EA000531B1 - Active substance for production of pharmaceutical preparation for treatment of traumatic brain injury - Google Patents

Abstract

1. Use of 5-(2-ethyl-2H-tetrazol-5-yl)-1,2,3,6-tetrahydro-l-methylpyridine or a pharmaceutically acceptable salt thereof as an active substance for the manufacture of a pharmaceutical preparation for the treatment of traumatic brain injury. 2. Use according to Claim 1, characterized in that in said pharmaceutical preparation said active substance is used in a unit dosed form. 3. Use according to any of Claims 1-3, characterized in that the pharmaceutical preparation manufactured is for the treatment of traumatic brain injury caused by physical forces acting on the scull or spinal column, ischaemia, stroke, arrested breathing, cardiac arrest, cerebral thrombosis or emboism, neurological problem caused by AIDS, cerebral hemorrhage, encephalomyelitis, hydrocephalus, post-operative events, cerebral infections, concussions or elevated intracranial pressure and or for the treatment of the sequelae of such a condition.

Description

The present invention relates to the use of 5 - (2-ethyl-2H-tetrazol-5-yl) -1,2, 3,6-tetrahydro-1-methylpyridine for the manufacture of pharmaceutical preparations for the treatment of traumatic brain injury (TBI).

BACKGROUND OF THE INVENTION

Patent EP-A1-0 296 721 discloses a class of piperidine or 1,2,3,6tetrahydropyridine compounds substituted at the 5-position with a five-membered heterocyclic group, including a subclass of optionally substituted 5-tetrazoyl-1,2,3,6-tetrahydropyridine compounds. The compounds that have been described have high affinity for central cholinergic receptors, in particular high affinity for central muscarinic M ₁ receptors, thus being useful in the treatment of Alzheimer's disease, senile dementia and impaired learning and memory functions.

The structure-activity relationship of this subclass has been described by Moltzen et al., J. Med. Chem. 1994, 37, 4085-4099. One of the compounds, i.e. 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6tetrahydro-1-methylpyridine, has been reported to be selective for muscarinic receptors with affinities several times higher for Mi than for M ₂ and M ₃ receptors (Subtypes of Muscarinic Receptors, The Sixth International Symposium, Nov. 9-1 2, 1 994, Fort Lauderdale). Functionally, they have been described to behave like partial antagonists at the M ₁ receptors and antagonists at the M ₂ and M ₃ receptors. Moreover, the only known reported in vivo effect was the spatial memory gain effect of young and old rats, respectively.

TBI caused by physical or neurological conditions or various diseases is gaining increasing importance among the population and there is a significant need for effective and safe drugs to treat such disorders and their complications. Now, it has been unexpectedly discovered that the compound 5- (2-ethyl-2H-tetrazol-5yl) -1,2,3,6-tetrahydro-1-methylpyridine shows a positive effect in the treatment of TBI and its complications.

Summary of Entity

Accordingly, the present invention relates to the use of 5- (2-ethyl-2H-tetrazol5-yl) -1,2,3,6-tetrahydro-1-methylpyridine or a salt thereof with an acid

for the manufacture of pharmaceuticals for the treatment of traumatic brain damage or its complications. The term traumatic brain injury (TBI), as described herein, includes all conditions associated with a brain or spinal cord injury, for example, caused by physical effects on the skull or spine, ischemia, stroke, respiratory arrest, cardiac arrest, cerebral thrombosis or embolism , neurological problems caused by AIDS, cerebral hemorrhage, encephalomyelitis, hydrocephalus, postoperative events, cerebral infections, concussion or increased intracranial pressure epnym pressure.

Pharmaceutically acceptable salts resulting from the addition of an acid to the compounds used in the present invention are salts formed with non-toxic organic and inorganic acids.

