TREATMENT OF SYMPTOMS OF TRAUMATIC BRAIN INJURY

Abstract

Method of treating or reducing a symptom of traumatic brain injury (TBI) in a subject, comprising administering to the subject a therapeutically-effective amount of botulinum neurotoxin. Composition for use in treating or reducing a symptom of TBI in a subject comprising botulinum neurotoxin. Computer system programmed to receive information related to a subject's response to administration of botulinum neurotoxin, store that response in a database, and transmit the response to a medical practitioner. Non-transitory computer-readable storage medium storing instructions that, when executed by a computer system, causes the computer system to perform the aforementioned steps.

Claims

1. A method for treating a symptom of traumatic brain injury (TBI), the method comprising administering a therapeutically-effective amount of a botulinum neurotoxin to a subject diagnosed to be in need thereof.

2. A composition for use in a method of treating a symptom of traumatic brain injury, the composition comprising a therapeutically-effective amount of a botulinum neurotoxin.

3. The method or composition for use according to claim 1 or claim 2, wherein the administration is local administration to the subject's head.

4. The method or composition for use according to any one of the preceding claims, wherein the botulinum neurotoxin is administered in a single dose.

5. The method or composition for use according any one of the preceding claims, wherein the botulinum neurotoxin is administered subcutaneously.

6. The method or composition for use according to any one of the preceding claims, wherein the botulinum neurotoxin is administered pre-injury.

7. The method or composition for use according to any one of the preceding claims, wherein the botulinum neurotoxin is administered up to 4 days pre-injury.

8. The method or composition for use according to any one of the preceding claims, wherein the botulinum neurotoxin is administered between 1-3 days pre-injury; optionally wherein the botulinum neurotoxin is administered 3 days pre-injury.

9. The method or use according to any one of the preceding claims, wherein the treatment is effected at a site that is peripheral to the site of administration; preferably wherein the administration is local administration to the subject's head.

10. The method or composition for use according to any one of the preceding claims, wherein the botulinum neurotoxin is administered within 48 hours following injury; and/or wherein the botulinum neurotoxin is administered within 2 hours following injury; and/or wherein the botulinum neurotoxin is administered immediately following injury.

11. The method or composition for use according to any one of the preceding claims, wherein the botulinum neurotoxin is administered at least about an hour following injury.

12. The method or composition for use according to any one of the preceding claims, wherein: (a) the botulinum neurotoxin is administered at least about 2 hours following injury; (b) the botulinum neurotoxin is administered at least about 6 hours following injury; (c) the botulinum neurotoxin is administered at least about 12 hours following injury; (d) the botulinum neurotoxin is administered at least about 24 hours following injury; (e) the botulinum neurotoxin is administered at least about 48 hours following injury; and/or (f) the botulinum neurotoxin is administered 2 to 72 hours following injury.

13. The method or composition for use according to any one of the preceding claims, wherein the symptom is one or more selected from temporary loss of consciousness, headache, sensitivity to light, sensitivity to noise, nausea, vomiting, lack of motor coordination, dizziness, light headedness, difficulty balancing, blurred vision or tired eyes, ringing in the ears, bad taste in the mouth, fatigue or lethargy, changes in sleep patterns, abnormality in learning and memory, impulsivity, emotional reactivity, mood swings, depression, or emotional blunting; optionally wherein the symptom is a headache or sensitivity to light.

14. The method or use according to any one of the preceding claims, wherein the symptom is chronic sensitivity to light, sound, stress and/or fatigue.

15. The method or use according to any one of the preceding claims, wherein the symptom is chronic sensitivity to light.

16. The method or composition for use according to any one of the preceding claims, wherein the amount of botulinum neurotoxin administered is between 20-70 units, optionally wherein the amount of botulinum neurotoxin administered is about 50 units.

17. The method or composition for use according to any one of the preceding claims, wherein: (a) the amount of botulinum neurotoxin administered is less than about 30 units per kilogram of the total body weight of the subject; (b) the botulinum neurotoxin is administered parenterally; (c) the botulinum neurotoxin is administered through a suture of the skull of the subject; (d) the botulinum neurotoxin has at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to the amino acid sequence of a botulinum neurotoxin of type A; or type A1 or type A2; or type A3; or type A4; or type B; or ty[e C; or type C1 or type D, or type E; or type F; or type G; (e) the botulinum neurotoxin is: botulinum neurotoxin type A; botulinum neurotoxin type A1 botulinum neurotoxin type A2; botulinum neurotoxin type A3; botulinum neurotoxin type A4; botulinum neurotoxin type B; botulinum neurotoxin type C; botulinum neurotoxin type C1 botulinum neurotoxin type D, botulinum neurotoxin type E; botulinum neurotoxin type F; or botulinum neurotoxin type G, optionally wherein the botulinum neurotoxin is botulinum neurotoxin type A; (f) the botulinum neurotoxin is DYSPORT®; (g) the method further comprises measuring the severity of the symptom of the traumatic brain injury, optionally wherein the amount of botulinum neurotoxin administered is determined based in part on the level of severity of the symptom of traumatic brain injury; (h) the method further comprises measuring the severity of the traumatic brain injury, optionally wherein the amount of botulinum neurotoxin administered is determined based in part on the level of severity of the traumatic brain injury; (i) the amount of botulinum neurotoxin administered is determined based in part on the weight of the subject; (j) the amount of botulinum neurotoxin administered is determined based in part on whether the subject has previously experienced traumatic brain injury; (k) the amount of botulinum neurotoxin administered is determined based in part on the amount of time that has elapsed following injury; (l) the composition further comprises a diluent, optionally wherein the diluent comprises a lyophilized powder, and optionally wherein the composition is formed by reconstituting lyophilized powder comprising botulinum neurotoxin; and/or (m) the composition further comprises an excipient, optionally wherein the excipient is: a filler, a binder, a disintegrant, an anti-adherent, a solvent, a buffering agent, a preservative, or a humectant.

