KR19990067353A - Brain damage caused by trauma - Google Patents

Abstract

Formula I is useful for improving cognitive performance in the case of traumatic brain injury, attenuating the reduction in wound recovery of cholinergic neurons in traumatic brain injury models, and also for the preparation of pharmaceutical preparations for the treatment of brain injury due to trauma. 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine compound.

Formula I

Description

Treatment of brain damage due to trauma

EP-A1 0 296 721 discloses 1, substituted at the 5-position with a 5-membered heterocyclic group comprising a subclass of an optionally substituted 5-tetrazolyl-1,2,3,6-tetrahydro-pyridine compound 2,3,6-tetrahydropyridine compounds or piperidines are disclosed. The compounds have a high affinity for central cholinergic receptors and, in particular, high affinity for central muscarinic M ₁ receptors, and have been shown to be useful for the treatment of Alzheimer's disease, senile dementia and impaired learning and memory functions. .

The structure-activity relationship of this subclass is described by Moltzen et al., J. Med. Chem. 1994, 37, 4085-4099. One of the compounds, namely 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine, is responsible for the M ₂ and M ₃ receptors. It has been reported to be selective for muscarinic receptors with a few times higher affinity for M ₁ (Subtypes of Muscarinic Receptors, the Sixth International Symposium, Nov. 9-12, 1994, Fort Lauderdale). Functionally, it has been described that act as an antagonist (antagonist) In a partial agonist eogo (agonist) the M ₁ receptor, M ₂ and M ₃ receptors. Moreover, the only major in vivo effects reported were effects on spatial memory acquisition in young and old rats, respectively.

TBI caused by physical or neurological conditions or various diseases is of increasing importance among humans, and there is a high demand for safe and effective drugs for the treatment of such diseases and their sequelae.

It has now been found that compound 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine shows a surprisingly beneficial effect in treating TBI and its sequelae. .

The present invention provides 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro- for preparing a pharmaceutical formulation for traumatic brain injury (TBI). It relates to the use of 1-methylpyridine.

Accordingly, the present invention provides 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetra of formula (I) for the preparation of a pharmaceutical formulation for the treatment of brain injury or sequelae caused by trauma. And to the use of hydro-1-methylpyridine or acid addition salts thereof.

The term brain injury due to trauma (TBI) is used herein to refer to, for example, force exerted physically on the skull or spinal column, ischemia, stroke, dyspnea, heart failure, thrombosis or cerebral embolism, neuropathic abnormality by AIDS, cerebral hemorrhage. It includes all conditions related to trauma to the brain or spinal cord caused by encephalomyelitis, hydrocephalus, postoperative sequelae, infection of the brain, concussion, or increased intracranial pressure.

Pharmaceutically acceptable acid addition salts of the compounds used in the present invention are salts formed with non-toxic organic or inorganic acids. Examples of such organic salts include maleic acid, fumaric acid, benzoic acid, ascorbic acid, pamoic acid, succinic acid, oxalic acid, bis-methylenesalicylic acid, methanesulfonic acid, ethanedisulfonic acid, acetic acid, propionic acid, tartaric acid, salicylic acid, citric acid, gluconic acid, lactic acid, Malic acid, mandelic acid, cinnamic acid, citraconic acid, aspartic acid, stearic acid, palmitic acid, itaconic acid, glycolic acid, p-amino-benzoic acid, glutamic acid, benzene sulfonic acid or thiophyllic acetic acid, for example 8-promo Salts with 8-halothiophylline, such as thiophylline. Examples of such inorganic salts are hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, salts with nitric acid and the like.

The compounds used according to the invention have been found to be useful for the treatment of TBI. So, for example, it improves cognitive performance after brain injury due to minor trauma and weakens the reduction in wound recovery of cholinergic neurons. Moreover, it has been found that it does not cause adverse side effects to the heart and other at doses that are believed to be clinically relevant.

In another aspect, the invention provides treatment for patients in need of 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine or acid addition salts thereof. Provided are methods for the prevention and treatment of human TBI, comprising administering a pharmaceutically effective amount.

The compounds used according to the invention and their pharmaceutically acceptable acid addition salts can be administered, for example, by oral or parenteral appropriate methods, and the compounds can be, for example, tablets, capsules, powders, Syrups or injectable solutions or suspensions may be provided in a form suitable for such administration.

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

The compounds used according to the invention are described in EP-A1 0 296 721, the acid addition salts of which can be readily prepared by methods well known in the art.

Pharmacology

The compounds used according to the invention were tested by the following methods which were recognized and reliable.

