USE OF A BENZODIAZEPINE DERIVATIVE AND METHOD OF TREATMENT OF TRAUMATIC BRAIN INJURY

Abstract

Present invention aims to protect a new use of JM-20, a benzodiazepine derivative, in the treatment of and recovery from traumatic brain injury (TBI) and its related symptoms. Additionally, it provides a method of treatment for TBI-induced brain damage.

Claims

1. (canceled)

2. The method of treatment according to claim 6, wherein said administering is therapeutically effective to improve the patient's recovery rate after a TBI.

3. The method of treatment according to claim 6, wherein said administering is therapeutically effective to improve the patient's sensory-motor recovery after a TBI.

4. The method of treatment according to claim 6, wherein said administering is therapeutically effective to improve the patient's cognitive recovery after a TBI.

5. The method of treatment according to claim 6, wherein said administering is therapeutically effective to improve the patient's spatial memory after a TBI.

6. A method of treatment to reduce and/or reverse traumatic brain injury known as TBI in a patient, said method comprising administering to the patient a therapeutically effective dose of (3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-4,11-dihydro-1H-pyridol[2,3-b][1,5]benzodiazepine) known as JM20.

7. The method of treatment according to claim 6, wherein said administering is therapeutically effective in reducing and/or reversing behavioral, morphological and biochemical changes of the patient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1. Effects of TBI and JM-20 treatment on latency to fall during rotarod test.

[0015] FIG. 2. Effects of TBI and JM-20 treatment on crossings events during the open field test.

[0016] FIG. 3. Effects of TBI and JM-20 treatment on rearing events during the open field test.

[0017] FIG. 4. Effects of TBI and JM-20 treatment on immobility time during the open field test.

[0018] FIG. 5. Effects of TBI and JM-20 treatment on time to complete the beam-walk test.

[0019] FIG. 6. Effects of TBI and JM-20 treatment on brain water content.

[0020] FIG. 7. Effects of TBI and JM-20 treatment on p-Akt expression in cortex by Western blotting analysis.

[0021] FIG. 8. Effects of TBI and JM-20 treatment on p-Akt expression in hippocampus by Western blotting analysis.

[0022] FIG. 9. Effects of TBI and JM-20 treatment on iba-1 expression in cortex by Western blotting analysis.

[0023] FIG. 10. Effects of TBI and JM-20 treatment on iba-1 expression in hippocampus by Western blotting analysis.

[0024] FIG. 11. Effects of TBI and JM-20 treatment on GFAP expression in cortex by Western blotting analysis.

[0025] FIG. 12. Effects of TBI and JM-20 treatment on GFAP expression in hippocampus by Western blotting analysis.

DETAILED DESCRIPTION OF THE INVENTION

[0026] Inventors of the present invention surprisingly found that the use of the JM20 compound has biological activity in the treatment of TBI.

[0027] When assessing the effects of administering JM-20 on the following parameters in animal models after a TBI, namely: motor performance; cerebral edema; astrocyte reactivity; microglial activation; and pro-survival pathway activation, it proved to be effective in reducing and, in some cases, reversing TBI adverse effects, including behavioral, morphological and biochemical changes.

[0028] During behavioral tests, it was observed that animals who suffered a TBI showed a reduction of the first fall latency time on the rotarod test in contrast with the other groups (p<0.05). In FIG. 1 the values are expressed as mean±SEM (n=8-11, per group), where (*) are the differences of the control, JM-20 and TBI+JM-20 groups, p<0.05 (by Kruskal-Wallis test followed by the Dunn post hoc test).