18. The method or composition for use according to any one of the preceding claims, the method further comprising: (a) measuring the subject's response to the administration of botulinum toxin; and/or (b) measuring a dosing regimen for botulinum neurotoxin based on the subject's response to a previous administration thereof, optionally wherein a subsequent administration of botulinum neurotoxin is based on the dosing regimen determined.

19. The method or composition for use according to any one of the preceding claims, further comprising recording the subject's response to an administration of botulinum neurotoxin, optionally wherein the subject's response is recorded on a first computer device and is accessed on a second computer device.

20. The method or composition for use according to claim 19, wherein the subject's response is recorded into a software program that is configured to receive the information, and/or further comprising storing the subject's response in a database.

21. The method or composition for use according to any one of the preceding claims, the method further comprising determining whether the subject has or is likely to develop a symptom of traumatic brain injury before administering the botulinum neurotoxin.

22. The method or composition for use according to claim 21, wherein the step of determining whether the subject has or is likely to develop a symptom of traumatic brain injury comprises measuring the level of a biomarker in the blood of the subject, the level being indicative of whether the subject has or is likely to develop the symptoms of traumatic brain injury, optionally wherein the biomarker is: ubiquitin carboxy-terminal hydrolase L1, glial fibrillary acidic protein and calcitonin gene related peptide, TNF-α, or NF-L.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0244] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

[0245] FIG. 1 shows a schematic of the experimental time-line for the pilot study (green), the originally proposed main study (exp. 1-3, red) and additional experiments (exp. 5-6, blue).

[0246] FIG. 2 shows Dysport administered 2 hours after mTBI prevents acute mTBI-induced allodynia and latent BLS-induced allodynia. After measuring baseline periorbital and hindpaw responses mice were given mTBI or sham injury and 2 h following the injury/sham-injury were administered saline or Dysport (0.5 U). (A) Periorbital and (D) hindpaw responses were measured in mice periodically over 15 days after mTBI or sham injury. At day 15, all mice were exposed to BLS and (B) periorbital and (E) hindpaw tactile allodynia was measured over a 5-h time course. At day 28, all mice were again exposed to BLS and (C) periorbital and (F) hindpaw tactile allodynia was measured over a 3-h time course. Two-way repeated measures ANOVA with Sidak's multiple comparison test shows significantly elevated frequency of tactile responses in the mTBI/saline group compared to sham/saline mice at times indicated by red asterisk (*), and significantly higher responses in the mTBI/0.5 U group compared to sham/0.5 U mice indicated by blue asterisk (*). Allodynia was significantly reduced in the mTBI/0.5 U group compared to mTBI/saline group at times indicated by blue hashtag (#). Data are plotted as means and SEM; N=5-10 mice/group.

[0247] FIG. 3 shows Dysport administered 2 hours after mTBI prevents acute mTBI-induced allodynia and latent BLS-induced allodynia. After measuring baseline periorbital and hindpaw responses mice were given mTBI injury and 2 h following the injury mice were administered saline or Dysport (0.5 U). (A) Periorbital and (D) hindpaw responses were measured in mice periodically over 14 days. At day 14/15, all mice were exposed to BLS and (B) periorbital and (E) hindpaw tactile allodynia was measured over a 5-h time course. At day 26/27, all mice were again exposed to BLS and (C) periorbital and (F) hindpaw tactile allodynia was measured over a 5-h time course. Two-way repeated measures ANOVA with Sidak's multiple comparison test shows significantly reduced frequency of tactile responses in the mTBI/0.5 U group compared to mTBI/saline group at times indicated by blue hashtag (#). Data are plotted as means and SEM; N=8 mice/group.

[0248] FIG. 4 shows Dysport administered 2 hours after mTBI prevents acute mTBI-induced allodynia and latent BLS-induced allodynia. After measuring baseline periorbital and hindpaw responses mice were given mTBI injury and 2 h following the injury mice were administered saline or Dysport (0.5 U). (A) Periorbital and (D) hindpaw responses were measured in mice periodically over 14 days. At day 14/15, all mice were exposed to BLS and (B) periorbital and (E) hindpaw tactile allodynia was measured over a 5-h time course. At day 26/27, all mice were again exposed to BLS and (C) periorbital and (F) hindpaw tactile allodynia was measured over a 5-h time course. Two-way repeated measures ANOVA with Sidak's multiple comparison test shows significantly reduced frequency of tactile responses in the mTBI/0.5 U group compared to mTBI/saline group at times indicated by blue hashtag (#). Data are plotted as means and SEM; N=8 mice/group.

[0249] FIG. 5 shows levels of CGRP in blood. (A) Blood was collected from naïve (BL) mice and at indicated times after mTBI injury and CGRP levels in plasma were analyzed. One way ANOVA shows no significant difference between groups. N=4 mice/group. (B) Mice were given mTBI or sham injury, development of allodynia in mTBI but not sham mice was verified. On day 14 mice were exposed to BLS or no stress as indicated and blood was collected 30 minutes after BLS. CGRP levels in plasma were analyzed. One way ANOVA shows no significant difference between groups. N=3-4 mice/group. (C) Mice were given mTBI or sham injury, development of allodynia in mTBI but not sham mice was verified. On day 14 mice were exposed to BLS or no stress as indicated and blood was collected 4 hours after BLS. CGRP levels in plasma were analyzed. One way ANOVA with Tukey's post test shows significant difference between Sham/No BLS and Sham/BLS groups. N=3 mice/group. (D) Mice were given mTBI or sham injury, and were administered saline or Dysport 2 h post injury. Development of allodynia in the mTBI/saline group and decreased allodynia in the other groups was verified (See FIG. 2). On day 15, all mice were exposed to BLS. On day 28 all mice were again exposed to BLS and blood was collected 2-3 hours after BLS as indicated. CGRP levels in plasma were analyzed. One way ANOVA with Tukey's post test shows significant difference between Sham/saline and mTBI/saline groups. N=8 mice/group.