1. Cognitive function after TBI

Brain damage from trauma to the central fluid percussion in rats, as described in Dixon, C., et al., J. Neurosurgery, 67 (1987) 110-119. Coated. Damaged animals start with 24 hours post-injury and daily for 1-15 days post-injury, either saline or 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro -1-methylpyridine, 3.6 μmol / kg or 15 μmol / kg was treated by subcutaneous injection.

Righting reflex suppression after TBI is described by Dixon et al. 1987, (see above).

Body weights are recorded before and 1-5 days after injury, and rotarod performance is performed 1-5 days after injury, Hamm, RJ, et al., J. Neuurotrauma, 11 (1994) determined according to the method of 187-196. The rotarod test was used to measure athletic performance after TBI.

Finally, mean latency (+ S.E.M.) To find the target platform in the Morris water maze was measured for 11-15 days after injury. The results were analyzed by ANOVA. All animals were injected 10 minutes prior to evaluation with water.

A comparative example includes a shaminjured animal (an animal prepared for damage but not delivered with a fluid pulse).

2. Quantification of ChAT Neurons After TBI

Mice were injured by brain injury due to central fluid percussion trauma and treated as described above. Saline (n = 5) or test compound (5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine, 15 μmol / kg) ( n = 5) was injected subcutaneously. False-impaired mice were injected subcutaneously with saline (n = 4) or 15 μmol / kg of test compound (n = 4).

Stereotactic reflection inhibition was determined as described above.

Possible loss of cholinergic neurons after TBI was determined by quantification of choline acetyltransferase (ChAT) immunoreactive neurons in the basal brain. At the 15th post-injury, all animals were anesthetized with pentobarbital (90 mg / kg, intraperitoneal) and at a rate of 500 ml / 30 min at room temperature, 200-250 ml of isotonic and then 0.1 M 500 ml of 4.0% paraformaldehyde / 0.2% picric acid in phosphate buffer was perfused through the aorta. After perfusion, the brain was dissected into two masses and postfixed for 24 hours in the same solution at 10 ° C. 40 μm slices in the coronal suture direction were collected on vibratome through the whole brain nucleus, and every fifth slice was processed for ChAT immunoreactivity. Side-by-side flakes were stained for nistle bodies with cresyl violet to quantify any possible cholinergic or noncholinergic neuronal loss in the basal ganglia.

Free floating whole brain slices were incubated in final ChAT antibody at 1.0 μg / ml concentration (1:50) in 0.01 M phosphate buffered saline solution (PBS) containing 0.1% Triton X-100. Whole brain slices were dissected and incubated in primary antibody for 24 hours at room temperature in culture dishes (4 slices / 300 μL / well).

After washing four times in PBS, the flakes were incubated for 1 hour at 37 ° C. in a secondary antibody (horse antimouse IgG: Vecter Laboratories, Burlingame, Calif.). After washing three times in PBS, the sections were transferred to mouse avidin-biotin-peroxidase complex (ABC) (vector) technology (Shu et al. J. Histochem. Cytochem. 29, 1981, 577-580). Incubation was for 2 hours at ℃. After washing three times with PBS and once with saline (TBS) buffered with 0.1 M Tris, free-floating flakes were removed. Neurosci. Lett. 85 Processed by the glucose oxidase-diaminobenzidine-nickel method described in 1988, 169-171. The reaction was stopped by transferring the slices to TBS. The flakes were placed on gelatin-chromium alum coated glass slides, dried overnight at room temperature, dehydrated in increasing concentrations of ethanol and xylene, and then coverglassed with Permount.

ChAT immunoreactive neurons in the medial septal nucleus (MSN), the vertical limb nucleus of the diagonal band (VDB), and the enlarged cell basal ganglia (NMB) can be microcomputer imaging device systems (MCID). (Imaging Research Inc., Ontario, Canada). The boundary between MSN and VDB was defined by an anterior commisure. NMB was defined as immunolabeled neurons located within the contiguous regions of the globulus pallidus and the internal capsule. Cell counts were obtained from each animal at 0.2 mm intervals through the whole brain nucleus. The number of cells was reported as the group average per 10,000 μm 2 for each forebrain nucleus.

Neurons of MSN, VDB and NMB from parallel flakes stained with cresyl violet were also counted on MCID. Because of the numerous total cholinergic and noncholinergic whole neurons stained with Cresyl Violet throughout this region, left and right neuronal samples were counted within each nucleus. Grit with the desired area was used to count cells for each nucleus. For MSN and VDB nuclei, three 2,000 μm 2 areas were counted on each side (thus a total area of 12,000 μm 2 was sampled for each nucleus in each of the four lamellas counted). For NMB, one 12,000 μm 2 area was counted on each side (total area is 24,000 μm 2 in each of the four counted flakes). Only large cells (> 20 μm diameter) with visible body wall nistle bodies and distinct neuronal nuclei were quantified.

result

Inhibition of stereotactic reflexes after TBI was not significantly different between injured animals. This means that in each experiment the damaged group suffered equally severe damage. Falsely injured animals were located significantly faster than any of the animals wearing TBI (in each P <0.0001 comparison). Stereotactic suppression of falsely damaged groups is due to the gas anesthesia used before the damage.