Examples of such organic salts are those of maleic, fumaric, benzoic, ascorbic, pamic, succinic, oxalic, bismethylene salicylic, methanesulfonic, ethanedisulfonic, acetic, propionic, tartaric, salicylic, citric, gluconic, milk, apple, cinnamic, almond, almond stearic, palmitic, itaconic, glycolic, paminobenzoic, glutamic, benzenesulfonic and theophylline acetic acids, as well as 8-halotheophylline, for example 8-bromteophylline. Examples of inorganic salts are salts of hydrochloric, hydrobromic, sulfuric, aminosulfonic, phosphoric and nitric acids.

The compound used according to the present invention, as found, is useful in the treatment of TBI. So, for example, it improves cognitive function after moderate traumatic brain injuries, and it weakens the decrease in cholinergic neurons caused by trauma. Moreover, it was found to not cause adverse cardiac or other side effects at doses that are considered clinically acceptable.

In another aspect, the present invention provides a method of preventing or treating TBI in humans, comprising the step of administering a therapeutically effective amount of 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro1-methylpyridine or a salt thereof attaching an acid to a patient in need thereof.

The compound used according to the present invention and its pharmaceutically acceptable salts with acids can be administered in any convenient manner, for example orally or parenterally, and these compounds can be presented in any convenient form for such administration, for example in the form of tablets, capsules, powders, syrups or solutions or dispersions for injection.

An effective daily dose of a compound of the present invention or a pharmaceutically acceptable salt thereof is from 10 μg / kg to 10 mg / kg body weight, preferably from 25 μg / day / kg body weight to 1.0 mg / day / kg body weight. Accordingly, a suitable daily dose is from 500 μg to 600 mg / day, preferably from 1.0 mg to 100 mg.

The compound used according to the present invention can be obtained as described in EP-A1 0 296 721, and its acid addition salts are readily prepared using methods well known in the art.

Pharmacology

The compound used according to the present invention was tested by the following recognized and reliable method.

Cognitive function after traumatic brain damage

Rats were subjected to central fluid percussion traumatic brain injury, as described by Dixon, C.E. et al., J. Neurosurgery, 67 (1987) 110119. Injured animals were injected subcutaneously daily 1 to 5 days after the injury, starting 24 hours after the injury, saline, or 5- (2-ethyl-2H-tetrazole5 -yl) -1,2,3,6-tetrahydro-1-methylpyridine, 3, 6 μmol / kg or 1 5 μmol / kg.

The suppression of the reflex after TBI was determined according to the method of Dixon et al. 1987, supra.

Body weight was recorded before the injury, and 1–5 days after the injury and rotarod activity was determined 1–5 days after the injury according to the method of Hamm, R.J., et. al., J. Neurotrauma, 11 (1994) 187-196. The Rotarod test was used to measure motor activity after TBI.

Finally, the average latency (+ S.E.M.) Required to locate the target platform in the Morris water maze was measured 11–1 5 days after the injury. The results were analyzed by ANOVA. During the test in the water maze, all animals were injected 10 minutes before being evaluated in the water maze.

Animals with simulated injury (animals prepared for injury but not given a liquid impulse) were included for comparison.

Determination of the number of ChAT neurons after TBI

Rats were subjected to central liquid shock TBI and treatment as described above. He was injected subcutaneously with a salt (n = 5) or test compound (5- (2 ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro -1 methylpyridine, 15 μmol / kg) (n = 5). Rats with simulated injured were injected with saline (n = 4) or test compound 1 5 μmol / kg (n = 4). The suppression of the installation reflex was determined as described above.