[0250] FIG. 6 shows Dysport administered 24 hours after mTBI reverses acute mTBI-induced allodynia and prevents latent BLS-induced allodynia. After measuring baseline periorbital and hindpaw responses, mice were given mTBI or sham injury and (A) periorbital and (D) hindpaw tactile responses were measured 24 h later. Following the Day 1 measurements, mice were administered saline or Dysport (0.5 U) and tactile responses were measured periodically over 13 days after mTBI or sham injury. At day 13, all mice were exposed to BLS and (B) periorbital and (E) hindpaw tactile allodynia was measured over a 5-h time course. At day 17, all mice were again exposed to BLS and (C) periorbital and (F) hindpaw tactile allodynia was measured over a 5-h time course. Two-way repeated measures ANOVA with Sidak's multiple comparison test shows significantly elevated frequency of tactile responses in the mTBI/saline group compared to sham/saline mice at times indicated by red asterisk (*), and significantly higher responses in the mTBI/0.5 U group compared to sham/0.5 U mice indicated by blue asterisk (*). Allodynia was significantly reduced in the mTBI/0.5 U group compared to mTBI/saline group at times indicated by blue hashtag (#). Data are plotted as means and SEM; N=5-10 mice/group.

[0251] FIG. 7 shows Dysport administered on day 11 after mTBI (72 hours before BLS) prevents latent BLS-induced periorbital allodynia. Baseline periorbital (A) and hindpaw (B) responses were measured; mice were then given mTBI or sham injury and tactile responses were measured at Days 1, 4, 6 and 11 after the injury. Following the Day 11 measurements, mice were administered saline or Dysport (0.5 U) and tactile responses were measured on Day 14. At day 14, all mice were exposed to BLS and periorbital (C) and hindpaw (D) tactile allodynia was measured over a 5-h time course. Two-way repeated measures ANOVA with Sidak's multiple comparison test shows significantly elevated frequency of tactile responses in the mTBI/saline group compared to sham/saline mice at times indicated by red asterisk (*), and significantly higher responses in the mTBI/0.5 U group compared to sham/0.5 U mice indicated by blue asterisk (*). Allodynia was significantly different between the mTBI/0.5 U and mTBI/saline groups at times indicated by blue hashtag (#). Data are plotted as means and SEM; N=4-9 mice/group.

[0252] FIG. 8 shows Dysport administered 72 hours before mTBI prevents acute mTBI-induced allodynia and latent BLS-induced allodynia. Mice were pre-treated with saline or Dysport (0.5 U); 3 days (72 h) later (A) periorbital and (C) hindpaw responses were measured and mice were given mTBI injury and tactile responses were assessed periodically over 12 days. At day 12, all mice were exposed to BLS and (B) periorbital and (D) hindpaw tactile allodynia was measured over a 5-h time course. Two-way repeated measures ANOVA with Sidak's multiple comparison test shows significantly reduced frequency of tactile responses in the mTBI/Dysport group compared to mTBI/saline group at times indicated by blue hashtag (#). Data are plotted as means and SEM; N=4 saline and 9 Dysport mice.

[0253] FIG. 9 shows Dysport administered 72 hours after mTBI partially reverses acute mTBI-induced periorbital allodynia and prevents latent BLS-induced periorbital allodynia. Baseline periorbital (A) and hindpaw (C) responses were measured; mice were then given mTBI or sham injury and tactile responses were measured at Day 1 and Day 3 after the injury. Following the Day 3 measurements, mice were administered saline or Dysport (0.5 U) and tactile responses were measured periodically over 14 days after mTBI or sham injury. At day 14, all mice were exposed to BLS and (B) periorbital and (D) hindpaw tactile allodynia was measured over a 5-h time course. Two-way repeated measures ANOVA with Sidak's multiple comparison test shows significantly elevated frequency of tactile responses in the mTBI/saline group compared to sham/saline mice at times indicated by red asterisk (*), and significantly higher responses in the mTBI/0.5 U group compared to sham/0.5 U mice indicated by blue asterisk (*). Allodynia was significantly reduced in the mTBI/0.5 U group compared to mTBI/saline group at times indicated by blue hashtag (#). Data are plotted as means and SEM; N=8-10 mice/group.

[0254] FIG. 10 A-F depicts the effect of DYSPORT® (0.5 units/mouse) treatment in mice when DYSPORT® is administered immediately after mild traumatic brain injury (mTBI). Facial allodynia (FIG. 10A) and hindpaw allodynia (FIG. 10B) are measured 0, 3, 5, 7, 10, 12, and 14 days post-injury. Facial alloydynia (FIG. 100) and hindpaw allodynia (FIG. 10D) were measured again for 5 hours following bright light stress (BLS) on day 15 post-injury. Facial alloydynia (FIG. 10E) and hindpaw allodynia (FIG. 10F) were also measured again for 5 hours following further exposure to BLS on day 32 post-injury. Asterisks denote statistically significant differences between the TBI/saline and sham/saline groups and crosses denote statistically significant differences between the TBI/saline and TBI/0.5 U DYSPORT® groups (p<0.05).

[0255] FIG. 11 A-F depicts the effect of DYSPORT® (0.5 units/mouse) treatment when DYSPORT® is administered two hours after mild traumatic brain injury (mTBI). Facial allodynia (FIG. 11A) and hindpaw allodynia (FIG. 11B) are measured 0, 1, 2, 3, 8, 10, 11, 15 days post-injury. Facial alloydynia (FIG. 11C) and hindpaw allodynia (FIG. 11D) were measured again for 5 hours following bright light stress (BLS) on day 15 post-injury. Facial alloydynia (FIG. 11E) and hindpaw allodynia (FIG. 11F) were also measured again for 3 hours following further exposure to BLS on day 28 post-injury. Asterisks denote statistically significant differences between the TBI/saline and sham/saline groups and crosses denote statistically significant differences between the TBI/saline and TBI/0.5 U DYSPORT® groups (p<0.05).

