmol.2017.00284 (2017). 34 Golden, J. P. et al. RET signaling is required for survival and normal function of nonpeptidergic nociceptors. J Neurosci 30, 3983-3994, doi:10.1523 / JNEUROSCI.5930-09.2010 (2010). 35 Ma, M., Basso, D. M., Walters, P., Stokes, B. T. & Jakeman, L. B. Behavioral and histological outcomes following graded spinal cord contusion injury in the C57Bl / 6 mouse. Exp Neurol 169, 239-254, doi:10.1006 / exnr.2001.7679 (2001). 36 Hoschouer, E. L., Finseth, T., Flinn, S., Basso, D. M. & Jakeman, L. B. Sensory stimulation prior to spinal cord injury induces post-injury dysesthesia in mice. J Neurotrauma 27, 777-787, doi:10.1089 / neu.2009.1182 (2010). 37 Dias, D. O. et al. Reducing Pericyte-Derived Scarring Promotes Recovery after Spinal Cord Injury. Cell 173, 153-165.e122, doi:10.1016 / j.cell.2018.02.004 (2018). 38 Brennan, F. H., Hall, J. C. E., Guan, Z. & P.G., P. Microglia limit lesion expansion and promote functional recovery after spinal cord injury in mice. Cold Spring Harbor Laboratory, doi:https: / / doi.org / 10.1101 / 410258 (2018). 39 Brenner, D. S., Golden, J. P. & Gereau, R. W. A novel behavioral assay for measuring cold sensation in mice. PLoS One 7, e39765, doi:10.1371 / journal.pone.0039765 (2012). 40 Oudega, M. Molecular and cellular mechanisms underlying the role of blood vessels in spinal cord injury and repair. Cell Tissue Res 349, 269 - 288, doi:10.1007 / s00441-012-1440-6 (2012). 41 Zhu, Y. et al. Hematogenous macrophage depletion reduces the fibrotic scar and increases axonal growth after spinal cord injury. Neurobiol Dis 74, 114 - 125, doi:10.1016 / j.nbd.2014.10.024 (2015). 42 Khennouf, L. et al. Active role of capillary pericytes during stimulation-induced activity and spreading depolarization. Brain 141, 2032-2046, doi:10.1093 / brain / awy143 (2018). 43 Li, Y. et al. Pericytes impair capillary blood flow and motor function after chronic spinal cord injury. Nat Med 23, 733-741, doi:10.1038 / nm.4331 (2017).

Claims

Claim 1 A pharmaceutical composition of eletriptan hydrobromide for use in improving locomotor function after spinal cord injury. Claim 2 The pharmaceutical composition of eletriptan hydrobromide for use according to claim 1, wherein the spinal cord injury is in the acute or subacute phase. Claim 3 The pharmaceutical composition of eletriptan hydrobromide for use according to claim 1 or 2, wherein the eletriptan hydrobromide is administered starting from the first hour after injury.

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