[0029] They also had a lower number of crossings and a longer immobility time during the open field test in comparison with the control group (p<0.05 y p<0.01). In FIG. 2 the values are expressed as mean±SEM (n=8-11, per group) where (*) are the differences of the control groups, p<0.05 (one-way ANOVA followed by the Tukey post hoc test). While in FIG. 3, the data are reported as mean±SEM (n=8-11, per group), where (*) are the differences between the control and TBI+JM-20 group, p<0.05 (by one-way ANOVA followed by the Tukey post hoc test). Additionally, a higher number of rearings was observed in comparison with the control and the TBI+JM-20 group (p<0.05). In FIG. 4 the data are reported as mean±SEM (n=8-11, per group). (**) Different from the control, p<0.01 (by one-way ANOVA followed by the Tukey post hoc test).

[0030] In the beam-walking test, rats in the TBI group took longer to complete the course than the control and JM-20 groups (p<0.05). In FIG. 5 the data are expressed as mean±SEM (n=6, per group). (*) Different from the control and JM20 group, p<0.05 (by one-way ANOVA followed by the Tukey post hoc test.) The content of water in the brain increased in the TBI group in contrast with the control and TBI +JM-20 groups (p<0.05). In FIG. 6 the data are reported as mean±SEM (n=6). (*) Different from the control, #different from the TBI group p<0.05 (by one-way ANOVA followed by the Tukey post hoc test).

[0031] In the Western blotting, the p-Akt expression decreased in TBI groups, both in the cerebral cortex and the hippocampus in comparison with the control group (p<0.05), and the hippocampus in comparison with the control and TBI+JM-20 groups (p<0.05). In FIG. 7, the data are reported as mean±SEM (n=6). (*) In contrast with the control, p<0.05 (by unidirectional ANOVA followed by the Tukey post hoc test). While in FIG. 8 the data are reported as mean±SEM (n=6). (*) Different from the control, p<0.05; #different from TBI, p <0.05 (by one-way ANOVA followed by the Bonferroni post hoc test).

[0032] The iba-1 expression increased in TBI groups, both in the cerebral cortex in comparison with all the groups (p<0.01), and in the hippocampus in comparison with the control group (p<0.01). In FIG. 9, the data are reported as mean±SEM (n=6). (**) Different from the control, JM-20 and TBI+JM-20 p<0.01 (by one-way ANOVA followed by the Newman-Keuls post hoc test). While in FIG. 10, the data are reported as mean±SEM (n=6). (**) Different from the control, p<0.01 (by one-way ANOVA followed by the Tukey post hoc test).

[0033] The GFAP expression showed no changes in any of the groups. Both in FIGS. 11 and 12, the data are reported as mean±SEM (n=6).

Examples

[0034] Animals

[0035] Male Wistar rats (200-250 g), with an average age of 60 days were used, conditioned in boxes with food and water at will. Procedures were performed pursuant to the rules of the Animal Ethics and Welfare Committee UFSM (9426190418).

[0036] TBI Induction

[0037] Performance Tests

[0038] TBI was induced through the weight-drop model (MANNIX, R. et al. Chronic Gliosis and Behavioral Deficits in Mice Following Repetitive Mild Traumatic Brain Injury. Journal of Neurosurgery. 2014) A 54 g weight was used released from a 100 cm height freely falling on the animals head. Animals were suspended on aluminum foil, with small cuts to ensure it would break with the impact of the weight allowing for the sudden acceleration of the movement, thus tearing the aluminum foil and falling over a sponge. TBI-induced animals were previously anesthetized with 2% isoflurane. In addition, topical lidocaine was applied to the animals' head to minimize posttraumatic pain (MYCHASIUK, R. et al. A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats. Journal of Visualized Experiments. 2014)

[0039] Euthanasia

[0040] Animals were beheaded by shearing and their brains were immediately extracted and their hippocampi and cortices were dissected.

[0041] Performance Tests

[0042] Rotarod

[0043] Animals were trained for 5 minutes before the TBI so they could adapt to the apparatus. The testing session comprised 5 trials and concluded when animals fell off the rotor (3.7 cm diameter, velocity 25-30 rpm) or after the cutoff time, i.e. 300 s. (Whishaw et al., 2003). The first fall latency was analyzed.