[0256] FIG. 12 depicts the effects of DYSPORT® (0.5 U/mouse) administration two hours after mild traumatic brain injury (mTBI) on blood concentrations of CGRP 2 or 3 hours after exposure to bright light stress (BLS) on post-TBI day 28. *p<0.05 in comparison to the corresponding TBI/saline group.

[0257] FIG. 13 depicts the effect of DYSPORT® (0.5 units/mouse) treatment when DYSPORT® is administered 24 hours after mild traumatic brain injury (mTBI). Facial allodynia (FIG. 13A) and hindpaw allodynia (FIG. 13B) are measured 0, 1, 2, 3, 6, 8, and 13 days post-injury. Facial alloydynia (FIG. 13C) and hindpaw allodynia (FIG. 13D) were measured again for 5 hours following bright light stress (BLS) on day 15 post-injury. Facial alloydynia (FIG. 12E) and hindpaw allodynia (FIG. 12F) were also measured again for 5 hours following further exposure to BLS on day 17 post-injury.

EXAMPLES

[0258] The invention will now be described, by way of example only, with reference to the following Examples.

SUMMARY

[0259] Presented here are the results of a study in a preclinical model of TBI (e.g. post-traumatic headache). Three main experiments were conducted to determine if acute administration of BoNT/A (Dysport) after a single mTBI blocks the expression of mTBI-induced cephalic pain (Aim 1) and to characterize the possible efficacy of Dysport treatment after the initial cephalic pain has resolved, but latent sensitization to environmental stressors demonstrated as post traumatic headache (PTH) is still present (Aim 2).

[0260] Further experiments tested if pre-treatment of mice with Dysport three days (72 h) prior to mTBI (Exp. 5) can prevent pain symptoms and 2) if treatment with Dysport 72 h after mTBI can reverse established mTBI-induced allodynia in mTBI animals (Exp 6). These experiments have generated important insights into the neural mechanisms of mTBI-associated allodynia and the mechanisms of anti-allodynic actions of Dysport. Experiment 5 established an efficacy of Dysport as a preventive treatment during activities where mTBI injuries are likely, including competitive sports or military mTBI injuries (e.g., falls). Experiment 6 widened a therapeutic window for Dysport efficacy after the mTBI.

INTRODUCTION

[0261] Traumatic brain injury (TBI) is a serious public health issue that can result from sports or accidents in the general population or from combat activities in military personnel. Sports-related (e.g., football, soccer, ice hockey) TBI is very common and negatively impacts the lives of youth, collegiate and professional male and female athletes. Such TBI is commonly described as mild (mTBI). Mild TBI exhibits little or no apparent damage but is thought to cause direct injury to the skull, brain, meninges, vasculature, or neck with different severity and manifestations, which can result in psychiatric, cognitive, and physiological disorders later in life. A complicating factor is that successive injuries, including sub-concussive ones, may synergize with previous ones resulting in collectively greater effects on neurological and cognitive consequences.

[0262] Following mTBI, patients can experience a sustained and unrelenting headache that often has migrainous features including pain, and hypersensitivity to light, sound and touch (i.e., photophobia, phonophobia and allodynia). This post-traumatic headache (PTH) is debilitating and can be continuous or episodic with initiation of the headache linked to life events including exercise and most significantly, stress. These events are common as athletes return to competition, the classroom or work environments.

[0263] At present, therapies for TBI and for PTH have not been clinically validated. A critical impediment to the discovery of effective and well-tolerated therapeutics for PTH has been a relative lack of understanding of the molecular, biochemical and physiological mechanisms of this disorder.

[0264] The inventors utilised a mouse model of single or repetitive mild TBI (rmTBI) injury where mice experience mild, closed head injury without resistance and without observable extracranial damage. This model produces symptoms that are consistent with migraine-like pain that is commonly experienced following single or repeated mild concussive injuries. This model has several important advantages: (a) the forces can be controlled without producing observable damage to the skull and to the underlying brain tissues; (b) the model allows for the assessment of the consequences of repeated head impacts modeling the repetitive nature of concussive injuries experienced by many athletes; and (c) this model closely mimics the most critical factors in producing mild concussive brain injuries, which are high velocity impact and rapid head acceleration and rotation. These studies have shown that this approach can mimic PTH for up to 60 days following a single mTBI. In this model, mice previously exposed to a single mTBI demonstrate hypersensitivity to stress and to light, consistent with clinical experience. The availability of this model allows assessment of potential efficacy of pharmacological interventions in PTH.

[0265] Objectives: [0266] Determine if acute administration of Dysport after a single mTBI blocks the expression of mTBI-induced cephalic pain and prevents development of latent sensitization revealed by stress-induced reinstatement of cephalic allodynia. [0267] Characterize the efficacy of Dysport treatment after the initial cephalic allodynia has resolved, but latent sensitization to environmental stressors demonstrated as post traumatic headache (PTH) is still present.

Materials and Methods:

Animals

[0268] Male, C57Bl/6J adult mice weighing 17-22 grams were housed five to a cage on a 14/10-hour light/dark cycle (5 am-7 pm lights on) with food and water ad libitum. Experiments were conducted during the light cycle phase. All experiments were performed in accordance with the ARRIVE reporting guidelines and with the approval of the Mayo Clinic Institutional Animal Care and Use Committee and Teva Pharmaceuticals. Group size requirements to obtain significance at the α=0.05 and statistical power 0.9 were determined from previous experiments using power analysis.