Body weight loss after TBI was not significantly different between the injured groups, indicating once again that the injured groups were equally severely injured.

There was no significant difference between the injured groups in rotarod performance for 1-5 days after injury. Since the test compound was administered for 1-5 days after the injury, these tests also indicate that the drug did not affect body weight or rotarod performance after the injury.

Anova has shown that injured animals injected daily with subcutaneous test compounds perform better in the Morris water maze than injured animals given saline. Injured animals treated with 15 μmol / kg of test compound significantly improved performance with P <0.01.

TBI significantly reduced the number of ChAT-IR neurons in VDB and NMB in mice treated with saline and test compounds, respectively. However, the compounds of the present invention significantly attenuated the wound recovery reduction in ChAT-IR (32% reduction in VDB and 51% reduction in NMB compared to the group administered saline). Parallel flakes stained with cresyl violet showed no decrease in cell numbers in MSN, VDB or NMB, indicating that the loss in ChAT-IR neurons was not due to cell death.

The pharmaceutical compositions of the present invention may be formulated by conventional methods in the art.

For example, tablets may be formulated by mixing the active ingredient with common adjuvants and / or diluents and compressing the mixture in a conventional tableting machine. Examples of adjuvants or diluents include corn starch, lactose, talcum, magnesium stearate, gelatin, gums and the like. Other auxiliaries or additives, flavors, preservatives and the like can be used provided they are compatible with the active ingredients.

Injectable solutions are dissolved in an excipient, preferably sterile water, in an excipient, preferably in sterile water, to a desired volume, and the solution is sterilized and then placed in an appropriate ampoule or vial. Suitable additives customarily used in the art can be added, for example tonics, preservatives, antioxidants and the like.

Typical examples of prescriptions of the preparations of the invention are as follows (the amount of active ingredient calculated as the free base).

1) Tablets:

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

Lactose 60mg

Corn Starch 30mg

Hydroxypropylcellulose 2.4mg

Microcrystalline Cellulose 19.2mg

Croscarmellose Sodium Type A 2.4mg

Magnesium Stearate 0.84mg

2) Tablets:

10 mg of 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine

Lactose 46.9mg

Cornstarch 23.5mg

Povidone 1.8mg

Microcrystalline Cellulose 14.4mg

Croscarmellose Sodium Type A 1.8mg

Magnesium Stearate 0.63mg

3) Syrups:

5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine 5.0 mg

Sorbitol 500mg

Hydroxypropylcellulose 15mg

Glycerol 50mg

Methyl-paraben 1 mg

Propyl-paraben 0.1mg

Ethanol 0.005ml

0.05mg fragrance

Saccharin Sodium 0.5mg

Water ad 1ml

4) Solution:

1.0 mg of 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine

Sorbitol 5.1mg

Acetic acid 0.08mg

Injectable Water ad 1ml

Claims (4)

5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine or a medicament for the preparation of a pharmaceutical formulation for the treatment of brain injury due to trauma The use of its acid addition salts, which is scientifically acceptable. Formula I The pharmaceutical preparation according to claim 1, wherein the prepared pharmaceutical preparation is 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine or a medicament in unit dosage form. Uses characterized in that it comprises acidically acceptable acid addition salts thereof. The pharmaceutical preparation according to claim 1, wherein the prepared pharmaceutical preparation is 5- (2-ethyl-2H-tetrazol-5-yl)-in an amount of 500 μg to 600 mg / day, preferably 1.0 mg to 100 mg / day. And 1,2,3,6-tetrahydro-1-methylpyridine or a pharmaceutically acceptable acid addition salt thereof. The pharmaceutical preparation according to any one of claims 1 to 3, wherein the pharmaceutical preparation produced is physically exerted on the skull or spinal column, ischemia, stroke, dyspnea, heart attack, thrombosis, cerebral embolism, neuropathic abnormality by AIDS, Use for the treatment of cerebral hemorrhage caused by cerebral hemorrhage, encephalomyelitis, hydrocephalus, postoperative symptom, brain infection, concussion, or trauma caused by increased intracranial pressure and / or treatment of sequelae of the condition .