[0044] Open Field Test

[0045] Animals were placed in the central area of an open field (56 cm diameter) that had its surface divided into equal parts. The duration of the test was 300 seconds. The number of times animals walked around the quadrants, the number of their exploratory responses (rearings) and their immobility time were analyzed.

[0046] Beam-Walking Test

[0047] The beam-walking test (HAUSSER, N. et al. Detecting Behavioral Deficits in Rats After Traumatic Brain Injury. J. Vis. Exp. (131), e56044, doi:10.3791/56044. 2018.) consists in animals walking on a hanging wooden beam (2.5 cm wide and 100 cm long) to reach a black wooden box placed at the end of the apparatus. First, the rats were placed in the wood box for one minute for acclimation. Shortly after, they were placed on the other end and encouraged to walk along the beam to reach the opposite end. Three attempts were analyzed. The rats were trained prior to the TBI for familiarization with the apparatus. The day of the test, the times of each attempt were recorded and their values were averaged.

[0048] Cerebral Edema

[0049] The cerebral edema was determined measuring the water content of the brain using the wet-dry method described by Chen et al (2014) (CHEN, W. et al. Neuroprotective effect of allicin against traumatic brain injury via Akt/endothelial nitric oxide synthase pathway-mediated anti-inflammatory and anti-oxidative activities. Neurochemistry International. 68:28-37. 2014) Twenty-four hours after the TBI, the animals were sacrificed and their brains were rapidly removed and weighed to determine their wet weight. Then they were dried in the oven at 100° C. for 48 hours, the tissues were then weighted again until they were dry. The water content of the brain was calculated using the following formula:

% H2O=(1−dry weight/wet weight)

[0050] Western Blotting

[0051] The Western blot test was performed according to Gerbatin et al (2017) with some adjustments (GERBATIN, R. D. R. et al. Guanosine Protects Against Traumatic Brain Injury-Induced Functional Impairments and Neuronal Loss by Modulating Excitotoxicity, Mitochondrial Dysfunction, and Inflammation. Molecular Neurobiology. 54(10):7585-7596. 2017.). The hippocampus and cerebral cortex tissue samples were lysed in the RIPA (radioimmunoprecipitation assay) and centrifuged for 20 minutes at 12.700×g and 4° C. The protein concentration of each sample was determined by the bicinchonininc acid protein assay (Thermo Fisher Scientific). The samples (30 μg of protein) were subjected to SDS-polyacrylamide gel electrophoresis at 4-12% and transferred to a nitrocellulose membrane using the Trans-Blot® Turbo™ transference system and the protein load was confirmed by Ponceau S solution (Sigma Aldrich-P7170). After the specific blockade, the transferences were incubated for one night at 4° C. with one rabbit anti-Iba-1 ionized calcium-binding adaptor molecule (1: 400; Santa Cruz Biotechnology, Santa Cruz, Calif., EE. UU.), rabbit anti-glial fibrillary acidic protein (GFAP) (1: 1000; Dako), Phospho-Akt (1: 1000; Cell signaling).

[0052] The mouse anti-β-actin antibody (1: 10,000, Santa Cruz Biotechnology, Santa Cruz, Calif., EE. UU.) was dyed as an additional control to the protein load. After the primary antibody was incubated, the membranes were rinsed in TBS-T (TBS+Tween 20 at 0.1%) twice at room temperature for 10 minutes and incubated with anti-rabbit (Sigma Aldrich-A6154) or anti-mouse (Santa Cruz Biotechnology-sc-2005) secondary antibodies conjugated to horseradish peroxidase (HRP) (1: 5000) for two hours at room temperature. The bands were visualized by enhanced chemioluminescence using ECL Western Blotting substrate (Pierce ECL, BioRad) and the signals were registered with the photodocumentation system ChemiDoc XRS+(BioRad). Then the bands were quantified using the Image Lab software (Bio-Rad).