Induction of Mild Traumatic Brain Injury

[0269] The mouse model of experimental mTBI was adapted from Kane et al (J Neurosci Methods 2012; 203: 41-49. DOI: 10.1016/j.jneumeth.2011.09.003). Briefly, mice were lightly anaesthetized with 3% isofluorane and then laid with their ventral surface on an elevated tissue paper stage capable of supporting body weight with the head unrestrained. The paper stage was situated over a plexiglass apparatus with a soft sponge at the bottom. A metal guide tube was directed to the top of the mouse skull between the ears to ensure standardized placement of the weighted drop. The weight (100 g), released from a height of 94 cm, results in a concussive impact to the head, pushing the mouse through the tissue paper and flipping it down to land on the soft sponge. All mTBI mice in this study experienced both rotational and linear head forces. After impact the weight falls through the apparatus thereby avoiding a second impact with the animal. Following the procedure, mice were returned to their home cages and allowed to recover. Sham animals were anaesthetized and placed on the tissue paper stage but did not undergo the weighted drop or rotational flip. All mice awoke within 5 minutes of the procedure and were observed to confirm that no visual signs of neurological complications arose. Animals remained grouped in their same cohorts following the procedure.

Bright Light Stress (BLS) Challenge

[0270] Unrestrained mice were exposed to BLS induced by LED strips (1000 lux output) that were placed on both sides of their home Plexiglass cages for 15 minutes. The parameters of BLS protocol were modified from our previous studies in Sprague Dawley rats to produce mild stress that arises from endogenous mechanisms and does not elicit significant CA in naïve or sham mice.

Dysport Administration

[0271] Dysport was provided by Ipsen and was diluted with saline to concentration of 0.5 U/50 μl. One injection of 0.5 U/50 μl Dysport or vehicle (saline) was administered subcutaneously along the lambdoid suture at indicated times following the mTBI or sham injury. This dose and route of administration was well tolerated and the animals did not show any visible adverse effects.

Behavioral Assessment of Cutaneous Allodynia

[0272] Prior to baseline behavioral assessment, mice were placed individually in elevated Plexiglass chambers with mesh flooring, located in a quiet, non-trafficked area and allowed to acclimate for 3 days for 2 hours. Starting on day 0 (pre-mTBI baseline) and periodically thereafter, cephalic (periorbital) and extracephalic (hindpaw) allodynia was measured in the same mice following a 2-hour acclimation period. For assessment of periorbital allodynia, a 0.4 g (3.61) von Frey filament was applied with just enough pressure to cause the filament to display a slight arch to the periorbital region 10 times with a space of 20-30 sec between each application. A positive response was considered swiping of the face, shaking of the head, and/or turning away from the stimuli. Running away or rearing up were not considered as positive responses. For assessment of hindpaw allodynia, a 0.6 g (3.84) von Frey filament was applied with just enough pressure to cause the filament to display a slight arch to the left hindpaw 10 times with a space of 20-30 sec between each application. Sharp withdrawal of the paw, shaking and/or licking the paw were considered a positive response, while lifting of the paw with the filament or running away were not. Frequency response was calculated as [(number of positive responses/10)*100%]. Following mTBI induction, CA was measured for a time course of up to 14 days and in response to Dysport and control/vehicle administration.

CGRP Measurements

[0273] Concentrations of CGRP in plasma were measured with commercially purchased mouse enzyme-linked immunosorbent assay kits (Cayman Chemical, Ann Harbor, Mich., USA). Mice were anesthetized and whole blood was collected from the heart at indicated times and stored in ethylenediaminetetraacetic acid-coated vials. Blood was centrifuged at 3000 rpm for 20 minutes at 4° C. in order to separate the plasma. Plasma was stored at −80° C. and analyzed within 1 week of collection.

Study Design

[0274] Aim 1: Determine if acute administration of Dysport after a single mTBI prevents the expression of mTBI-induced cephalic pain and latent sensitization revealed by stress-induced allodynia. Efficacy of Dysport was assessed at four different acute time-points following mTBI: 1) immediately after mTBI (Exp. 5); 2) 2 h after mTBI (Exp. 1); 3) 24 h after mTBI (Exp. 2); 4) 72 h after mTBI (Exp. 6).

[0275] Aim 2: Characterize the possible efficacy of Dysport treatment after the initial cephalic pain has resolved, but latent sensitization to environmental stressors demonstrated as post traumatic headache (PTH) is present (Exp. 3).

Example 1—Experiments 1-7

Experiment 1 (Outline of Experiment):

[0276] Dysport (0.5 U/mouse) was administered at 2 hours following the mTBI.

[0277] A. Baseline cutaneous allodynia was determined for male, C57/Bl6 mice using calibrated von Frey filaments. Following mTBI and Dysport administration, cutaneous allodynia was followed intermittently for 14 days.

[0278] B. On day 15, mice were exposed to environmental stress (bright light stress, BLS) for 15 min and cutaneous allodynia was assessed over a 5 h time period following BLS.

[0279] C. On day 28 the mice were again exposed to BLS for 15 min followed by assessment of cutaneous allodynia over a 4 h time period. On day 29, the animals were exposed to the same BLS protocol, blood was collected at the peak of post BLS cutaneous allodynia. Plasma was prepared and used to estimate levels of CGRP using CGRP EIA kit (Phoenix Pharmaceuticals, Inc.).

[0280] D. In a separate cohort of animals exposed to mTBI and Dysport, blood was collected 4 h and 26 h post mTBI (i.e.; 2 h and 24 h post Dysport) and the levels of CGRP in plasma were analyzed.

Experiment 2 (Outline of Experiment):

[0281] Dysport (0.5 U/mouse) was administered at 24 h following the mTBI. Experiments 2A-D was performed in the same manner as the Experiment 1 (1A-D), but Dysport was administered 24 h after induction of mTBI when neuronal changes (increased CGRP levels) and cutaneous allodynia are likely to be developed.

Experiment 3 (Outline of Experiment):

[0282] Dysport (0.5 U/mouse) was administered at day 11 following the mTBI, when von Frey responses have returned to baseline and we investigated if on day 14, BLS-induced allodynia would be blocked.

[0283] A. On day 14 following the TBI, cutaneous allodynia will be determined before BLS and during a 5 h time period following BLS. BoNT-A will be administered at the end of the 4 h period.

[0284] B. On day 21, cutaneous allodynia will be measured before BLS and during a 5 h time period following BLS.