The possible loss of cholinergic neurons after TBI was determined by quantifying cholinacetyltransferase (ChAT) immunoreactivity in the basal front of the brain. On the 15th day after the injury (2-4 hours after the last injection), all animals were anesthetized with pentobarbital (90 mg / kg, intraperitoneally) and perfusion through an aorta of isotonic saline, 200-250 ml, followed by 500 ml 4.0% paraformaldehyde / 0.2% picric acid in 0.1 M phosphate buffer at a rate of 500 ml / 30 min at room temperature. After perfusion, the brain was cut into two blocks and after that it was secondarily fixed for 24 hours in the same solution at 10 ° С. Coronal 40 μm sections were collected on a vibratome through the cell nucleus of the anterior part of the brain and every fifth section was processed for ChAT immunoreactivity. Parallel sections were stained for Nissi with cresyl violet to quantify the possible cholinergic and non-cholinergic neuronal losses in the cell nuclei of the basal anterior part of the brain.

Free-floating sections of the anterior part of the brain were incubated in a final ChAT antibody concentration (1:50) of 1.0 μg / ml in 0.01 M phosphate-buffered saline (PBS) solution containing 0.1% X-100 Triton. Sections of the anterior part of the brain were cut and incubated in the primary antibody for 24 hours at room temperature in culture pallets (4 sections / 300 μl / well).

After four washes in PBS, sections were incubated in a secondary antibody (horse antibody to mouse IgG JgG; Vector Laboratories, Burlingame, CA) for 1 h at 37 ° C. After three washes in PBS, sections were incubated with murine avidin biotin peroxidase complex (ABC) (Vector procedure) (Hsu et al., J. Histochem. Cytochem. 29, 1981, 577-580) for 2 hours at 37 ° C. After three washes in PBS and one in 0.1 M Tris-buffered saline (TBS), three floating sections were treated using the glucose oxidase-diaminobesidene-nickel method described by Shu et al. Neurosci. Lett. 85, 1988, 169-171. The reaction was stopped by transferring the slices to TBS. Slices were fixed on glass slides coated with chromium-alum in gelatin and allowed to dry overnight at room temperature, dehydrated at increasing concentrations of ethyl alcohol and xylene, and then closed with Permount sliding covers.

ChAT immunoreactive neurons in the medial septal nuclei (MNS), the vertical terminal diagonal ligament nuclei (VDB) and the basal nuclei magnosellulares (NMB) nuclei were counted using the Microcomputer Imaging Device System (MCID) (Imaging Research Inc., Ontario, Canada). The boundary between MSN and VDB has been defined as a front commissure. NMBs have been defined as immunolabeled neurons located in globulus pallidus and the adjacent inner capsule. Cell counts were performed in each animal at 0.2 mm intervals through the nucleus of the brain cell. The number of cells is reported as a group average of 10,000 μm ² for each nucleus of a brain cell.

Cresyl violet stained MSN, VDB, and NMB neurons from parallel sections were also counted on MCID. Due to the large number of common cholinergic and non-cholinergic neurons that are stained with cresyl violet throughout this area, a bilateral sample of neurons was counted in each nucleus. A grid with predefined areas was used to count cells for each nucleus. For MSN and VDB nuclei, three areas of 2000 μm ² were counted on each side (thus, a total area of 12000 μm ² was tested for each core in each of the four counted sections). For NMB, one 1 2000 μm ² region was counted on each side (total area = 24,000 μm ² in each of the four counted sections). Only large cells (> 20 μm in diameter) with visible Nissi somatic bodies and with a clearly neural type of nuclei were quantified.

results

No significant differences were observed between any of the injured animals in suppressing the installation reflex after TBI. This shows that in each experiment, the injured groups received an equivalent seriousness injury. Animals with simulated injuries were rehabilitated significantly faster than any of the TBI animals (p <0.0001 for each comparison). Suppression of rehabilitation in a simulated injured group is due to gas anesthesia used before injury.

There was no significant difference between any of the injured groups in terms of body weight loss that followed the TBI, again showing equivalence in the severity of injuries between the injured groups.

No significant differences were observed in rotarod activity 1-5 days after injury between any injured groups. Since the test compound was applied 1–1 to 5 days after the injury, these tests also show that this drug does not affect body weight or rotarod activity after injury.