[0285] C. The following day (day 22), the animals will be exposed to the same BLS protocol, blood will be collected at the peak of post BLS cutaneous allodynia (determined on day 21) and the levels of CGRP will be determined.

Experiment 4 (Outline of Experiment):

[0286] Dysport will be administered at 2 h following the TBI using subcutaneous administration route (results omitted). The dose, volume, number of injections and the site will be provided by Ipsen.

[0287] A. Baseline cutaneous allodynia will be determined for all mice using calibrated von Frey filaments. Following TBI and BoNT-A administration, cutaneous allodynia will be followed daily (work days) for 14 days.

[0288] B. At day 15 we will expose the mice to environmental stress (bright light stress, BLS) for 15 min and assess cutaneous allodynia over a 4 h time period following BLS.

Experiment 5 (Outline of Experiment):

[0289] Dysport (0.5 U/mouse) was administered 3 days (72 h) before the mTBI.

[0290] A. Baseline cutaneous allodynia was determined and mice were exposed to mTBI or sham injury. Following mTBI, cutaneous allodynia was followed intermittently for 12 days.

[0291] B. At day 12, mice were exposed to environmental stress (bright light stress, BLS) for 15 min and cutaneous allodynia was assessed over a 5 h time period following BLS.

Experiment 6 (Outline of Experiment):

[0292] Dysport (0.5 U/mouse) was administered immediately (at 0 hours) following the mTBI (before the animals wake up from anesthesia) (results omitted).

[0293] A. Baseline cutaneous allodynia was determined for male, C57/Bl6 mice using calibrated von Frey filaments. Following mTBI and Dysport administration, cutaneous allodynia was followed intermittently for 14 days.

[0294] B. At day 15, mice were exposed to environmental stress (bright light stress, BLS) for 15 min and cutaneous allodynia was assessed over a 5 h time period following BLS.

[0295] C. On day 32 the mice were again exposed to BLS for 15 min followed by assessment of cutaneous allodynia over a 5 h time period.

Experiment 7 (Outline of Experiment):

[0296] Dysport (0.5 U/mouse) was administered at 72 h following the mTBI.

[0297] A. In these experiments we first confirmed the development of acute allodynia on days 1-3 after mTBI and then we treated the animals with dysport to investigate if acute allodynia can be reversed.

[0298] B. At day 15, mice were exposed to environmental stress (bright light stress, BLS) for 15 min and cutaneous allodynia was assessed over a 5 h time period following BLS.

Results of Experiments 1-7

Experiment 1 (Results)

[0299] Exp 1A-C (FIGS. 2, 3, 4): Three independent experiments were performed to demonstrate that Dysport administration at 2 h post mTBI significantly reduces mTBI-induced allodynia and also prevents BLS-induced allodynia at 2-4 weeks post mTBI (FIGS. 2, 3, 4). The results of all three experiments are consistent and each experiment shows statistically significant effects of mTBI on allodynia as well as statistically significant reduction of allodynia with Dysport treatment. Significant effects of mTBI and reduction by Dysport were also observed at 2 weeks (FIGS. 2, 3, 4) and 4 weeks (FIGS. 2, 3) after mTBI in stress-induced allodynia. The findings demonstrate that one administration of Dysport early after a mTBI event prevents mTBI-iduced pain as well as prevent central sensitization that leads to chronification of pain and development of persistent post-traumatic headache.

[0300] Exp 1D (FIG. 5): We did not detect significant CGRP release in mTBI mice at 4 h and 24 h after mTBI, thus, the effects of Dysport on CGRP levels were not evaluated (FIG. 5 A). However, our pilot studies in mice exposed to BLS on day 14 show that BLS promotes CGRP release at 4 h (but not 30 min) post BLS (FIGS. 5 B, C). We therefore evaluated if Dysport administered at 2 h post mTBI could prevent BLS-induced CGRP release (FIG. 5 D). The results of this study show that stress (BLS) leads to increased CGRP blood levels in mTBI, but not sham, mice and Dysport administration at 2 h post mTBI may prevent the CGRP increase.

Experiment 2 (Results)

[0301] Exp 2A-C (FIG. 6): FIG. 6 shows the experiments with Dysport administered 24 h post mTBI. Importantly, we demonstrated development of both periorbital and hindpaw allodynia at 24 h post mTBI prior to Dysport or saline administration. Subsequent Dysport administration resulted in significant reduction of both periorbital and hindpaw allodynia in Dysport—but not saline—treated mTBI mice. The beneficial effects of Dysport were also evident on day 13, when all mice were exposed to bright lights. While BLS reinstated allodynia in saline treated mTBI mice, Dysport treated mTBI mice had significantly diminished allodynia. Re-exposure of the mice to another BLS episode again on day 17 resulted in periorbital and hindpaw allodynia in saline treated mTBI mice. Dysport treated mice showed reduced periorbital, but not hindpaw allodynia. The findings demonstrate that the window of the efficacy of Dysport in preventing mTBI-induced allodynia extends at least 24 h after a mTBI event (in mice). Thus, one administration of Dysport 24 h after mTBI can be used to prevent mTBI-iduced pain as well as prevent central sensitization that leads to chronification of pain and development of persistent post-traumatic headache.

Experiment 3:

[0302] Exp 3A-C (FIG. 7): The experiment 3 was designed to evaluate if Dysport can block BLS-induced allodynia when administered on day 11, 3 days (72 h) before BLS.

[0303] Following mTBI or sham injury, mTBI animals developed allodynia that resolved by day 11. Dysport was then administered on day 11 post mTBI and 72 hours before BLS. Dysport prevented latent sensitization revealed by BLS-induced periorbital allodynia. The sham and mTBI animals that previously received saline were given Dysport on day 25 and were again exposed to BLS on day 27.

[0304] Overall, results of the experiment 3 demonstate that even after initial acute mTBI-induced allodynia subsides, Dysport is effective in blocking stress-induced allodynia. Thus, Dysport can be used to be effective in patients with persistent post-traumatic headache.