The ANOVA method showed that injured animals that were given a (daily) subcutaneous test compound were better oriented in the Morris water maze than injured animals receiving saline. Injured animals treated with the test compound, 1 5 μmol / kg, significantly improved their activity with p <0.01.

TBI caused a significant decrease in the number of ChAT-IR neurons in VDB and NMB in rats treated with saline and test compound, respectively. However, the compound of the present invention significantly attenuated the decrease in ChAT-IR caused by trauma (32% reduction compared to the injured group treated with saline in VDB and 51% in NMB). Parallel cresyl-violet stained sections showed no decrease in the number of cells in MSN, VDB and NMB, showing that the loss of ChAT-IR neurons did not occur due to cell death.

Examples of finished dosage forms

The pharmaceutical formulations of the present invention can be prepared by conventional methods known to those skilled in the art.

For example, tablets can be prepared by mixing the active ingredient with adjuvants and / or diluents and then compressing the mixture in a tablet machine. Examples of adjuvants and / or diluents include corn starch, lactose, talc, magnesium stearate, gelatin, lactose, gums and the like. Any other adjuvant or coloring agent, flavoring, preservatives, etc., may be used provided that they are compatible with the active ingredient.

Injectable solutions can be prepared by dissolving the active ingredient and possible additives in a portion of the carrier, preferably sterilized water, bringing the solution to the desired volume, sterilizing the solution and filling in suitable ampoules or vials. Any suitable additive conventionally used in the art may be added, such as tonicity agents, preservatives, antioxidants, etc.

Typical examples of formulations of the present invention are as follows (amounts of active ingredient calculated as free base):

) Tablets

5- (2-ethyl-2H-tetrazol-5-yl) 1,2,3,6-tetrahydro-1-methylpyri - 20 mg din

Lactose 60 mg Corn starch 30 mg Hydroxypropyl cellulose 2.4 mg Microcrystalline cellulose 19.2 mg Croscarmellose Sodium Type A 2.4 mg Magnesium stearate 0.84 mg 2) Pills 5- (2-ethyl-2H-tetrazol-5 yl) -1,2,3,6-tetrahydro-1 methylpyridine 10 mg Lactose 49.9 mg Corn starch 23.5 mg Povidone 1, 8 mg Microcrystalline cellulose 14.4 mg Croscarmellose Sodium Type A 1, 8 mg Magnesium stearate 0, 63 mg 3) Syrup 5- (2-ethyl-2H-tetrazol-5-yl) 1,2,3,6-tetrahydro-1-methylpyri- 5.0 mg dean Sorbitol 500 mg Hydroxypropylcell lulose 1 5 mg Glycerol 50 mg Methylparaben 1 mg Propylparaben 0.1 mg Ethanol 0.005 ml Flavoring 0.05 mg Sodium Saccharin 0.5 mg Water Add 1 4) solution 5- (2-ethyl-2H-tetrazol-5-yl) 1,2,3,6-tetrahydro-1 methylpyridine 1.0 mg

Sorbitol 5.1 mg

Acetic acid 0.08 mg

Water for injection Add 1 ml

Claims (3)

CLAIM 1. Use of 5- (2-ethyl-2H-tetrazol-5yl) -1,2,3,6-tetrahydro-1-methylpyridine or its pharmaceutically acceptable salt as an active substance in the manufacture of a pharmaceutical preparation for treating traumatic brain damage. 2. The use according to claim 1, characterized in that in said pharmaceutical preparation said active substance is used in a standard dosage form. 3. The use according to any one of claims 1 to 2, characterized in that said pharmaceutical preparation is intended to treat traumatic brain damage caused by physical effects on the skull or spine, ischemia, sudden brain stroke, respiratory arrest, cardiac arrest, cerebral thrombosis or embolism, neurological problems caused by AIDS, cerebral hemorrhage, encephalomyelitis, hydrocephalus, postoperative events, cerebral infections, concussion or elevated trichenic pressure and / or for the treatment of complications of such conditions.