[0305] Results of the experiment 3 demonstrate that even after initial acute mTBI-induced allodynia subsides, Dysport is effective in blocking stress-induced allodynia. Thus, Dysport is effective in subjects with persistent post-traumatic headache.

Experiment 5 (Results)

[0306] Exp. 5 (FIG. 8): Dysport was administered 3 days (72 hours) prior to mTBI to establish a possible efficacy of Dysport as a preventive treatment during activities where mTBI injuries are likely, including competitive sports or military mTBI injuries (e.g., falls). FIG. 11 shows that Dysport administered 72 hours before mTBI effectively prevented both acute mTBI-induced allodynia and latent BLS-induced allodynia. Thus, Dysport can be used several days prior to a high risk event to prevent the consequences of a potential head injury.

Experiment 7 (Results)

[0307] Exp 6 (FIG. 9): To determine if the efficacy window may be extended even beyond 24 h after mTBI, we performed another experiment when Dysport was administered 72 h post mTBI. Development of both periorbital and hindpaw allodynia at 1 day and 3 days post mTBI was verified. Then, Dysport or saline were administered. Compared to saline-treated mTBI mice, Dysport-treated mTBI mice showed significant reduction in periorbital but not hindpaw allodynia. Similarly, following the exposure to BLS, Dysport treated mice showed reduced periorbital but not hindpaw allodynia. Thus, the Dysport efficacy window for the treatment of acute allodynia and stress-induced allodynia is extended up to 72 h post mTBI. Together with the results of Exp. 3 (Dysport administration an day 11 at 72 h before BLS), these results suggest that central sensitization develops 3 days after mTBI resulting in all body allodynia and stress-induced allodynia. However, Dysport is able to block periorbital allodynia.

Example 2—Further Tests of BoNT to Treat a Symptom of TBI

[0308] The efficacy of botulinum neurotoxin, when administered immediately after mild traumatic brain injury (mTBI), in treating the mTBI was evaluated in mice.

[0309] The mice were subjected to either mTBI or sham injury on day 0. DYSPORT® (resuspended in 0.9% NaCl from a lyophilisate) was infused through the sagittal suture of each mouse at 0.5 U/mouse immediately following injury.

[0310] Facial allodynia and hindpaw allodynia were measured 0, 3, 5, 7, 10, 12, and 14 days post-injury. On day 15, all mice were exposed to bright light stress (BLS) for 15 minutes and facial allodynia and hindpaw allodynia were measured for 5 hours following BLS. On day 32, all mice were exposed again to BLS for 15 minutes and facial allodynia and hindpaw allodynia were again measured for 5 hours following BLS. The results are depicted in FIG. 10.

[0311] The treatment prevented the onset of acute cephalic and hindpaw allodynia and caused a long-term prevention of sensitization to bright light stress-induced allodynia.

[0312] A single mTBI injury resulted in time dependent tactile facial and hindpaw allodynia after mTBI. The administration of 0.5 U DYSPORT® 24 hours immediately following mTBI reversed tactile facial and hindpaw allodynia. Exposure to BLS on Day 15 post-mTBI promoted tactile allodynia in mTBI-injured mice but not sham-injured mice. Allodynia was significantly attenuated in mTBI-injured mice treated with 0.5 U DYSPORT® immediately after mTBI. The second exposure of the mice to BLS promoted tactile allodynia in mTBI-injured mice but not in sham-injured mice. Facial and hindpaw allodynia were significantly attenuated in mTBI-injured mice treated with 0.5 U DYSPORT®.

[0313] DYSPORT® administered at immediately following TBI reverses acute TBI-induced allodynia as well as stress-induced allodynia in TBI-primed animals.

Example 3—Further Tests of BoNT to Treat a Symptom of TBI

[0314] The efficacy of botulinum neurotoxin, when administered two hours after mild traumatic brain injury (mTBI), in treating the mTBI was evaluated in mice.

[0315] The mice were subjected to either mTBI or sham injury on day 0.

[0316] DYSPORT® was infused through the sagittal suture of each mouse at 0.5 U/mouse two hours following injury.

[0317] Facial allodynia and hindpaw allodynia were measured 1, 2, 3, 8, 10, 11, and 15 days post-injury. On day 15, all mice were exposed to bright light stress (BLS) for 15 minutes and facial allodynia and hindpaw allodynia were measured for 5 hours following BLS. On day 28, all mice were exposed again to BLS for 15 minutes and facial allodynia and hindpaw allodynia were again measured for 3 hours following BLS. The results are depicted in FIG. 11.

[0318] A single mTBI injury resulted in time dependent tactile facial and hindpaw allodynia after mTBI. The administration of 0.5 U DYSPORT® 2 hours immediately following mTBI reversed tactile facial and hindpaw allodynia. Exposure to BLS on Day 15 post-mTBI promoted tactile allodynia in mTBI-injured mice but not sham-injured mice. Allodynia was significantly attenuated in mTBI-injured mice treated with 0.5 U DYSPORT® 2 hours after mTBI. The second exposure of the mice to BLS promoted tactile allodynia in mTBI-injured mice but not in sham-injured mice. Facial and hindpaw allodynia were significantly attenuated in mTBI-injured mice treated with 0.5 U DYSPORT®.

[0319] DYSPORT® administered 2 hours after TBI reverses acute TBI-induced allodynia as well as stress-induced allodynia in TBI-primed animals.

[0320] Levels of CGRP 2 and 3 hours following exposure to BLS on day 28 following injury were measured. The results are shown in FIG. 12. The levels of CGRP were increased in untreated mice that suffered mTBI injury as compared with sham injured mice. However, levels were reduced in mTBI-injured mice that were treated with DYSPORT® 2 hours following injury.

Example 4—Further Tests of BoNT to Treat a Symptom of TBI

[0321] The efficacy of botulinum neurotoxin, when administered 24 hours after mild traumatic brain injury (mTBI), in treating the mTBI was evaluated in mice.

[0322] The mice were subjected to either mTBI or sham injury on day 0.

[0323] On day 1, facial and hindpaw allodynia were assessed. After that, and 24 hours after injury, DYSPORT® was infused through the sagittal suture of each mouse at 0.5 U/mouse.

[0324] Facial allodynia and hindpaw allodynia were measured 2, 3, 8, and 13 days post-injury. On day 13, all mice were exposed to bright light stress (BLS) for 15 minutes and facial allodynia and hindpaw allodynia were measured for 5 hours following BLS. On day 17, all mice were exposed again to BLS for 15 minutes and facial allodynia and hindpaw allodynia were again measured for 5 hours following BLS. The results are depicted in FIG. 13.

[0325] As in Examples 1 and 2, a single mTBI injury resulted in time dependent tactile facial and hindpaw allodynia as demonstrated on day 1 after mTBI and returning to baseline levels by day 13 post-mTBI. The administration of 0.5 U DYSPORT® 24 hours after mTBI reversed tactile facial and hindpaw allodynia. Exposure to BLS on Day 13 post-mTBI promoted tactile allodynia in mTBI-injured mice but not sham-injured mice. Allodynia was significantly attenuated in mTBI-injured mice treated with 0.5 U DYSPORT® 24 hours after mTBI. The second exposure of the mice to BLS promoted tactile allodynia in mTBI-injured mice but not in sham-injured mice. Facial allodynia was significantly attenuated in mTBI-injured mice treated with 0.5 U DYSPORT®. Hindpaw allodynia was reduced in mTBI-injured mice treated with DYSPORT®, but this reduction was not statistically significant.

[0326] DYSPORT® administered at 24 hours post TBI reverses acute TBI-induced allodynia as well as stress-induced allodynia in TBI-primed animals.

Example 5—Further Tests of BoNT to Treat a Symptom of TBI

[0327] 2.5 mL of preservative-free 0.9% sodium chloride solution is drawn into a syringe and then injected into a vial containing 500 units of DYSPORT®. The vial is tilted side-to-side (and not shaken) for approximately one minute to ensure solution homogeneity.

[0328] Using a second syringe, 0.5 mL of the reconstituted DYSPORT® is drawn up without inverting the vial.

[0329] 2.0 mL of preservative-free 0.9% sodium chloride solution is drawn into a third syringe. The third syringe is then connected to the second syringe with a connector. The plunger of the second syringe is pulled to transfer the 2.0 mL of preservative-free 0.9% sodium chloride solution into the second syringe. The syringe now contains 2.5 mL of solution containing 100 units of DYSPORT®.

[0330] A 1 mL syringe is then connected using a connector to the syringe containing the DYSPORT® solution. The plunger of the 1 mL syringe is pulled to transfer 0.8 mL of the DYSPORT® solution to the 1 mL solution. The syringe is removed and capped.

[0331] The solution is stored in the syringe in a refrigerator at 2 to 8° C. and protected from light. The composition is used within 24 hours and, if not used, is discarded. Before use in injection, the cap is removed and a needle attached.

Example 6—Further Tests of BoNT to Treat a Symptom of TBI

[0332] A patient suffering from a blow to the head is immediately seen by a physician.

[0333] The physician measures the severity of the patient's injury using the Glasgow Coma Scale (GCS). The patient's GCS score is determined to be 6 and thus severe.

[0334] The likelihood of the patient developing a symptom of TBI is then determined by measuring the level of UCH-L1 in his blood with higher than normal amounts indicating a high likelihood that he would develop a symptom of TBI.

[0335] Higher than normal amounts were measured and thus a higher dosage amount of 25 units/kg per treatment session is determined to be appropriate.

[0336] The weight of the patient is determined to be 80 kg. As such, 2,000 units of Dysport® are administered within an hour of the TBI.

[0337] The patient's response to the administration is then assessed using Rancho Los Amigos scale after administration. The patient's response exhibits a Level VIII response. As such, administration of botulinum neurotoxin is discontinued.

Example 7—Further Tests of BoNT to Treat a Symptom of TBI

[0338] A patient that has lost consciousness after a fall is examined by a physician.

[0339] The patient regains consciousness after 3 hours. As LOC was 3 hours, the patient has suffered a moderated case of TBI.

[0340] The likelihood of the patient developing a symptom of TBI is then determined by measuring the level of GFAP in his blood. The level is determined to be only slightly above normal, indicating a moderate likelihood that he would develop a symptom of TBI.

[0341] In view of the above, a moderate dosage amount of 15 units/kg per treatment session is determined to be appropriate.

[0342] The weight of the patient is determined to be 60 kg. As such, 900 units of Dysport® are administered (20 hours after TBI).

[0343] The patient's response to the administration is then assessed using the Rancho Los Amigos scale. The patient's response exhibits a Level VI response. As such, a further administration is scheduled but at a lower dose of 10 units/kg.

Example 8—Further Tests of BoNT to Treat a Symptom of TBI

[0344] A patient that has suffered from post-traumatic amnesia (PTA) after being a car accident is examined by a physician.

[0345] The patient regains continuous memory after 15 minutes. As PTA lasted less than 20 minutes, the patient has suffered a mild case of TBI.

[0346] The likelihood of the patient developing a symptom of TBI is then determined by measuring the level of CGRP in her blood. The level is determined to be normal, indicating a low likelihood that she would develop a symptom of TBI.

[0347] In view of the above, a low dosage amount of 5 units/kg per treatment session is determined to be appropriate.

[0348] The weight of the patient is determined to be 50 kg. As such, 250 units of Dysport® are administered (10 hours after TBI).

[0349] The patient's response to the administration is then assessed using Rancho Los Amigos scale. The patient's response exhibits a Level IV response. As such, a further administration of botulinum neurotoxin is scheduled but at a higher dose of 10 units/kg per treatment session.

[0350] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.